INVESTIGATING THE COUPLING MECHANISM IN THE E. COLI MULTIDRUG TRANSPORTER, MdfA, BY FLUORESCENCE SPECTROSCOPY

N. Fluman, D. Cohen-Karni, E. Bibi

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

In bacteria, multidrug transporters couple the energetically favored import of protons to export of chemically-dissimilar drugs (substrates) from the cell. By this function, they render bacteria resistant against multiple drugs. In this work, fluorescence spectroscopy of purified protein is used to unravel the mechanism of coupling between protons and substrates in MdfA, an E. coli multidrug transporter. Intrinsic fluorescence of MdfA revealed that binding of an MdfA substrate, tetraphenylphosphonium (TPP), induced a conformational change in this transporter. The measured affinity of MdfA-TPP was increased in basic pH, raising a possibility that TPP might bind tighter to the deprotonated state of MdfA. Similar increases in affinity of TPP also occurred (1) in the presence of the substrate chloramphenicol, or (2) when MdfA is covalently labeled by the fluorophore monobromobimane at a putative chloramphenicol interacting site. We favor a mechanism by which basic pH, chloramphenicol binding, or labeling with monobromobimane, all induce a conformational change in MdfA, which results in deprotonation of the transporter and increase in the affinity of TPP. PHENOTYPE CHARACTERIZATION OF AZOSPIRILLUM BRASILENSE Sp7 ABC TRANSPORTER (wzm) MUTANT

A. Lerner1,2, S. Burdman1, Y. Okon1,2

1Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, 2The Otto Warburg Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel

Azospirillum, a free-living nitrogen fixer, belongs to the plant growth promoting rhizobacteria (PGPR), living in close association with plant roots. These bacteria are able to exert beneficial effects on plant growth and yield of many crops of agronomic importance under a variety of environmental and soil conditions. These positive effects are mainly as a result of morphological and physiological changes of the inoculated plant roots, with enhanced water and mineral uptake. Plant growth promoting substances produced by the bacteria seems to be at least partially responsible for these effects. Azospirillum cells are surrounded by a thick, dense, and tightly cell-bound layer of capsular polysaccharide (CPS) and by an outer lighter exopolysaccharide (EPS) layer bound to the cell. The EPS is involved in the attachment of Azospirillum to the plant root. Several genes involved in the Azospirillum brasilense-plant root interaction are carried on a 90 MDa plasmid called p90. p90 carries also genes involved in motility, adsorption to roots, colony morphology and genes belonging to the glycosyl- or mannosyl transferase, sugar dehydratase families and genes involved in the ABC transporter-dependent pathway (wzm and wzt). These ATP binding cassette (ABC) superfamily transporters (or traffic ATPases) are frequently involved in the translocation of complex carbohydrates across the cytoplasmic membrane. An A. brasilense wzm mutant was generated and its phenotype in comparison with the wild type strain Sp7 was evaluated. The wzm mutant was more resistance to heat, osmotic shock, osmotic pressure, desiccation and starvation but was more sensitive to elevated levels on NaCl, UV radiation and hydrogen peroxide. Differences in sensitivity to antibiotics and growth on different carbon sources were observed between the two strains. The wzm mutants also exhibited changes in cell morphology and motility. IDENTIFICATION AND CHARACTERIZATION OF THE CARBOHYDRATE ABC TRANSPORTERS IN CLOSTRIDIUM THERMOCELLUM

Y. Nataf1, S. Shulami1, S. Yaron1, F. Stahl2, R. Lamed5, E.A. Bayer4, T.H. Scheper2, A.L. Sonenshien3, Y. Shoham1

1Department of Biotechnology and Food Engineering, Technion–Israel Institute of Technology, Haifa, Israel, 2Institute of Technical Chemistry, University of Hannover, Germany, 3Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA, 4Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 5Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Clostridium thermocellum is an anaerobe thermophilic bacterium which efficiently degrades crystalline cellulose to soluble cellodextrins and is capable of producing ethanol. This bacterium produces a unique enzymatic complex, termed cellulosome, which integrates many glycoside hydrolases and mediates the attachment of the complex to cellulose. C. thermocellum is capable of assimilating cellodextrins which undergo phosphorylitic cleavage within the cell. This metabolic pathway allows the cell to obtain more ATP per sugar unit, since only one ATP molecule is required for the uptake of relatively large cellodextrins. Based on the recent published genome sequence of C. thermocellum, seven putative extracellular sugar binding proteins (SBP1-7) which are part of an ABC transport systems were identified. These His-tags fused proteins were purified and their ability to bind different sugars was tested using Isothermal Titration Calorimeter (ITC), Differential Scanning Calorimeter (DSC) and fluorescence quenching. Four of the binding proteins bind cellodextrins and one SPB binds laminaribiose. The ITC results indicated that all of the binding interactions were exothermic. The highest Kb values of SBP3, SBP4 and SBP5 analyses were gained with cellopentaose, suggesting that these proteins prefer to bind five sugar units. SBP7 binds only cellotriose and SBP6 interacts best with laminarbiose. Northern blot analysis indicated that the sbp3, sbp4 and sbp5 genes are part of 3.1kb, 4.0kb, and 3.3kb transcriptional units, respectively. Based on the genome sequence and the Northern blot results it appears that the ABC transporter genes are cotranscribed. E. COLI MULTIDRUG TRANSPORTER MdfA IS A MONOMER

N. Sigal, O. Lewinson, S.G. Wolf, E. Bibi

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

MdfA is a 410-residue-long secondary multidrug transporter from E. coli. Cells expressing MdfA from a multicopy plasmid exhibit resistance against a diverse group of toxic compounds, including neutral and cationic ones, because of active multidrug export. As a prerequisite for high-resolution structural studies and a better understanding of the mechanism of substrate recognition and translocation by MdfA, we investigated its biochemical properties and overall structural characteristics. To this end, we purified the beta-dodecyl maltopyranoside (DDM)-solubilized protein using a 6-His tag and metal affinity chromatography, and size exclusion chromatography (SE-HPLC). Purified MdfA was analyzed for its DDM and phospholipid (PL) content, and tetraphenylphosphonium (TPP+)-binding activity. The results are consistent with MdfA being an active monomer in DDM solution. Furthermore, an investigation of two-dimensional crystals by electron crystallography and 3D reconstruction lent support to the notion that MdfA may also be monomeric in reconstituted proteoliposomes. PROTEIN ACETYLATION/DEACETYLATION IN THE EXTREMELY HALOPHILIC ARCHAEON HALOFERAX VOLCANII

N. Altman-Price, S. Barak

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Protein acetylation and deacetylation reactions are known for sometime to be involved in many regulatory processes in eukaryotes. Recently it was shown that similar reactions have regulatory roles also in bacteria and archaea Sequence analysis of the haloarchaeon Haloferax volcanii genome enabled the identification of three putative protein acetyltransferases of the GCN5 family (Pat1, Pat2 and Elp3) and two deacetylases; (Sir2 and HdaI). Intriguingly, the gene that encodes for HdaI shares an operon with an archaeal histone homologue. We have used a gene knockout method to determine whether these putative genes are essential, and found that whereas Sir2 knockout strain can grow normally, HdaI deletion is lethal. Moreover, the specific HdaI inhibitor Trichostatin A inhibits cell growth. We also showed that Pat2 and Elp3 are “synthetic lethals”. Genetic analysis of the histone gene has shown that it is essential for growth. Site directed mutagenesis of the two unique lysine residues of the histone established a link between the histone and the acetylation/deacetylation processes in Haloferax volcanii. Changing any of the lysine residues to glutamine made the cells more sensitive to Trichostatin A while mutagenesis of the lysine in the C-terminal domain to arginine rendered the cells more resistant to that drug. IDENTIFICATION AND CHARACTERIZATION OF EXTREMOPHILE YEAST STRAINS

L. Avrahami, A. Grabelsky, D. Engelberg, S. Braun

Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Israel

Given the global climate changes, a most urgent challenge of biotechnology research is finding extremophile strains. In the agriculture industry, for example, there is a concerning decrease in yields due to global warming. Obtaining extremophile strains is not trivial because their properties may be obtained by a combination of many genetic and epigenetic modifications. In natural extremophiles, the properties are a result of hundreds of millions of years of evolutionary selection. It is clearly impossible to mimic such a process in the laboratory, particularly for plants and other organisms of economical value. It should be emphasized that thermotolerant strains that are able to withstand temporary stress, frequently by ceasing cell cycle, have been obtained, but those are not extremophiles. To facilitate the isolation of extremophiles we initiated the current project aimed at finding extremophile yeast strains, studying the mechanism that rendered them extremophile and applying the knowledge to other organisms. The premise is that it would be possible to identify the desired properties in yeast by applying the powerful genetic and molecular systems available for this organism. To achieve these goals we are performing a battery of high-throughput screening applying several modes of selection. Central to our study is a specifically- designed chemostat selection system, which is fine-tuned for gradual improvement in the desired phenotype (i.e., small increase in temperature or alcohol concentrations). We have created library of transposon-induced mutations in yeast and subjected it to the chemostat selection at the growth temperature of forty four degrees Celsius. During the selection process we collected several theremopliles including one strain that is resistant to both heat and high ethanol concentration. Also, we recently isolated a few mutants from the yeast deletion project that show high growth rates. Molecular analysis of the strains isolated is currently in progress. GERMINATION AND SEEDLING SURVIVAL FOLLOWING PERIODS OF DESICCATION IN SCHISMUS ARABICUS (Poaceae)

T. Gendler, Y. Gutterman

Ben-Gurion University of the Negev, Desert Research Institutes, Beer-Sheva, Israel

Seedlings of Schismus arabicus (Poaceae), may survive after several weeks of desiccation. They have the ability to continue to grow and produce seeds when rewetted, following a period of desiccation. Such ability is very important for survival under extreme desert conditions, where there are periods of draught following the rain that engender germination. Nearly a 100% of S. arabicus caryopses germinated when caryopses are wetted each month during the different seasons of the year. However there are big fluctuations in the percentages of seedling survival after desiccation during the year. It depends on the month of the year when caryopses were germinated. When caryopses were germinated in January - February, close to a 100% of the seedlings survived after 7, 14, 21, 28, 35 and 42 days of desiccation. However, when caryopses were germinated in July, no one of the seedlings survived after desiccation. These results where repeated during 4 years of monthly germination experiments, in which more than 4800 seedlings were germinated in July, desiccated and rewetted. There was a gradual increase in seedling survival percentages of seedlings that germinated from August to September – October, and a gradual decrease in seedling survival percentages in seedlings that germinated from March to July. Such annual fluctuations in the percentages of seedling survival were found during 4 years of monthly experiments. Maternal, Environmental and annual changes in internal factors, in the dry caryopses, are involved in the ability of their seedlings to survive periods of desiccation including: caryopsis age and size. The larger caryopses germinated faster in a larger range of temperatures to higher percentages with the higher percentages of seedling survival following desiccation. No seedlings survived from caryopses germinated during the first year after seed maturated, however 2 to 4 years after seed maturation a 100% may survive periods of desiccation. GENE EXPRESSION AT LOW TEMPERATURE IN THE FUNGUS BOTRYRIS CINEREA

S. Ish-Shalom, A. Lichter

Department of Postharvest Science, The Volcani Center, Ministry of Agriculture, Bet-Dagan, Israel

The fungus Botrytis cinerea is a filamentous necrotrph, known to attack hundreds of plant species especially after harvest and during cold storage. It is an efficient postharvest pathogen due to its capacity to grow and sporulate under cold conditions C). Our research goal is to understand the mechanisms that enable the fungus toֶ¡0~) grow and develop at 0¡ֶC. Our hypothesis is that in order to adjust and grow at low temperature, the fungus needs to change its gene expression pattern and this change will reflect on relevant mechanisms. Two strains of this fungus, BO5.10 and T4 were sequenced recently and this enabled us to screen literature for cold-induced genes and study their expression pattern. Additionally, a cDNA subtraction library was used to identify several cold-induced cDNAs. The expression profile was examined by qPCR analysis of cDNA prepared at 4 time points, following a shift from growth at 20¨C to 4¨C. The results demonstrate robust cold induction of 4 genes isolated from the subtraction library, among them is a bacterio-Rhodopsin-like gene. Several genes which were isolated based on current literature, display a strain-specific cold induction pattern while the expression of other genes did not change or was repressed at low temperature. One of the important technical aspects of this study involves the tubulin-ג¥ use of normalizers for qPCR analysis. Three genes, 18S rRNA, actin and were used as normelizers. However, their expression pattern did not necessarily act predictably, requiring multiple comparison of the expression in order to reach firm conclusions. This study lays the infrastructure for confirmation of results from global expression under low temperature which is expected to be carried out in the near future. In addition, functional analysis of genes that were strongly expressed at low temperature is in progress. CHARACTERIZATION OF N-GLYCOSYLATION SIGNALS IN THE ARECHAEA HOLOFERAX VOLCANII

H. Magidovich, J. Eichler

Department of Life Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel

N-linked glycosylation is an important post-translational modification in all three domains of life. The process is best understood in Eukarya, where it occurs at select Asn residues within Asn-X-Ser/Thr sequons, where X is any residue but Pro. While it has long been known that eukaryal proteins experience N-glycosylation, it was only recently shown that Bacteria are also capable of performing this processing event. To date, the bacterial N-glycosylation pathway has been best characterized in the food- borne pathogen, Campylobacter jejuni. Here too, the Asn-X-Ser/Thr sequon is recognized, although the additional requirement for an acidic residue two positions upstream of the Asn residue exists. In contrast to relatively well-defined pathways of N-glycosylation in Eukarya and Bacteria, only little is known of the steps involved in archaeal N-glycosylation. This lack of understanding also pertains to the amino acid sequences recognized by the archaeal N-glycosylation apparatus. N-glycosylation occurs at Asn-X-Ser/Thr sequons, although the presence of Ser/Thr may not be essential. Moreover, as in the other two domains, not all archaeal sequons are modified. Towards understanding the rules governing N-glycosylation in Archaea, three approaches are being followed. In the S-layer glycoprotein of the halophilic archaeon Haloferax volcanii, the sequon including Asn-370 is not modified, whereas those sequons based on Asn-13 and Asn-83 are glycosylated. Site-directed mutagenesis will be used to convert the region bordering Asn-370 into one resembling those surrounding the modified Asn residues. Secondly, site-directed mutagenesis will be used to introduce sequons into a heterologously-expressed chimeric secretory protein, SP-CBD, with the aim of attaining an N-glycosylated version of this reporter protein. Finally, sequons will be introduced into native H. volcanii secreted enzymes so as to obtain modified versions of these proteins, possibly exhibiting modified properties. Relying on these approaches, rules governing glycosylation in Archaea will be defined. THE TOPOLOGY OF PROTEINS PUTATIVELY INVOLVED IN N- GLYCOSYLATION IN THE ARCHAEA HALOFERAX VOLCANII

N. Plavner, J. Eichler

Department of Life Sciences, Ben Gurion University, Beer Sheva, Israel

N-linked glycosylation is recognized as an important post-translational modification across all domains of life. While in Eukarya and Bacteria the process is well-defined, numerous questions concerning archaeal N-glycosylation remain. Towards redressing this situation, the location and orientation of the proteins putatively involved in N- glycosylation process in Archaea will be defined, using the halophilic archaea, Haloferax volcanii, as a model organism. Bioinformatics provided topologies of five proteins considered, i.e. Alg5A, Alg5B, Dpm1A, Dpm1C and Mpg1D. To confirm these predictions, the proteins were fused to the cellulose-binding domain (CBD) of the Clostridium thermocellum cellulosome, allowing for cellulose-based protein isolation in hypersaline conditions. To date, plasmids encoding CBD-Alg5A, CBD- Alg5B, CBD-Dpm1A, CBD-Dpm1C and CBD-Mpg1D were used to transform H. volcanii and soluble and membrane fractions were separated and purified with cellulose. These experiments showed that Alg5B and Mpg1D are soluble proteins, whereas Alg5A, Dpm1A and Dpm1C can be equally found in both fractions. To confirm the topology of the five fusion proteins, proteinase K accessibility was assessed by determining the amount of fusion protein digested over time. Alg5B and Mpg1D and their CBD-moieties are protected from proteinase K. Alg5A and Dpm1A were partially digested while their CBD-moieties fused to their N-terminal were protected, pointing to their membrane association and internal orientation of their N- terminus. While Dpm1C was expected to be partially digested, steric considerations may explain the failure of the protease to cleave the protein. Here too, the N-terminal CBD moiety was protected. An additional approach to define the topology involves 4- acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid (AMS) and [H3] N- ethylmaleimide (NEM). AMS binds only to externally-oriented cysteines while NEM penetrates the membrane and binds to any available cysteine. Using single cysteine mutants, pre-incubation with AMS will prevent subsequent [H3] NEM binding, thus delineating the topology of the proteins being studied. SENSING AND RESPONDING TO HYPERCAPNIA IN C. ELEGANS

K. Sharabi1, J. Sznajder2, Y. Gruenbaum1

1Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Department of Medicine, Northwestern School of Medicine, Northwestern University, Chicago, IL, USA

High CO2 levels (hypercapnia) occur in several lung diseases such as COPD and ARDS, and are associated with the worse outcomes. Previous studies showed that in lung cells, hypercapnia, but not changes in extra or intracellular pH impairs alveolar fluid reabsorption, suggesting a cellular mechanism that senses hypercapnia. Our major goal is to identify and characterize the sensing and response mechanisms to hypercapnia, using the Caenorhabditis elegans as a model animal. Growing Caenorhabditis elegans in 5% CO2 had no apparent effects. In contrast, chronic exposure to 9%-19% CO2 caused reduced fertility, delayed development, extension of lifespan and reduced motility. Changing the external pH had no effect, suggesting that the response to hypercapnia is due to sensing the CO2/HCO3- levels. Hypercapnic conditions also lead to a dramatic change in gene regulation. Early responding genes include genes of the hormone receptor family and genes that are involved in chemo- sensing, transcriptional regulation, protein degradation and innate immune response. Later responding genes include the carbonic anhydrase genes, many collagen genes and the MSP family genes. Strains homozygous for deletion in Jun kinase or protein kinase C showed a mild rescue of progeny size as compared with control, while deletion in the rhesus genes showed a further reduction in progeny size. The strain aqp-2 also failed to lay eggs and had a slower developmental time. PHYSIOLOGICAL AND MOLECULAR EVIDENCE OF HEAT ACCLIMATION MEMORY ASSOCIATED WITH CHROMATIN MODIFICATION: A LESSON FROM THE HEART

A. Tetievsky1, O. Cohen1, L. Eli-Berchoer1, G. Gerstenblith3, M.D. Stern4, I. Wapinski2, N. Friedman2, M. Horowitz1

1Laboratory of Environmental Physiology, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, 2School of Computer Science and Engineering, Hebrew University of Jerusalem, Israel, 3Departments of Medicine and Cardiology, Johns Hopkins University, Baltimore, MD, USA, 4Laboratory of Cardiology and Science, Gerontology Research Center, Johns Hopkins University, Baltimore, MD, USA

Sporadic findings in humans suggest that re-induction of heat acclimation (AC) after its loss occurs markedly faster than during the initial AC session. Animal studies substantiated that the processes underling acclimation are molecular. Goals: (i) To test the hypothesis that faster re-acclimation (re-AC) implicates ‘molecular memory’ and is linked to epigenetic modifications, (ii) To identify the suite of stress responsive genes involved in AC memory. Using our male Rattus norvegicus AC model physiological and molecular/cellular aspects of deacclimation (de-AC) for 30 and 60 days and subsequent re-AC for 2 days were assessed. The physiological criteria for de-AC/re-AC, integrative, organ and cellular levels, were: Tc-plateau, at 41 C, infarct size following ischemia/reperfusion insult and time to rigor contracture in isolated anoxic cardiomyocytes. A stress-associated gene chip analysis and qPCR were used to detect genomic responses. Protein levels were detected using Western immunoblotting (WB). Histones acetylation and phosphorylation were measured using Chromatin immunoprecipitation (ChIP) and WB. In vivo measurements of Tc profiles during heat-stress and ex vivo assessment of cross-tolerance to ischemia/reperfusion or anoxia demonstrated that re-AC only needs 2d vs the 30d required for the initial development of AC. Gene profiling highlighted a group of transcriptionally activated genes (37%), among which HSPs, antiapopototic and chromatin remodeler genes, did not resume pre-acclimation levels after de-AC of 30d despite the return of the physiological phenotype to its pre-acclimation state, suggesting a dichotomy between geno- and the pheno- types. Detection of hsp70 and hsf1 transcripts, HSP72, HSF1/P-HSF1 and BCLxl protein profiles and levels of H4 acetylation and Histone H3 phosphorylation (Ser10) agreed with the molecular changes observed in the AC, de-AC and re-AC groups. We argue that chromatin remodeling emerging with AC and retained during DeAC is an upstream regulator of the transcription state, preconditioning to faster cytoprotective acclimatory-memory. THE COPING OF SOIL MICROBIAL POPULATION DIVERSITY WITH GLOBAL CLIMATE CHANGE

I. Shamir, Y. Steinberger

Bar-Ilan University, Ramat Gan, Israel

The spatial structuring of soil properties and biota has been studied in man-made, natural, and secondary succession processes, with little attention given to a climatic gradient. We hypothesized that variation in a microbial community and its functional diversity along a climate gradient will be accompanied by a decrease in spatial structuring of organic matter resources, leading to decrease in microbial community parameters and increase in microbial functional diversity. Deceleration in microbial activity rate caused by extreme conditions lead to decrease in soil mineralization and decomposition processes. The study site was composed of four experimental stations along a climatic gradient in Israel. These experimental sites represent humid Mediterranean, Mediterranean, semi-arid, and arid climates. Community level physiological profile and microbial functional diversity were determined using the MicroRespTM method, which was also used for the calculation of basal respiration, soil microbial biomass, and qCO2 index evaluation. The results obtained in this study demonstrate a continuous and clear increasing trend in soil microbial biomass amount as well as organic carbon content, from south to north. While the lowest microbial biomass population was found in the south, the qCO2 index showed a clearly opposite trend, with high levels found in the south, decreasing toward the north. This evidence is supported by the fact that the northernmost site is a humid Mediterranean climate with lower heterogeneity, while the southernmost site symbolizes the harsh arid climate with extreme environmental conditions and relative higher heterogeneity. We can assume that there is a decreasing incline of the soil microbial population running from north to south, followed by correlation to climate change. Understanding the changes in community functional physiological profile of the soil microbial population will help us to estimate climatic changing processes and to decode the magnitude of the effect of climatic change on the soil biota community. Met-INDUCED MEMBRANE BLEBBING AND ITS ROLE IN CELL MIGRATION

A. Laser1, O. Kot1, J.H. Resau2, L. Mittelman1, I. Tsarfaty1

1Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 2Van Andel Research Institute, Grand Rapids, MI, USA

Hepatocyte Growth Factor/Scatter Factor (HGF/SF) induces mitogenic, motogenic and morphogenic changes mediated by the Met receptor tyrosine kinase. Met-HGF/SF signaling is also associated with protrusive cell motility during embryogenesis, invasiveness and metastasis. In this work we examine HGF/SF-induced cell motility in live cells expressing fluorescent-tagged Met and its signaling molecules using confocal microscopy. HGF/SF induces a significant increase in Met receptor dimerization as soon as 1 minute after treatment (FRET). HGF/SF also induces immediate, highly dynamic membrane blebbing. Elevation of medium osmolarity to a final concentration of 100mM sorbitol completely inhibits membrane blebbing. Met and actin spatio-temporal localization within blebs show that both proteins accumulate where blebs retract. Bleb retraction generates microspike membrane structure, leading to high concentration of Met, actin and the membrane markers YFP- Mem and YFP-GPI. Fluorescent recovery after photo-bleaching (FRAP) demonstrated increased Met lateral mobility after HGF/SF stimulation. Modification of the actin cytoskeleton by jasplakinolide (jaspamide) or latrunculin A attenuated Met lateral mobility both in untreated and HGF/SF treated cells. Fluorescence resonance energy transfer (FRET) analysis revealed that Met and actin interaction is oscillatory, suggesting a periodical attachment and detachment of Met from the actin cortex. Based on these results we suggest a bi-phasic model for Met signaling: Met initial HGF/SF-dependent phosphorylation induces membrane blebbing. Bleb retraction generates membrane spikes containing high Met concentrations (Met Activation Domains), leading to a profound, HGF/SF-independent increase in Met phosphorylation and signaling. This model may shed a light on the subcellular, spatial and temporal mechanism underlying Met-induced cell motility as a first step of metastasis. Key words: Met; HGF/SF; Actin; cell migration; FRAP; FRET; GFP TWO-PHOTON IMAGING OF HEMATOPOIETIC CELL MOBILIZATION FROM THE BONE MARROW OF LIVE MICE

P. Goichberg, I. Milo, G. Shakhar

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

The bone marrow (BM) is the primary hematopoietic organ sustaining blood cell production throughout life. Immature and mature hematopoietic cells interact in the BM with a variety of non-hematopoietic cell types, including endothelium. The chemokine CXCL12 and its receptor CXCR4 play important role in retention and development of hematopoietic cells. Mobilization of hematopoietic progenitor cells (HPCs) from the BM to the circulation is induced during stress, injury and in clinical settings by chemotherapy or cytokines. Recently, a new molecule, AMD3100, which is a CXCR4 antagonist, was shown to induce rapid mobilization of HPCs. We set out to determine, using two-photon imaging of BM in live mice, how disrupting the CXCL12-CXCR4 anchorage signal leads to mobilization. We aim to clarify whether it affects the directionality and velocity of HPC motility, HPC compartmentalization in the BM or their capacity to adhere to the endothelium and cross it. At this stage of the project, we have developed a chimeric mouse model to specifically image hematopoietic and non hematopoietic cells, succeeding in visualizing the motility of these cells in the BM cavities in skulls of anesthetized mice. Furthermore, we visualized hematopoietic cell movement in BM during AMD3100 induced mobilization. Thus, our preliminary observations indicate that the model is useful for intravital real-time imaging interactions of hematopoietic cells with the BM components and will enable us to decipher cellular and molecular pathways regulating hematopoietic cell trafficking. DEVELOPMENT OF COMBINED ANALYSIS OF MORPHOLOGY, FLUORESCENCE IN SITU HYBRIDIZATION (FISH) AND DNA PLOIDY IMAGE CYTOMETRY OF CANCER CELLS

S. Oren1, T. Shani2, A. Hirshberg2, G. Rechavi1, N. Amariglio1, L. Trakhtenbrot1

1Chaim Sheba Medical Center, Tel-Hashomer, Israel, 2The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Ramat Aviv, Israel

Abnormal nuclear DNA content is a well-recognized and common feature of human cancer and may be an indicator of aggressive biologic behavior in neoplastic cells. DNA content analysis of cancer cells using DNA flow cytometry and quantitative DNA image cytometry is performed in clinical practice in attempt to predict prognosis and appropriate treatment. The sensitivity of DNA flow cytometry method is reduced since it doesn't separate tumor cells from normal cells and as a result, small populations of non-diploid pathological cells may not be detected. The interpretation of DNA image cytometry histograms is not always straightforward, and there is a gray zone between a true diploid histogram and an aneuploidy pattern. We suggest that parallel analysis of morphology, FISH and DNA ploidy image cytometry based on measurements of DAPI intensity, can increase the sensitivity of the detection of cancer cells. Combination of morphology and FISH on the same cell enables clear distinction between pathological and normal cells. In addition, FISH analysis permits the discernment of non-diploid cells from proliferating mitotic cells. Depending on fluorescent signals, measured cells were classified into two classes, class 1 for normal and class 2 for pathological cells. DAPI intensity was measured and DNA index (DI) was defined as the mean value of class 2 measurements divided by the respective mean value of class 1. To analyze the accuracy of the combined analysis 30 samples of hematological and oral malignancies were analyzed by standard flow cytometry, image cytometry using Velocity Grid Confocal system as well as by new method. We found that DI values obtained by all three methods were highly correlated. It can be concluded that combined analysis is highly accurate to determine the DNA ploidy. Thus it enables the identification of small populations of aneuploid cells, thereby enhancing the specificity of pathological cell detection. THE LAMIN AND LEM-DOMAIN-BINDING PROTEIN BARRIER-TO- AUTOINTEGRATION FACTOR (BAF) BLOCKS PREMATURE CELL FUSION BY REGULATING SPECIFIC GENES AND MAINTAINS ADULT MUSCLE INTEGRITY IN C. ELEGANS

M. Davidovich-Cohen1, A. Margalit1, E. Neufeld1, N. Feinstein1, B. Podbilewicz2, Y. Gruenbaum1

1Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

Mutations in either lamin A/C or emerin cause Emery Dreifuss muscular dystrophy (EDMD). The mammalian BAF is present in protein complexes together with both lamin A and emerin,. However, the role of BAF in EDMD is unknown. Protein complexes containing BAF, lamin and emerin and the requirement of specific residues in BAF for these complexes are evolutionarily conserved in C. elegans. In C. elegans early embryos BAF-1 is required for chromatin organization, capture segregated chromosomes within the nascent nuclear envelope and to assemble lamin and emerin proteins in reforming nuclei. In turn, BAF-1 requires lamin and emerin proteins to localize at the nuclear periphery. Animals homozygous for a deletion of the entire baf-1 ORF, in which the maternal supply of BAF-1 is sufficient to complete embryogenesis and larval stages, revealed that BAF-1 is required for nuclear organization and several different tissue-specific pathways including maturation and survival of the germline, the second and third phases of distal tip cell migration, vulva formation and prevention of premature fusion of cells, suggesting specialized roles for BAF-1 in gene expression. BAF-1 also inhibits premature fusion of the seam cells by binding to the promoters of EFF-1 and AFF-1 fusogens. We are currently using the Tiling Array (ChIP-chip) technique to map all specific C. elegans promoters that bind BAF-1. Interestingly, BAF-1 is also required to maintain the integrity of specific muscles in the mid-body and tail regions of the adult animal. The requirement of BAF-1 interactions with emerin, lamin and DNA for these functions is currently being investigated. These results probably have direct implications on the mechanism of human muscular dystrophies caused by mutations in either A-type lamins or the protein emerin. MATEFIN/SUN-1, A NUCLEAR ENVELOPE PROTEIN, IS ESSENTIAL FOR EMBRYONIC AND GERMLINE DEVELOPMENT AND HOMOLOGOUS PAIRING IN CAENORHABDITIS ELEGANS MEIOSIS

A. Fridkin1, E. Kaplan1, A. Penkner2, V. Jantsch2, Y. Gruenbaum1

1Department of Genetics, Hebrew University of Jerusalem, Israel, 2Department of Chromosome Biology, Max F. Perutz Laboratories, Vienna, Austria

The C. elegans mtf-1/sun-1 gene encodes matefin/SUN1, which is a nuclear membrane SUN-domain protein. Matefin/SUN1 has a germline-specific expression. Animals homozygous for mtf-1/sun-1 deletion allele show that matefin/SUN1 is essential for germline maturation and/or survival. Matefin/SUN1 is also required for early embryogenesis since mtf-1/sun-1 (RNAi) embryos die around the ~300-cell stage with defects in nuclear structure, DNA content and chromatin morphology. Previous studies (Malone et al, Cell, 2003) have shown that matefin/SUN-1 is required for centrosomes attachment to nucleus in the 2-cell embryos. Staining gonad of C. elegans hermaphrodite, homozygous for a gk199 deletion allele of mtf/sun-1 revealed that this is probably not the case at later stages of development. Recently a highly specific mutation (jf18) in mtf-1/sun-1 was identified. It provided direct evidence for active involvement of the nuclear envelope in homologous chromosome pairing in C. elegans meiosis. Our data show that mtf-1/sun-1 mediates a novel nuclear lamina pathway(s) required for embryonic development, for germline proliferation and survival and for homologous chromosome pairing. ALTERNATIVE SPLICING OF A PUTATIVE TUMOR SUPPRESSOR GENE PRODUCING NUCLEAR AND NUCLEOLAR PROTEINS

N. Kinor, Y. Shav-Tal

Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

Loss of heterozygosity (LOH) represents the sequel of genetic instability that can lead to the loss of tumor suppressor genes that promote cell growth and tumor development. The product of a novel gene has recently been implicated as a tumor suppressor in cervical carcinoma cells as a result of LOH. Our laboratory has identified three alternatively spliced forms of this gene, each exhibiting a different subcellular distribution. Using GFP fusions we found that SP1 is predominantly nucleolar, SP2 is mainly nucleoplasmic, and SP3 is distributed throughout the whole cell. SP2 is a shuttling protein while SP1 does not shuttle. The nucleolar SP1 protein localizes to the granular component of the nucleolus, a region responsible for the assembly of rRNA with the ribosomal subunits. Treating cells with RNase and DNase did not change the distribution of SP1 indicating that it might be associated with protein complexes and not with nucleic acids. Using photobleaching techniques (FRAP) we measured the dynamics of these proteins. SP1 molecules showed slow kinetics of movement in and out of the nucleolus, and also included a fixed nucleolar fraction that does not leave the nucleolus. Transcriptional inhibition changed SP1 mobility in the nucleolus. These types of kinetics are common to many nucleolar proteins. SP2 molecules diffused rapidly throughout the nucleoplasm. Time-lapse live-cell imaging throughout the cell cycle demonstrated that both SP1 and SP2 proteins distributed diffusely in the cell during mitosis. After division, SP1 first localized to pre-nucleolar bodies, which later on form the nucleolus. We are now identifying the proteins that interact with SP1 in order to define whether this is the form that plays a role as a tumor suppressor in cervical carcinoma cells. LAMINOPATHIC MUTATIONS INTERFERE WITH THE LOCALIZATION AND DYNAMICS OF NUCLEAR LAMINS IN C. ELEGANS

A. Mattout1, N. Wiesel1, S. Melcer1, N. Melamed-Book1, O. Medalia2, U. Aebi3, Y. Gruenbaum1

1Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Department of Life Sciences and NIBN, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 3M.E. Mueller Institute for Structural Biology, Biozentrum, University of Basel, Switzerland

Lamins are nuclear-specific intermediate filament proteins (IFs) that, together with a complex set of inner nuclear membrane proteins, form a filamentous meshwork between the inner nuclear membrane and the peripheral chromatin. Lamins are involved in most nuclear activities; and mutations in human lamins or their associated proteins cause laminopathic diseases ranging from muscular dystrophies to accelerated ageing. Previous studies showed that lamin dimers form filamentous structures through ‘head-to-tail’ interactions, which then form paracrystalline structures in vitro. C. elegans has a single evolutionarily conserved lamin protein, termed Ce-lamin. This lamin is unique in its ability to form under specific conditions 10-nm filaments, similar to other IFs. We have used negative staining to determine the structure of the 10-nm filaments and to find how mutations in residues, which are conserved in human lamin A and lead to laminopathic diseases, affect lamin filaments assembly. We have further analyzed the effects of these mutations on lamin organization and dynamics in vivo, by creating mutated gfp::Ce-lamin transgenic strains and by performing FRAP experiments. Most of these mutants show an abnormal nuclear localization and /or an abnormal nuclear morphology, and two of the tested mutants (Y59C and R64P) show also a change in the dynamics properties of the Ce-lamin. Our results show for the first time the structure of the lamin filament as well as how mutations affect the filament assembly both in vitro and in vivo. NET-ACT PROLOGUE – A NOVEL NUCLEAR ENVELOPE PROTEIN

S. Melcer1, H. Hermann2, Y. Gruenbaum1

1Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Division of Cell Biology, DKFZ, Heidelberg, Germany

The nuclear envelope (NE) is an active barrier between the cytoplasm and the nuclear interior, harboring many proteins that play a major role in structure and most known functions in the nucleus. We confirmed NET-AcT, previously noted in NE proteomic studies, to be a novel NE protein in C.elegans. NET-AcT is essential for worm fertility and has in vitro acetyl-transferase activity, highly suggestive of a possible chromatin remodeling function. NET-AcT thus promises to be an intriguing addition to the growing family of NE proteins. MAPPING THE NUCLEAR PORE COMPLEX COMPONENTS (NUCLEOPORINS) IN PLANTS

S. Sultan1,2, T. Tzfira2, M. Elbaum1

1Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel, 2Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

Nuclear pore complexes (NPCs) are large proteinacious structures that cross the nuclear envelope (NE), the double membrane surrounding the eukaryotic cell nucleus. They render the NE barrier permeable, allowing passive diffusion of small molecules, and selective, bidirectional traffic of proteins, RNA, and foreign DNA. Although the NPC has many roles in the cell, and while almost all of the nucleoporins (Nups) have been characterized in vertebrates and yeast, the plant NPC composition remains largely unknown. Bioinformatic approaches have yielded several putative Nups orthologues in the Arabidopsis genome. We now analyze protein-protein interactions between putative Nups using the yeast two hybrid system. Clones have been PCR- amplified from a cDNA library of Arabidopsis thaliana. These clones include, for example the Sar3 (mos-3) sequence that showed high homology to vertebrate Nup196 and to Nup145 from yeast. Data about the search and the protein-protein interaction between components of the plant NPC will be presented. We believe that understanding how nucleoporins interact with each other to form the plant NPC structure will shed light on a variety of plant cell processes LAMINOPATHIC MUTATIONS INTERFERE WITH THE ASSEMBLY AND STRUCTURE OF NUCLEAR LAMINS IN C. ELEGANS

N. Wiesel1, A. Mattout1, K. Ben-Harush2, H. Herrmann3, U. Aebi4, O. Medalia2, Y. Gruenbaum1

1Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Department of Life Sciences, NIBN, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 3Division of Cell Biology, DKFZ, Heidelberg, Germany, 4M.E. Mueller Institute, Biozentrum, University of Basel, Switzerland

In the metazoan cell, underlying the inner nuclear membrane is a protein meshwork termed the nuclear lamina. It is composed of lamin proteins and a growing number of lamin-associated proteins. The lamins are evolutionarily conserved nuclear intermediate filaments (IFs) that are involved in most nuclear activities including maintaining nuclear shape, spacing of nuclear pores, replication of DNA, regulation of gene expression, transcription elongation by Pol II, nuclear positioning, segregation of chromosomes, meiosis and apoptosis. Mutations in human lamins cause a variety of genetic hereditable diseases collectively termed laminopathies ranging from muscular dystrophies to accelerated ageing. Previous studies showed that lamin dimers form filamentous structures through ‘head-to-tail’ interactions, which then form paracrystalline structures in vitro. C. elegans has a single evolutionarily conserved lamin protein, termed Ce-lamin. This lamin is unique in its ability to form under specific conditions 10-nm filaments, similar to other IFs. We have used both negative staining and cryo-electron tomography to determine the structure of the 10- nm filaments and paracrystals and to find how mutations in residues, which are conserved in human lamin A and lead to laminopathic diseases, affect lamin filaments or paracrystals assembly. We have further analyzed the effects of these mutations on lamin organization and dynamics in vivo, by creating mutated gfp::lmn1 transgenic strains and by performing FRAP experiments. Our results show for the first time the structure of the lamin filament and paracrystal as well as how mutations affect the filament assembly both in vitro and in vivo. OVERPRODUCTION OF MazF CAUSES A GLOBAL CHANGE IN THE PATTERN OF PROTEIN SYNTHESIS OF ESCHERICHIA COLI

S. Amitai, I. Kolodkin-Gal, H. Engelberg-Kulka

Department of Molecular Biology, Hebrew University - Hadassah Medical School, Jerusalem, Israel

Pairs of genes consisting of toxin-antitoxin systems are located on the chromosomes of many bacteria. We were the first to describe a prokaryotic chromosomal toxin- antitoxin system, mazEF, which is regulatable and responsible for programmed cell death. This mazEF system encodes a stable toxin, MazF, and a labile antitoxin, MazE, that prevents the lethal effect of MazF. Death mediated by the chromosomally borne E.coli mazEF system is triggered by stressful conditions that inhibit mazEF expression. Here we report that MazF overexpression causes a global change in the pattern of protein synthesis of E.coli. It causes a reduction in the level of synthesis of proteins whose MW is above ~20kDa and increment in the level of synthesis of proteins whose MW is below ~20kDa. We identified 16 proteins, whose MW is below ~20kDa and their level of synthesis is increased after MazF induction. Deleting each of the corresponding genes revealed that these proteins can be divided into three classes regarding their involvement in mazEF-mediated cell death:(i) proteins which are required for death of most of the population; (ii) proteins which are necessary for the persistence of a small sub-population; (iii) proteins which are not involved in mazEF-mediated death, at least not under the stressful conditions which we examined. MazF was previously reported by other investigators to be an endoribonuclease, which preferentially cleaves single stranded mRNAs at ACA sequences in a ribosome-independent manner. According to our results, which show that MazF overproduction causes a global change in the pattern of protein synthesis, it seems that long mRNAs are more susceptible to MazF cleavage than shorter ones. Here we suggest a mechanism of protection of relatively short mRNAs which permits a preferential synthesis of relatively small proteins (less than ~20kDa) over larger ones after MazF induction. MITOCHONDRIA MEDIATED ANTI-APOPTOTIC CASCADE IS ENHANCED IN HEAT ACCLIMATED RATS

M. Assayag1, G. Gerstenblith2, M. Horowitz1

1Laboratory of Environmental Physiology, Hebrew University of Jerusalem, Israel, 2Department of Medicine and Cardiology, Johns-Hopkins University, Baltimore, MD, USA

Acclimation is a reversible "within lifetime" phenotypic adaptation to long-term environmental stress. The acclimatory steady state enhances the ability to cope with other stressors. Apoptosis is an evolutionarily conserved process, wherein cells die without provoking a significant inflammatory response. It is mediated via death ligands or through an intrinsic pathway. Mitochondria play an important role in the intrinsic pathway via the activation of the Bcl-2 protein family, cytochrome c, caspase 9 and finally caspase 3. Previous studies in our laboratory demonstrated a significant up-regulation in the expression of anti-apoptotic genes in heat acclimated animals. In this study we tried to determine whether heat acclimation decreases apoptosis following heat stress in the heart, to examine the differences in the kinetics of heat shock mediated apoptosis and to investigate the role of the mitochondria in the apoptotic cascade and cytoprotection. Two groups of males Rattus norvegicus normothermic (C, ambient temperature 24 C) and heat acclimated (AC, ambient temperature 33 C) were subjected to 2 hours of heat stress at 41 C and sacrificed following different recovery periods. mRNA and protein levels from rat hearts were evaluated by standard techniques. Following heat stress, the Bclxl / Bad protein ratio and cytochrome c protein levels were higher in the mitochondrial fraction of AC vs C groups. Significantly lower protein levels of caspase 9 and of the active form of caspase 3 were found in the AC compare to the normothermic animals. In contrast, no change in caspase 8 transcripts in both groups was observed. Tunel analysis of the normothermic and acclimated hearts 10 hours after heat stress showed a significant difference between heat acclimated and normothermic hearts. Our data indicate that heat acclimation enhances anti-apoptotic pathways in response to heat stress via shifting the balance between pro- and anti-apoptotic Bcl-2 proteins, thus implying mitochondrial mediated cytoprotective activity. GOLGI UDPase CONTRIBUTES TO E4orf4-INDUCED APOPTOSIS IN MAMMALIAN CELLS

M. Avital, R. Koren, R. Sharf, T. Kleinberger

Department of Microbiology, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Studies in mammalian tissue culture cells indicate that the adenovirus E4orf4 protein induces classical as well as non-classical (caspase-independent) cancer cell-specific apoptosis, which requires an interaction between the viral protein and an active protein phosphatase 2A (PP2A). Our laboratory is using various genetic model systems to investigate the mechanisms underlying induction of apoptosis by E4orf4. A genetic screen in yeast revealed an E4orf4-associating protein, Ynd1, which is required for E4orf4-induced toxicity. Ynd1 genetically interacts with PP2A, and both of them additively affect E4orf4 induced toxicity. Ynd1 and PP2A also interact physically. A mammalian homologue of Ynd1, Golgi UDPase, interacts with E4orf4 in human cells and its contribution to E4orf4-induced apoptosis is currently under investigation. We show that over-expression of the UDPase significantly increases E4orf4-induced apoptosis. Furthermore, pull-down experiments suggest that the UDPase physically interacts with the PP2A-B subunit, similarly to the findings in yeast. We also found that changes in PP2A-B subunit levels affected UDPase expression, suggesting a functional interaction between them. Our laboratory continues to study the physical and functional interactions between the UDPase, PP2A and E4orf4, which contribute to E4orf4-induced apoptosis, and the results will be discussed. REGULATION OF THE PRO-APOPTOTIC MASPIN GENE BY E2F1 ENHANCES CHEMOTHERAPY-INDUCED APOPTOSIS OF CANCER CELLS

B. Ben-Shachar, D. Ginsberg

The Mina and Everard Goodman Faculty of Life Science, Bar Ilan University, Ramat Gan, Israel

The transcription factor E2F1 regulates both cell proliferation and apoptosis. Our study shows that E2F1 also enhance chemotherapy-induced apoptosis. This synergy between E2F1 and chemotherapy in inducing apoptosis is transcription dependent. In a screen for genes that are synergistically regulated by E2F1 and chemotherapy in cancer cells we identified the tumor suppressor Maspin (Mammary Serine Protease Inhibitor) as a novel E2F1-regulated gene. Maspin expression is elevated also in response to chemotherapuetic agents and combining chemotherapy with activation of E2F1 leads to enhanced activation of Maspin. The tumor suppressive activity of Maspin is attributed, at least in part, to induction of apoptosis. Importantly, we show here that inhibition of Maspin expression significantly impairs the ability of E2F1 to enhance chemotherapy-induced apoptosis. Thus, our data indicate that regulation of Maspin expression by E2F1contributes to the efficient apoptotic response of transformed cells to chemotherapeutic agents. A RESPONSE REGULATOR CRITICAL FOR SURVIVAL OF NITROGEN STARVATION IN THE CYANOBACTERIUM SYNECHOCOCCUS ELONGATUS

A. Cohen, E. Sendersky, A. Perelman, R. Schwarz

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

Cyanobacteria are highly abundant in nature and contribute significantly to global primary production. Native populations of phytoplankton, including cyanobacteria, frequently encounter nitrogen starvation. We employ the model cyanobacterium Synechococcus elongatus to unravel the molecular mechanisms underlying acclimation to nitrogen starvation. A mutant in the response regulator NblR dies rapidly during nitrogen starvation in contrast to the wild type strain. Comparative analyses of these two strains during nitrogen starvation indicated that only the wild type strain completes DNA replication following the onset of starvation. Breakage of the DNA of the mutant was observed early in the course of starvation explaining its inability to replicate the genetic material. Wild type cells, on the other hand, started showing DNA breakage only upon prolonged starvation. Cellular nuclease activity correlating with the time of DNA fragmentation suggests the involvement of an active cellular process in DNA breakage and not merely chemical damage to the genetic material as a consequence of starvation. In an attempt to identify mechanisms required for survival during nitrogen starvation, we compared the global transcriptome of the mutant and the wild type strains. The central role of NblR in acclimation to nitrogen starvation will be discussed in light of our comparative biochemical and global transcriptome analyses. BORON TOXICITY MECHANISM IN PLANTS MIGHT BE RELATED TO NORMOTONIC SHRINKAGE AND PROGRAMMED CELL DEATH

N. Dahan, N. Moran, M. Moshelion

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel

Boron (B) is both an essential micronutrient required for higher plants growth and development, and a toxic element at elevated concentrations. B deficiency on one hand and toxicity on the other hand are significant agricultural problems due to physiological disorders that affect both yield and quality in many crops. B can be found naturally in the soil in different concentrations, but most of the B reaching crops in Israel comes from irrigation with treated waste water or purified water. The cellular mechanism of B toxicity is far of being understood, yet early reports linked B toxicity with proton pump inhibition. Our objectives were to measure B permeability rate at a single plant protoplast level and its impact on leaf disk proton extrusion. To this end, we exposed plant protoplasts or leaf disks (Arabidopsis thaliana) to 1-100 mM B normotonic solutions and monitored, respectively, their volume changes and external pH changes. Surprisingly, we observed normotonic shrinkage of protoplasts at 10 or more mM B. Adding B to the leaf disks even at the low concentration of 1 mM lead to significant alkalization of the external solution. In animal cells, normotonic shrinkage is an initial step leading to programmed cell death. The spontaneous shrinkage we observe in response to B, along with the alkalization suggests release of ions from the cell in addition to proton pump inhibition. The possible causative link between these phenomena will be studies in further experiments. HUMAN 1-8D GENE, A NEW PRO-APOPTOTIC GENE, IS OVEREXPRESSED IN COLON CANCER: CHARACTERIZATION OF AN ABSURD SITUATION

V. Daniel Carmi, E. Avraham, O. Goldberger, E. Vadai, E. Tzehoval, L. Eisenbach

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

Screening of peptides derived from 26 overexpressed genes in colon carcinoma was resulted in many HLA-A2 binding peptides. Seven peptides were shown to be immunogenic and induced CD8 cytotoxic T cells. Three of them were derived from the 1-8D gene, which belongs to the 1-8 interferon-inducible gene family. Three members of the family 1-8D, 1-8U and 9-27 are linked on an 18Kb fragment of chromosome 11 and are highly homologous. The human 1-8D gene was confirmed to be overexpressed in colon tumor tissues compared to paired normal tissue using Real Time PCR. Sequence comparison of the human 1-8D in pairs of tumor/normal colon tissues revealed six different alleles. Transient expression of the human 1-8D gene in several mammalian cell lines showed accumulation of cells in G1 phase followed by elevation in the subG1 phase. This increase was dependent on caspase activity but not on p53 expression. To further confirm this induction of apoptosis by 1-8D, Annexin V binding assays in combination with propidium iodiode staining and TUNEL assays were performed. These experiments showed that 1-8D expression induces apoptosis in a time-dependent manner. Cell cycle regulation was also observed with the murine homologue. Furthermore, transformation assays revealed a possible role of 1-8D gene as a cell growth inhibitor. Significant reduction in the number of foci after transfection was observed. A human 1-8D-GFP fusion protein was expressed in COS cells, the protein localized to a compartment identified as the Golgi body by labeling with p58 antibody and fluorescent microscopy. Our data suggest a novel role for the 1-8D as a pro-apoptotic gene. The study of the functions of 1-8D and its role in the carcinogenous process are of importance to determine the role of apoptosis in tumor cell cycle regulation and will provide new insights into the regulated cellular pathways to death. DAP-kinase REGULATES JNK SIGNALING BY BINDING AND ACTIVATING PROTEIN KINASE D UNDER OXIDATIVE STRESS

A. Eisenberg-Lerner, A. Kimchi

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

The stress-activated kinase JNK mediates key cellular responses to oxidative stress. Here we show that DAP-kinase (DAPk), a cell-death promoting Ser/Thr protein kinase, plays a main role in oxidative stress - induced JNK signaling. We identify protein kinase D (PKD) as a novel substrate of DAPk and demonstrate that DAPk physically interacts with PKD in response to oxidative stress. We further show that DAPk activates PKD in cells and that induction of JNK phosphorylation by ectopically expressed DAPk can be attenuated by knocking down PKD expression or by inhibiting its catalytic activity. Moreover, knock-down of DAPk expression caused a marked reduction in JNK activation under oxidative stress, indicating that DAPk is indispensable for the activation of JNK signaling under these conditions. Finally, DAPk is shown to be required for cell death under oxidative stress in a process which displays the characteristics of caspase-independent necrotic cell death. Taken together, these findings establish a major role for DAPk and its specific interaction with PKD in regulating the JNK signaling network under oxidative stress. GENERATING MOLECULAR PROBES FOR DETECTION OF CASPASE ACTIVITY IN SITU

A. Florentin, E. Arama

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Apoptosis is a major form of natural cell death executed by active caspases. Once activated, caspases typically cleave a variety of cellular substrates to ultimately lead to cell death. However, some cells can harness the “death” program to differentiate instead of die. During terminal differentiation, in a process also known as “individualization”, Drosophila spermatids normally lose their bulk cytoplasm and organelles in a restricted, apoptosis-like process which requires active caspases [1]. At this point, the mechanisms used by spermatids to restrict caspase activity and avoid death are poorly understood. Whereas the CM1 antibody, which detects the cleaved and hence the active form of effector caspase has proven to be a highly useful tool for visualizing activation of caspases in vivo, it does not necessarily reflect caspase activity, as CM1 also readily detects cleaved caspases bound to inhibitor of apoptosis proteins (IAPs). In order to monitor both initiator and effector caspase activity, we designed several DNA constructs, serving as “in vivo” molecular probes for caspase activity, and generated transgenic flies. These probes will enable us to closely monitor caspase activity and its strength, as well as the exact spatiotemporal distribution of caspase activity in spermatids and other tissues in the fly. In addition, we will use these probes to test the idea that spermatid individualization also serves as a quality control mechanism to eliminate defective sperm. We hypothesize that the role of caspases during spermatid individualization is not only to promote the removal of the cytoplasm and organelles, but also to execute the death of developmentally defective spermatids that otherwise might affect male fecundity. 1. Arama E., Agapite J., and Steller H. Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila. Dev Cell, 4: 687–97 (2003). SPINDLE MIDZONE ORGANIZATION DURING MID-ANAPHASE PAUSE INDUCED BY MUTATIONS IN KINESIN-5 MOTOR PROTEINS

V. Fridman1, I. Shumacher2, N. Movshovich2, B. Katz2, L. Gheber1,2

1Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Kinesin-5 proteins are bipolar mitotic kinesins that crosslink and move antiparallel spindle microtubules, thereby performing essential functions in spindle dynamics. S.cerevisiae express two kinesin-5 homologues, Cin8p and Kip1p, which overlap in function during spindle assembly, metaphase and anaphase B. Although the role of kinesin-5 proteins in spindle assembly and maintenance is well established, little is known about the function of these proteins in anaphase B spindle elongation. To address this issue, we monitored anaphase B kinetics by Nuf2-GFP imaging in cells that carry KIP1 chromosomal deletion and/or Cin8 motor-domain mutation (Cin8- FA), which reduces the ability of Cin8p to bind microtubules in vitro. Our results indicate that in WT and kip1D cells, cin8-FA mutation causes the appearance of a pause/delay phase between fast and slow phases of anaphase B, during which spindle length remains constant. Since the timing of the pause phenotype correlates with the activation of Cdc14 release (FEAR pathway), we examined Cdc14 release and involvement of Slk19 and Spo12, components of FEAR pathway, in cells exhibiting pause phenotype. By tagging Cdc14 with GFP, we demonstrated that Cdc14 is released from nucleolus during the pause. We also found that in cin8-FA cells deletion of SLK19 or SPO12 significantly reduces occurrence of the mid-anaphase pause and in that in kip1D cin8-FA cells, SLK19 deletion leads to elimination of pause phenotype, while SPO12 deletion leads to spindle collapse after fast phase. In addition, we examined midzone organization in kinesin-5 mutant cells by tagging midzone-localizing proteins Ase1 and Slk19 with GFP. We found that in kip1D cin8- FA cells, the midzone occupies larger portions of the spindle, compared to kip1D cells. We conclude that in kinesin-5 mutant cells, the mid-anaphase pause occurrence is related to interference in spindle midzone organization and dependent on FEAR pathway. THE ROLE OF dBRUCE, A GIANT UBIQUITIN - CONJUGATING ENZYME, IN APOPTOSIS AND SPERMATID TERMINAL DIFFERENTIATION

L. Gibbs, E. Arama

Department of Molecular Genetics, Weizmann Institute for Science, Rehovot, Israel

In both insects and mammals, caspase activation plays a pivotal role in promoting programmed cell death or apoptosis. However, caspase activation does not necessarily lead to cell death. For example, active caspases and other apoptotic factors are required for spermatid maturation in Drosophila. During this process, also known as “individualization”, spermatids eliminate most of their organelles and cytoplasm, while leaving several structures intact, such as the flagellum, mitochondria, and nucleus. Drosophila dBruce is an evolutionary conserved, giant, anti apoptotic protein that may be required for nuclear integrity during individualization and dbruce loss-of- function mutants are male-sterile. dBruce contains an N-terminal BIR domain, a hallmark of the inhibitor of apoptosis protein (IAP) family, and a C-terminal UBC domain, characteristics of E2 ubiquitin conjugating enzymes. Whereas the BIR domain of another Drosophila IAP, Diap1 can interact with both the pro-apoptotic RHG (Reaper, Hid and Grim) proteins and caspases, it is unclear how dBruce is regulated and how it promotes apoptosis and spermatid maturation. The goal of my research is to elucidate the roles and mode of regulation of dBruce during spermatid individualization and conventional apoptosis. NOVEL NAPHTHOQUINONE COMPOUNDS, AS ANTI-LEUKEMIC AGENTS THAT MEDIATE CELL DEATH THROUGH ROS FORMATION, MITOCHONDRIAL DYSFUNCTION AND PROTEASE ACTIVATION IN MYELOID LEUKEMIC CELLS

M. Hallak1, S. Bittner2, Y. Granot3, I. Nathan1

1Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 3Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Leukemia is still mostly an uncured disease and there is still need for developing new drugs. Naphthoquinones are derivatives of quinones which contain a naphthyl group attached to the quinone ring. A mostly known naphthoquinone compound is menadione which was found to have lower toxicity than the quinone derived cytotoxic drugs. To this aim new antileukemic amino-phenyl-naphthoquinone compounds, were synthesized and their SAR was studied. Among the forty compounds synthesized and screened, two groups of compounds were found to be most active (1) the chloro- amino-phenyl-naphthoquinones as well as (2) the -pyrolidino-amino-phenyl- naphthoquinone. Within the first group, derivatives with m-methyl substituent alone (TW-74) and p-amino-m-methyl substituent on the phenyl ring (TW-92) were found to be most active. In the second group, the most active compounds were those with p- methyl (TW-85) and p-hydroxyl substituent on the phenyl ring (TW-96). These compounds killed leukemic cells by induction of apoptotic or necrotic cell death as dependent on dose and time of exposure. The mechanism of cell death induction was studied. TW-92 and TW-96 induced rapid accumulation of H2O2 which occurred within a few minutes of exposure and was inhibited by diphenylene iodonuim (DPI), an inhibitor of NADPH-oxidase, and accompanied by reduction in glutathione level as well, indicating the involvement of ROS generation mediated by NADPH oxidase. All four compounds induced swelling of isolated rat liver mitochondria that can be inhibited by cyclosporine A, indicative of permeability transition pore opening. In intact cells disruption of mitochondrial transmembrane potential was followed by cytochrome c release and caspases activation. We demonstrate here for the first time that TW-92 as well as menadione, activate serine protease-dependent apoptosis. Our findings indicate that naphthoquinones provoke a cascade of events, involving various proteases activation, culminating in apoptotic or necrotic cell death. IDENTIFICATION OF BAX-INTERACTING PROTEINS AT THE MITOCHONDRIA

G. Hen, A. Gross

Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

Pro-apoptotic BAX is a downstream effector of tBID, which leads to permeabilization of the mitochondrial outer membrane. BAX is thought to act as a homo-oligomer, and we believe that additional proteins are involved in regulating this process. Identification of mitochondrial resident proteins that interact with BAX might reveal the mechanism by which membrane permeabilization is regulated. To identify such interactors, 293 cells were infected with HA-BAX adenoviruses, mitochondria were prepared from these cells and treated with cross-linkers. Mitochondria were then lysed and HA-BAX was immunoprecipitated using anti-HA antibodies. Proteins were resolved using SDS-PAGE and observed bands were excised from the gel and analyzed by mass-spectrometry. Several mitochondrial outer membrane proteins were identified, including proteins of the TOM-receptor family and the voltage-dependent anion channel (VDAC). In a separate study, we found that pre-treatment of mitochondria with proteinase K significantly decreased the mitochondrial targeting of BAX. Taken together, these studies suggest that additional mitochondrial proteins (probably surface-exposed) are involved in the mitochondrial-targeting of BAX and in its ability to induce apoptosis. Further studies will determine whether the proteins, which we found to be cross-linked to BAX, play a critical role in executing its activity at the mitochondria. REGULATION OF CASPASE ACTIVATION BY THE UBIQUITIN SYSTEM DURING DROSOPHILA SPERMATOGENESIS

Y. Kaplan, E. Arama

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Caspases are a unique type of proteases function to execute apoptosis. However, these enzymes can also exert nonlethal roles, for example, during the process by which Drosophila spermatids eliminate their bulk cytoplasm and organelles [1, 2]. In a genetic screen for mutants that abrogate effector caspase activation in Drosophila spermatids, we identified two components of a Cullin-3–dependent (E3) ubiquitin ligase complex, including a testis-specific isoform of Cullin-3 and the BTB-Kelch- domains protein Klhl10 [3]. Based on our genetic data and the fact that Cullin- dependent complexes target proteins for degradation by the 26S proteasome, we suggest a model in which this E3 enzyme activates caspases by degrading a caspase inhibitor. This inhibitor, therefore, should interact physically with the substrate recognition protein Klhl10. For this, we screened a yeast-two-hybrid cDNA library using Klhl10 as bait. We identified a novel, small protein expressed in developping spermatids, which we called Sik1. Introduction of three different point mutations into the Kelch domain of Klhl10, which correspond to the mutations identified in our genetic screen, blocked the binding between Klhl10 and Sik1, demonstrating the specificity of this interaction. Currently, we are analyzing the role and mode of regulation of Sik1 in spermatids. Interestingly, recent data from our lab shows that the giant inhibitor of apoptosis (IAP)-like protein, dBruce, can interact with Klhl10 in S2 cells, suggesting that dBruce is another substrate candidate for the Culin-3 complex [3]. 1. Arama E, Agapite J, Steller H (2003) Dev Cell 4: 687–697. 2. Arama E, Bader M, Srivastava M, Bergmann A, Steller H (2006) EMBO J 25: 232–243. 3. Arama E, Bader M, Rieckhof GE, Steller H (2007) Plos Biol 5: e251. THE EXTRA-CELLULAR DEATH FACTOR (EDF): A LINEAR PENTA- PEPTIDE REQUIRED FOR mazEF-MEDIATED CELL DEATH IN ESCHERICHIA COLI

I. Kolodkin-Gal1, A. Gaathon2, H. Engelberg-Kulka1

1Department of Molecular Biology, Core Research Facility, Hebrew University - Hadassah Medical School, Jerusalem, Israel, 2Core Research Facility, Hebrew University-Hadassah Medical School, Jerusalem, Israel

Gene pairs specifying for a toxin and its antitoxin (TA) are called toxin-antitoxin modules and are found on the chromosomes of many bacteria. The most studied of these modules is Escherichia coli mazEF in which mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF (1). We have shown that mazEF-mediated cell death is a population phenomenon requiring a quorum sensing peptide that we call the Extracellular Death Factor (EDF). We characterized the chemical nature of EDF to be a novel linear penta-peptide whose amino acid sequence is NNWNN (1). Our results indicate that EDF is specified by a larger precursor, the zwf gene product, carrying the amino acid sequence NNWDN. In the case of zwf , an amidation step should be involved in the generation of the NNWNN sequence of EDF. The amidation step may occur either before of after the cleavage of the precursor by one of E.coli proteases. Such a step may be contributed by either aspargine synthetase A and/or B . Our results indicate that aspargine synthetase A is involved. Our recent experiments also revealed that the protease ClpXP is the e. coli protease involved in the generation of EDF. We also found that there was a positive feedback loop between the mazEF module and EDF signaling. On one hand, EDF permits the activation of MazF , and EDF action was dependent on the presence of the mazEF module in the recipient cells. On the other hand, MazF activation by stressful conditions led to an increase in the production of EDF , resulting in an increase in cell death. Therefore, EDF is an integral part of the mazEF system.

1. Kolodkin-Gal, I., Hazan,R., Gaathon,A., Carmeli,S., and H. Engelberg-Kulka. (2007). Science . 318: 652-5. DEATH ASSOCIATED PROTEIN 1 (DAP1): A NEW PLAYER IN AUTOPHAGY?

I. Koren, A. Kimchi

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Autophagy is a physiological and evolutionarily conserved phenomenon that maintains homeostatic functions like protein degradation and organelle turnover. It is rapidly upregulated under conditions leading to cellular stress, such as nutrient or growth factor deprivation, providing an alternative source of intracellular building blocks and substrates for energy generation to enable continuous cell survival. In some cellular settings autophagy is recruited towards self killing. Although studies of autophagy have rapidly progressed over the past decade, many gaps in the molecular mechanisms underlying the process still exist. In this work we investigated the involvement of Death Associated Protein 1 (DAP1) in the autophagy process. DAP1 is a small, ubiquitously expressed protein, highly conserved in evolution. It was isolated by an anti-sense-based genetic screen aimed at identifying positive mediators of gamma-IFN-induced cell death in a mammalian system. Here we show that DAP1 is a phospho-protein under nutrient-rich culture conditions, but undergoes rapid dephosphorylation in response to amino acid starvation. This modification is accompanied by up-regulation of the protein steady state levels. shRNA mediated knock-down of DAP1 enhanced starvation induced autophagosome accumulation, as demonstrated by LC3 immunoblotting and GFP-LC3 fluorescent microscopy, suggesting a possible role for DAP1 protein in the autophagy pathway. IDENTIFICATION AND CHARACTERIZATION OF THE SIGNAL TRANSDUCTION PATHWAY INDUCED BY THE VIRAL PROTEIN E4orf4 IN DROSOPHILA

M. Lahav1, E. Bitman1, A. Salzberg2, T. Kleinberger1

1Unit of Microbiology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel, 2Unit of Genetics, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

E4orf4 (E4 open reading frame 4) is a 14kDa protein encoded by adenovirus. This protein is involved in several cellular processes, which include down regulation of signal transduction pathways induced by adenovirus, control of gene expression, and induction of p53-independent apoptosis in transformed cells. E4orf4-induced apoptosis can occur without activation of known caspases, although E4orf4 induces caspase activity in some cell lines. Transformed cells are more susceptible to E4orf4 induced apoptosis than normal cells. Since acquired resistance to apoptosis is a hallmark of cancer, E4orf4-induced apoptosis may contribute to cancer therapy. We chose Drosophila melanogaster as a model to investigate E4orf4 function in a whole organism which can develop cancer, several transgenic flies containing a GAL4- inducible E4orf4 transgene have been generated and E4orf4 effects were found to be dosage-dependent in various normal fly tissues. Wing imaginal discs expressing E4orf4 under control of the engrailed driver were labeled with an antibody recognizing activated caspase 3. The stained cells were organized in a unique pattern, consisting of a cloud of cells, which appeared to be outside the engrailed domain. The discs were also stained with an antibody to E-cadherin, a cell-to-cell adhesion molecule. The results demonstrated that the "clouded sphere" region stained by the caspase antibody was not stained by the E-cadherin antibodies. It is possible that the apoptotic cells lost their adhesion to the tissue and were excluded from it. This phenomenon is not exclusive to engrailed. Expression of E4orf4 under the regulation of MS1096 also led to accumulation of caspase labeled cells in a distinct pattern. These results suggest that E4orf4-induced apoptosis is associated with decreased cell adhesion, which is reminiscent of E4orf4 effects in mammalian cells. ISG12 - A NOVEL PRO-APOPTOTIC GENE, OVER EXPRESSED IN BREAST CANCER

E. Makovitzki-Avraham, V. Daniel Carmi, E. Vadai, E. Tzehoval, L. Eisenbach

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

Interferon Stimulated Gene 12 (ISG12) is a member of a family of small intracellular non-secreted proteins (10-20 kDa), mainly induced by IFNalpha and slightly induced by IFNgamma and was originally cloned as an estrogen induced gene in the human breast carcinoma cell line, MCF7. It has high homology to the 6-16 protein, to which there are indications of a direct role in growth inhibition. ISG12 encodes a hydrophobic protein of 12 kDa, which was shown to be localized to the nuclear membrane. In our lab, ISG12 was found to be over expressed in breast cancer cell lines compared to normal breast cell lines originated in the same patients, by DNA chip, SSH (Suppressive Subtraction Hybridization) and RT-PCR. Using propidium iodide staining and FACS analysis, remarkable changes in cell cycle profiles were found, in cells expressing ISG12. ISG12 transfected to NIH3T3 and HeLa cells led to massive accumulation of cells in sub-G1 phase, as a function of time post transfection, which is considered to reflect apoptotic cells. The induction of apoptosis was further confirmed by performing Annexin V binding assays and TUNEL assays. The apoptotic activity of ISG12 was found to be caspase dependent, using a general inhibitor of caspases, a caspase-9 inhibitor and a caspase-3 and 7 inhibitor. Incubation of several human cell lines with Doxorubicin (influences the intrinsic apoptotic pathway) led to increased expression of endogenous ISG12. Incubation of the same cell lines with hTNFalpha and Cycloheximide (influences the extrinsic apoptotic pathway) did not elevate endogenous ISG12 expression yet, both treatments caused apoptosis in all of the cell lines. Taken together, our data suggest a pro-apoptotic role for the ISG12 gene, involving the intrinsic pathway of apoptosis. Further experiments are needed to shed a light on the apoptotic pathway, and for understanding the importance of ISG12 in breast cancer. THE ROLE OF BID IN INHIBITING TUMOR DEVELOPMENT

M. Maryanovich, A. Gross

Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

The pro-apoptotic BID protein is known to mediate cell death in the TNF/Fas death receptor pathway. In previous work conducted in our laboratory, it was demonstrated that BID also plays a pro-survival role in the DNA damage pathway. BID has been shown to be phosphorylated by the ataxia-telangiectasia mutated (ATM) kinase on Ser61 and Ser78, and that this phosphorylation is important for the ability of cells to temporarily arrest in S phase following DNA damage. A novel concept that has recently emerged in the DNA damage field is that oncogenes trigger a DNA damage response (DDR) in pre-tumoral lesions which serves as a barrier to tumor progression by inducing senescence or apoptosis. Recent studies have shown that oncogene- induced senescence is associated with signs of DNA replication stress, and inhibiting ATM suppressed the induction of senescence and led to an increase in tumor size in a mouse model. In addition, BID was shown to be phosphorylated on Ser61 in response to Myc-induced DDR in vivo. Strikingly, we recently found that breeding BIDS61A/S78A knock-in mice (mice carrying a non-phoshorylatable BID mutant) and wild type mice with Eµ-myc mice resulted in accelerated onset of Myc-induced B cell lymphomas in the BIDS61A/S78A background. Thus, BID may play a role in the DDR to suppress oncogene-induced tumorigenesis and our future studies will focus in exploring this possible role. THE ROLE OF ATM MEDIATED BID PHOSPHORYLATION IN THE DNA DAMAGE RESPONSE

G. Oberkovitz1, I. Kamer1, H. Niv1, S. Rienstein2, A. Aviram-Goldring2, A. Gross1

1Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel, 2Chaim Sheba Medical Center, Tel Hashomer, Israel

The BH3-only BID protein acts as a sentinel to interconnect specific death signals to the core apoptotic pathway. Our previous studies using BID knock-out cells demonstrated that BID is important for both S phase arrest and cell death following DNA damage, and that the cell cycle arrest function is regulated by its phosphorylation by the ATM kinase on serine 61 and 78. To define the importance of ATM-mediated BID phosphorylation, we generated a BID knock-in mouse, in which the endogenous BID gene has been replaced with a gene that drives the expression of a non-phosphorylatable BID protein (BIDS61A/S78A). Using cells from these mice we found that BIDS61A/S78A knock-in primary T cells demonstrate a defect in S phase arrest, increase chromosomal damage, and increased apoptosis in response to the DNA interstrand cross-linking agent Mitomycin C. Thus, BID's S phase arrest function seems to be critical for T cells to preserve genomic stability and to survive following replication stress. In a separated study we reveled that BID is found as part of a 50-kDa cross-linked complex in healthy cells and in cells treated with DNA damage, suggesting that BID may execute its function in the DNA damage pathway by a stable interaction with another protein. Taken together, our recent studies further strengthen the link between BID and the DNA damage response, and future studies will determine the exact role and importance of BID phosphorylation in mammalian organisms. Beclin1, AUTOPHAGY AND NEURODEGENERATION

R. Pinkas-Kramarsli1, S. Erlich1, L. Mizrahy1, T. Diskin1, P. Tal-Or1, O. Segev2, L. Lindenboim1, O. Zmira1, S. Harel1, J.A. Hirsch2, R. Stein1, E. Shohami3

1Department of Neurobiochemistry, Tel-Aviv University, Ramat-Aviv, Israel, 2Department of Biochemistry, Tel-Aviv University, Ramat-Aviv, Israel, 3Department of Pharmacology, Hebrew University of Jerusalem, Israel

Autophagy, a bulk degradation of sub-cellular constituents, represents the major pathway by which the cell maintains a balance between protein synthesis and protein degradation. Beclin1, a Bcl-2 interacting protein previously found to promote autophagy. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin1. Our results show that Beclin1 directly interacts with Bcl-2, Bcl-xL, Bcl-w and to a lesser extent with Mcl-1. Beclin1 does not bind the pro- apoptotic Bcl-2 proteins. The interaction between Beclin1 and Bcl-xL was inhibited by BH3-only proteins. Structural modeling suggests that Beclin1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-xL. Mutation of the Beclin1 amino acids predicted to mediate this interaction inhibited Beclin1/Bcl- xL association. Our results suggest that Beclin1 may modulate the interactions between anti- and pro-apoptotic Bcl-2 proteins. Autophagy was documented in several neurodegenerative diseases. Using the closed head injury (CHI) model we investigated the possible role of autophagy in neurodegeneration. We demonstrated that the levels of Beclin1 are dramatically increased near the injury site in neurons and astrocytes. Double staining of Beclin1 and TUNEL indicate that most of the cells that exhibit double staining are neurons and not astrocytes. These findings show for the first time that Beclin1 may play a role in brain responses to head trauma. These observations led us to the hypothesis that elevated autophagy following injury may represent a mechanism to discard injured components. To examine this hypothesis we used rapamycin (mTOR inhibitor and autophagy inducer). Rapamycin injection following CHI significantly improved the recovery as judged by neurological score. Thus, we suggest that enhanced autophagy following head trauma may serve as a neuroprotective mechanism, as well as a novel target for treatment of traumatic brain injury. MITOTIC CELL DEATH VERSUS MITOTIC CATASTROPHE AS DISTINGUISHED BY THE ACTIONS OF HYPERICIN AND DIMETHYL TETRAMETHOXY HELIANTHRONE IN GLIOBLASTOMA CELL LINES

G. Pinto1,2, M. Mandel2, T. Barliya2, Z. Hazan3, G. Lavie2

1Institute of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel, 2Blood Center, Sheba Medical Center, Tel-Hashomer, Israel, 3Hy Biopharma Inc., Rehovot, Israel

Perihydroxylated dianthraquinones are emerging as potent anti cancer agents. When administered to cancer cell lines in culture (in the dark) for intervals in the range of 72-96 hrs these compounds induce cytostatic effects at single digit micromolar concentrations and cancer cell death at double digit concentrations. Nevertheless, the mechanisms of induction of cell death differ and appear to define a novel distinction between mitotic cell death that is induced by hypericin and a mitotic catastrophe that is induced by its closely related structural analog dimethyl tetra methoxy helianthrone. Treatment of glioblastoma cells (and other tumor cell lines) with >10 microMolar hypericin results in G2 phase arrest and multinucleation, both of which are hallmarks of mitotic cell death. However, treatment of these same cell lines with dimethyl tetramethoxy helianthrone resulted in dysregulation of S-phase checkpoints followed by uncontrolled DNA re-replication. Very large amounts of DNA accumulate within the cells which develop dramatically enlarged nuclei. Cell cycle distribution analyses on propidium iodide stained cells analyzed on Coulter Multicycle software reveal existence of abnormal G4 and even G8 cell cycle phases. These cells die via a mitotic catastrophe as single cells, a mechanism which differs from the mitotic cell death mechanism. These two forms of cell death must be viewed as separate entities. E2F1 REGULATES AUTOPHAGY AND THE TRANSCRIPTION OF AUTOPHAGY GENES

S. Polager-Modan, M. Ofir, D. Ginsberg

The Mina and Everard Goodman Faculty of Life Science, Bar Ilan University, Ramat Gan, Israel

The RB pathway is often inactivated in human tumors resulting in deregulated E2F activity that can induce both proliferation and cell death. While the role of E2F in apoptosis is well characterized, little is known regarding its putative participation in other cell death pathways. We show here that activation of E2F1 up-regulates the expression of four autophagy genes - LC3, ATG1, ATG5 and DRAM. E2F1-mediated induction of LC3, ATG1 and DRAM is direct and indeed, endogenous E2F1 can be found bound to regions encompassing the promoters of these genes. Regulation of ATG5 by E2F1 is indirect. Importantly, we demonstrate that E2F1 activation enhances autophagy and conversely, reducing endogenous E2F1 expression inhibits DNA damage-induced autophagy. These studies identify E2F1 as a transcriptional regulator of autophagy, and for the first time establish a role for E2F1 in DNA damage-induced autophagy. THE ROLE OF MITOCHONDRIAL DYNAMICS FOR CASPASE ACTIVATION IN DROSOPHILA SPERMATIDS

L. Ravid, E. Arama

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Being at the core of the intrinsic apoptosis pathway, mitochondria have emerged as the central regulators of the apoptotic program in mammalian cells. During sperm differentiation, mitochondria undergo many structural changes. In early postmeiotic spermatids, mitochondria aggregate beside the nucleus and fuse into two giant mitochondrial derivatives that wrap around each other to form the Nebenkern. During flagellar elongation, the two mitochondrial derivatives unfurl from each other and elongate beside the growing axoneme. However, the purpose of these dramatic structural organizations of the mitochondria is poorly understood. To understand the regulation of caspases in spermatids, we screened a collection of more than 1,000 male-sterile mutant fly lines, defective in spermatid individualization, for mutants that lack CM1 staining, an antibody that detects processed effector caspase [1]. Twenty two different complementation groups were identified; among these are eight different genes that affect mitochondrial structural organization. We mapped one of these mutants to the testis specific cytochrome c1 (cyt1) gene, which encodes a component of complex III in the respiratory chain, and which functions to pass the electron out to cytochrome C. Whereas a role for cytochrome C in the activation of the apoptosome and apoptosis has been well established, no such role in caspase activation and apoptosis has been demonstrated for Cyt1. Furthermore, as opposed to cytochrome c mutant spermatids, which do not display any gross mitochondrial structural organization defects, fluorescent and electron microscopy analyses of cyt1 mutants show severe defects in the elongation of the mitochondrial derivatives. These findings suggest a role for Cyt1 in mitochondrial dynamics in spermatids, which is independent of its role in the energy metabolism of the cells. 1. Arama E., Bader M., Rieckhof G.E. and Steller H. A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila. PLoS Biol., 5(10): e251 (2007). A smARF WAY TO DIE: A NOVEL SHORT ISOFORM OF p19ARF IS LINKED TO AUTOPHAGIC CELL DEATH

S. Reef, E. Zalckvar, S. Bialik, A. Kimchi

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

The tumor suppressor functions of p19ARF have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. We describe a novel cellular function of p19ARF, involving a short isoform (smARF) that localizes to a proteinase-K resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, and lacks all known nucleolar and p53 related functional domains. smARF is maintained at low levels via proteasome mediated degradation, but increases in response to viral and cellular oncogenes. Ectopic expression of smARF impairs the structure of the mitochondria and dissipates the mitochondrial membrane potential, without causing cytochrome C release or caspase activation. The dissipation of mitochondrial membrane potential is p53 and Bcl-2 family independent. Notably, the human p14ARF orthologue is also capable of producing a rapidly degraded short form of ARF (hsmARF), which can localize to and impair the mitochondria, despite the retention of a second nucleolar localization signal within its sequence. smARF ultimately induces the formation of autophagosomes. Significantly, shRNA-mediated knockdown of two important elements in the autophagy pathways, ATG5 and Beclin- 1, partially compromised both autophagic activity and cell death, implying that smARF-induces type II autophagic cell death. Consistent with its localization to the mitochondria, smARF was found to interact with a previously characterized mitochondrial protein, p32. p32 physically interacts with both human and murine smARF, and co-localizes with each to the mitochondria. Remarkably, knock-down of p32 protein levels significantly reduced the steady state levels of smARF by increasing its turn-over. As a consequence, the ability of ectopically expressed smARF to induce autophagy and to cause mitochondrial membrane dissipation was significantly reduced. In contrast, the protein levels of the nucleular full length p19ARF, were not influenced by p32 depletion, suggesting that p32 exclusively stabilizes the mitochondrial smARF protein. DEATH ASSOCIATED PROTEIN 1: A NEW ER STRESS REGULATED PROTEIN WITH FUNCTIONAL IMPLICATIONS IN UPR AND CELL DEATH RESPONSES

E. Reem, A. Kimchi

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Accumulation of unfolded proteins induces endoplasmic reticulum (ER) stress. Excessive and prolonged periods of ER stress leads to apoptosis. However, the precise molecular mechanisms of ER stress-induced apoptosis have not been fully elucidated . We investigated in this work the involvement of Death Associated Protein 1 (DAP1) in ER stress-induced cell death pathways. DAP1 is a small, ubiquitously expressed proline rich protein, highly conserved in evolution. It was isolated by an anti-sense- based genetic screen aimed at identifying positive mediators of IFN-g - induced cell death in a mammalian system. Here we characterized the involvement of DAP1 in the ER cellular stress response. Using biochemical fractionations and immunofluorescent staining, we found that DAP1 is localized to the cytosolic side of the ER. It is a phospho protein which in response to ER stress undergoes rapid de-phosphorylation, followed by an up regulation of the protein steady sate levels. The knock down of DAP1 by transfection with shRNA enhances the caspase-mediated apoptotic cell death caused by ER stress. This is preceded by a faster and stronger activation of the unfolded Protein Response (UPR), as reflected by enhanced XBP1 splicing, detected as early as 2 hours after exposing the cells to ER stress inducers. As a result of this study, we propose that DAP1 is a novel regulator of the ER stress response. THE INTERACTIONS BETWEEN YND1 AND CDC55 AND THEIR ROLE IN THE SIGNAL TRANSDUCTION PATHWAY INDUCED BY THE ADENOVIRUS E4orf4 PROTEIN IN YEAST

K. Scharfstein, I. Ben-Ari, T. Maoz, R. Koren, T. Kleinberger

Unit of Microbiology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Adenovirus early region 4 open reading frame 4 (E4orf4) protein induces p53- independent, protein phosphatase 2A (PP2A)-dependent non classical apoptosis in mammalian cells and irreversible growth arrest in Saccharomyces cerevisiae. Growth arrest requires the interaction of E4orf4 with an active PP2A enzyme containing Cdc55p, the yeast PP2A-B subunit. The toxic pathway activated by E4orf4 has conserved properties in yeast and mammalian cells, suggesting that yeast genetics may serve as a powerful tool to identify more of its components. Deletion of YND1, encoding a yeast Golgi apyrase, confers partial resistance to E4orf4, although Ynd1p apyrase activity is not required for E4orf4-induced toxicity. Ynd1p and Cdc55p contribute additively to E4orf4-induced toxicity. Overexpression of Cdc55p inhibits yeast growth in a ynd1-delta background, and sensitizes yeast cells to E4orf4- mediated toxicity. In addition to their functional interaction, Ynd1p and Cdc55p interact physically, and their interaction is disrupted by E4orf4, which remains associated with both proteins. We continue to study the interactions between E4orf4, PP2A and Ynd1 in order to further dissect the E4orf4 network required for inducing growth arrest in yeast. MODULATION OF CELL FATE DURING STARVATION: CHARACTERIZATION OF TOXIN-ANTITOXIN SYSTEMS IN THE CYANOBACTERIUM SYNECHOCOCCUS ELONGATUS

E. Sendersky, R. Schwarz

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

Cyanobacteria are equipped with numerous responses allowing them to survive long periods of nutrient starvation. Continuous starvation, however, results eventually in cell deterioration. Cell death may occur by necrosis or via programmed cell death, two processes that may act in parallel. Previous studies suggested apoptotic-like mechanisms involving caspase-like proteolytic activity in unicellular algae as well as in cyanobacteria. Additional mechanisms that have been implicated in the modulation of cell fate, known as toxin-antitoxin (TA) systems, are ubiquitous in heterotrophic bacteria. These systems have not been studied in cyanobacteria. vapC of Synechococcus elongatus (hereafter S. elongatus), a homolog of the toxin of ‘vapBC TA systems’, emerged as a nitrogen-starvation-induced gene from a global transcriptome analysis, which we recently performed. This encouraged us to search the genome of S. elongatus for TA systems and to closely follow their transcription. We identified three potential TA systems in the unicellular cyanobacterium, S. elongatus, and revealed induction of their genes upon nitrogen and sulfur starvation. Furthermore, ectopic expression of VapC of S. elongatus by a strong promoter resulted in the death of the cyanobacterial cells under inducing conditions. These data show that VapC of S. elongatus has the potential to interfere with cell viability. Additionally, in the NblR-mutant, a strain impaired in a response regulator central to acclimation to nitrogen starvation, vapC is induced to higher levels as compared to the wild type strain. Intriguingly, this mutant dies rapidly during nitrogen starvation. The cellular function of the TA systems of S. elongatus will be discussed in light of inactivation of the operons encoding these systems and characterization of the mutants during starvation. THE ROLE OF MITOCHONDRIAL Mtch2 IN APOPTOSIS AND EMBRYOGENESIS

L. Shachnai, Y. Zaltzman, A. Gross

Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

Apoptosis is a crucial process for proper embryonic development and maintenance of tissue homoeostasis. In the extrinsic death pathway, apoptosis is initiated through activation of the TNF/Fas receptors. Activation of the TNF/Fas receptor results in the cleavage of pro-apoptotic BID into truncated BID (tBID), which translocates to the mitochondria to induce mitochondrial outer membrane permeabilization (MOMP) resulting in the release of apoptogenic factors such as cytochrome c. However, the mechanism by which tBID triggers MOMP is largely unknown. Mitochondria carrier homolog 2 (Mtch2) was identified in our lab as part of a complex with tBID in cells signaled to die by TNFalpha. Mtch2 is a novel and previously uncharacterized 33-kDa protein, which is related to a family of mitochondrial carrier proteins. We have revealed that knocking out Mtch2 in mice results in embryonic lethality, and analysis of timed pregnancies revealed that Mtch2-/- animals are not viable beyond E7.5. Histological analysis of the Mtch2+/+ and the Mtch2-/- embryos indicated that the null embryos are morphologically abnormal and do not seem to undergo complete gastrulation. Moreover, Mtch2 mRNA is highly expressed in the extraembryonic (ExEm) region of E7.5 wild type embryos, and this region is largely impaired in the Mtch2-/- embryos. Thus, Mtch2 might play a critical role in the formation of the ExEm region. To study the connection between Mtch2 and apoptosis we generated Mtch2-/- stable embryonic stem (ES) cell lines carrying either an empty vector or Mtch2. Using these lines we demonstrated that the presence of Mtch2 sensitizes cells to tBID-induced MOMP. Thus, we discovered that Mtch2 is critical for normal embryonic development, and is an important positive regulator of tBID-induced apoptosis at the mitochondria. Future studies using a newly generated Mtch2 conditional knockout mouse will unable us to further elucidate the role of Mtch2 in apoptosis and embryogenesis. THE TRANSLOCATOR PROTEIN MAY REGULATE THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE

Y. Shandalov1, L. Veenman1, S. Leschiner1, W. Kugler2, M. Lakomek2, M. Gavish1

1Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel, 2Pediatrie I, Universitatsklinikum Gottingen, Germany

Malignant gliomas are the most common brain tumors, which in most instances are resistant to radiation and chemotherapy and therefore remain largely incurable. The novel chemotherapeutic drug, Erucylphosphohomocholine (ErPC3) has shown potent antineoplastic activity on various malignant tumor cell lines. ErPC3 activates the mitochondrial apoptosis pathway. The mitochondrial apoptosis pathway is triggered by opening of the mitochondrial permeability transition pore (MPTP). By genetic manipulation, we found previously that the MPTP associated Translocator Protein (TSPO) is needed for apoptosis induction by ErPC3. Presently, we study ErPC3 induced apoptosis levels in the glioma cell lines, U118MG, A172 and U87MG: 1) by using the cell death detection ELISAplus kit of Roche, and 2) by Fluorescence Assisted Cell Sorting (FACS) determining the percentage of cells in the pre-G1 phase of the cell cycle. We found, for example, that the classical TSPO ligand, PK 11195 (25micromolar), reduces the levels of apoptosis triggered by ErPC3 in the U118MG glioma cells by 80%. We also show by viability experiments using Propidium Iodide that PK 11195 reduces the percentage of dead cells after ErPC3 treatment, e.g., PK 11195 (25micromolar) reduces the proportion of dead U118MG cells by 75%. Using the dye JC-1, we found that PK 11195 attenuates ErPC3 induced opening of the MPTP in the U118MG, A172 and U87MG glioma cell lines. For example, PK 11195 (25micromolar) causes a 57% reduction in the number of U118MG cells displaying opening of the MPTP. Thus, it appears that ErPC3 reduces viability of cancer cells via opening of the MPTP leading to cell death, including apoptosis. Furthermore, it appears that the TSPO ligand PK 11195 can prevent antineoplastic effects of ErPC3 by preventing the opening of the MPTP and cell death, including apoptosis. These data provide further evidence that the TSPO can be targeted for novel approaches to treat cancer. LEVELS OF THE PRO-APOPTOTIC PROTEIN ARTS, ARE TIGHTLY REGULATED THROUGH THE UBIQUITIN PROTEOSOME SYSTEM, WITH XIAP-SERVING AS ITS E3-LIGASE

A. Shekhtman1, Y. Gottfried1, H. Gonen2, A. Ciechanover2, S. Larisch1

1Apoptosis and Cancer Research Laboratory, Department of Pathology, Rambam Medical Center, Haifa, Israel, 2Department of Biochemistry and the Rappaport Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

ARTS is a potent pro-apoptotic protein, which is localized at the mitochondria. High levels of ARTS are sufficient to induce apoptosis in a large variety of cancer cells. ARTS main mechanism of pro-apoptotic action is through binding and antagonizing IAP (Inhibitor of Apoptosis Proteins). IAPs bind caspases in the cytosol and prevent their activation. During apoptosis, ARTS is released from mitochondria into the cytosol, binds to IAPs and reduces their levels through increased ubiquitin mediated degradation, leading to release of caspases and cell death. ARTS levels are regulated through the ubiquitin- proteasome system. Upon apoptotic induction, ARTS ubiquitination is inhibited, causing increased stability and up-regulation of ARTS cytosolic levels, causing apoptotic cell death. Since XIAP can function as E3- ubiquitin ligase we examined its involvement in regulating ARTS levels through ubiquitination and degradation. In-vitro and in-vivo ubiquitination assays have shown that COS cells overexpressing XIAP have higher levels of the ARTS ubiquitin conjugates as compared to those overexpressing XIAPDRING which lacks the E3 ligase domain. The N'-terminal sequence of ARTS contains a lysine residue, which might be the target of XIAP-E3 ligase activity. We therefore propose that XIAP serves as the E3- ligase for ARTS and the lysine residue at position 3 of ARTS is probably one of the target lysines, which are ubiquitinated by XIAP. Our working model suggests that in healthy cells, ARTS protein is kept at low levels in the cytosol through the ubiquitin proteosome system. Under these conditions XIAP serving as the E3–ligase for ARTS, binds to specific lysines at its N' terminal sequence and promote ARTS degradation. Under apoptotic conditions, the ARTS N' terminus is cleaved by caspases, and ARTS ubiquitination by XIAP is prevented. As a result, ARTS cytosolic protein levels are increased, allowing efficient binding to XIAP leading to XIAP down-regulation and caspase activation. ZIPk: A UNIQUE CASE OF MURINE-SPECIFIC DIVERGENCE OF A CONSERVED VERTEBRATE GENE

Y. Shoval, S. Pietrokovski, A. Kimchi

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Zipper Interacting Protein Kinase (ZIPk), a member of the Death Associated Protein Kinase family, is a positive mediator of programmed cell death. ZIPk regulates membrane blebbing by promoting myosin light chain phosphorylation, and also leads to autophagy. Contradictory findings regarding its intracellular localization and molecular mode of action have been reported, particularly upon comparison of the human and rodent orthologs. We therefore aligned the sequences of all available ZIPk orthologs, from fish to human. Surprisingly, rat and mouse sequences have diverged considerably from the consensus conserved in vertebrates. This sequence divergence correlated with several functional differences. While ectopically expressed human ZIPk localized to the cytoplasm and induced membrane blebbing, rat ZIPk localized exclusively within nuclei, mainly to PML bodies, and induced significantly lower levels of membrane blebbing. Additionally, rat ZIPk, but not human ZIPK, interacted with the PAR-4 protein. Co-expression of PAR-4 with rat ZIPk led to the latter’s accumulation in the cytoplasm and strong membrane blebbing induction. Thus, the murine protein’s unique interaction with PAR-4 provides a possible adaptive mechanism to compensate for its sequence divergence, ultimately enabling conservation of cellular function. THROMBOSPONDIN-1-N-TERMINUS DOMAIN (HEPARIN BINDING N- TERMINUS DOMAIN) INDUCES A PHAGOCYTIC STATE, AND THROMBOSPONDIN-1-C-TERMINUS DOMAIN INDUCES A TOLERIZING STATE IN DENDRITIC CELLS

A. Tabib1, A. Krispin1, M. Lebendiker2, D. Danieli2, D. Mevorach1

1Laboratory for Cellular and Molecular Immunology, Protein Expression and Purification Unit, Wolfson Centre for Applied Structural Biology, Hebrew University of Jerusalem, Israel, 2Hebrew University of Jerusalem, Hadassah University Medical Center, Israel

Thrombospondin-1 (TSP-1) is a homotrimeric, multifunctional protein, containing several domains within the molecule, such as calcium binding C-terminus domain, Heparin binding N-terminus domain (HBD) and several types of repeats in between. TSP-1 interacts with broad range of receptors and was first isolated from activated blood-platelets and characterized as a thrombin-binding protein. TSP-1 is secreted by various cell types, including apoptotic cells. We have recently shown (Krispin et al. Blood 2006), that cells undergoing an active process of programmed cell death (apoptosis), are expressing TSP-1 and secreting it to their extra-cellular matrix, presumably as an “eat me” signal, sent to professional antigen presenting cells (APC) of the immune system, such as dendritic cells. In the current study, we decided to examine the role of Heparin Binding Domain of TSP-1 in clearance of apoptotic cells. we applied a cloning approach, in which we have generated recombinant monomeric HBD (rHBD) using Human Throbospondin-1 N-Terminus domain c-DNA. HBD was expressed in Origami B bacterial strain under IPTG induction conditions and then purified from bacteria supernatant, using Q-Beads column followed by Heparin Agarose FPLC. Clearance of apoptotic cells was evaluated using flow cytometry and confocal microscopy and dendritic cells were generated from human monocytes. Results: rHBD has significantly elevated professional APC’s capability to engulf apoptotic monocytes in 40-100%, at a dose dependent pattern. rHBD was not able to induce a tolerizing phenotype of DCs, in contrast to TSP-1. Non-specific engulfment, carried-out with latex-beads, have shown similar results. In support of that, using specific antibodies we were able to demonstrate that whereas engulfment was induced by HBD, tolerized phenotype was induced by the N-terminal domain that includes CD47. Conclusion: HBD that is secreted both separately or as part of TSP-1 induce phagocyitc state whereas TSP-1 induces both pahgocytic and tolerizing states. ARSENIC INDUCED APOPTOSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS

A. Vanichkin, O. Bairay, O. Shpilberg

Institute of Hematology, Rabin Medical Center, Beilinson Hospital, Petah Tikva and Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel

CLL is accumulative disorder: the malignant cells increase in blood, resistance towards apoptosis is thus the key factor for the survival of CLL cells. Arsenic trioxide induces apoptosis in malignant cells by mechanisms different from those used by anti- cancer drugs. Peripheral blood mononuclear cells from 16 CLL patients were used for this study. Cells were incubated with increasing concentration of As2O3 from 0.25-4 µM for 2; 7 or 14 days. Cell viability was measured using the AlamarBlue Assay. Induction of apoptosis was analyzed by AnnexinV - PI kit. The expressions of caspase-3, Bcl-2 and Bax proteins were analyzed by flow cytometry. As2O3 reduced significantly (p<0.01) the viability in all patients cell examined in a time and dose dependently. At 2 days at 2 µM concentration the number of CLL cells in culture was reduced to 79±5.3% of control, whereas at 7 days the reduction was to 17±3.2%. We have found that As2O3 induced apoptotic CLL cell death in vitro. The percentage of viable cells decreased and of early and late apoptosis increased significantly with increasing dose and length of incubation. After 7 days of incubation control cells showed 10±4% early apoptosis and 12±1% late apoptosis while cells incubated with 0.5, 1 and 2 µM As2O3 demonstrated 25±6%, 31±8%, 32±7% early apoptosis and 15±2%, 30±4%, and 56±5% late apoptosis (p< 0.05, p< 0.01, p<0.001) respectively. After 7 days of incubation with As2O3 all patients showed significant decrease in the mean MFI of Bcl-2 levels which was also dose dependent. In control CLL cells Bcl-2 MFI was 62.1±5.1; while with incubation with 1.0 µM As2O3 it was reduced to 43.5±4.8 (p<0.01) and with 2.0µM As2O3 to 40.5+2.9 (P<0.01). Conclusions: Arsenic reduces the viability and induces apoptosis in CLL cell in vitro and may be a new approach for treatment of resistant CLL patients. REAL TIME CHARACTERIZATION ALA-PDT INDUCED CELL DEATH IN B16 MELANOMA AND NIH3T3 FIBROBLASTS

D. Darvish1, Z. Malik1, A. Weiss2

1School of Life Sciences, Bar Ilan University, Ramat Gan, Israel, 2School of Engineering, Bar Ilan University, Ramat Gan, Israel

5-Aminolevulinic acid (ALA) based photodynamic therapy (ALA–PDT) is a cancer treatment modality based on the enzymatic conversion of ALA into protoporphyrin IX (PpIX) by cancer cells, followed by light irradiation. The endogenous photosensitizer PpIX absorbs light, and the energy is scavenged by molecular oxygen to produce singlet oxygen, which kills the tumor cells. PpIX is synthesized and retained more efficiently in cancer cells than in normal cells, which allows this treatment to selectively target tumors. In order to understand the mechanism of ALA- PDT induced cell death, we used live cell imaging to observe the cells following ALA-PDT treatment. ALA-PDT consisted of incubation of the cells with ALA for 1-- 4 hours, followed by illumination with blue light for 30 seconds. The cells were imaged for one hour immediately following irradiation with the blue light. Incubation with ALA was shown to produce PpIX in a dose dependent manner. Illumination of ALA treated cells was shown to produce singlet oxygen, again in a dose dependent manner. Chromatin condensation was observed in cells which were transfected with a histone-mRFP fusion. JC1 was used to show that ALA-PDT treated cells suffered complete loss of mitochondrial potential within two minutes of illumination following ALA incubation. Cell morphology was observed, and exhibited characteristics of apoptosis in some cells. Membrane integrity was found to be compromised, both by testing with propidium iodide (PI), and by a novel test using the anionic dyes sulforhodamine B (SRB) or G (SRG). Finally, a detector of caspase activity was used to show that there was no caspase activity associated with our ALA-PDT treated cells. The lack of detectable caspase activity, together with the entry of PI and SRG/SRB into the cells, led us to conclude that apoptosis was not the mechanism of cell death in our experiments. GERM CELL DEATH IN DROSOPHILA

K. Yacobi Sharon, E. Arama

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Apoptosis is a morphologically distinct form of programmed cell death that serves to remove unwanted and potentially dangerous cells during the development and homeostasis of the animal. The execution of apoptosis depends on the action of caspases, the proteases that cleave numerous cellular proteins to promote cell death. In the testes of insects and mammals, a large number of early germ cells (spermatogonia) normally undergo apoptosis. The reason for this death and the exact mechanism that control this loss of germ cells are unclear. To better understand this phenomenon, we study apoptosis of early male germ cells in Drosophila. Using whole-mount immunofluoresence TUNEL staining for detection of apoptosis, we discovered that germ cell death largely occurs during the early pre-meiotic stages of spermatogonial development. We found that apoptosis of germ cells is dependent on the activity of the initiator caspase Dronc. Ectopic expression of the "killer" genes reaper and grim in the germ cells, increased this apoptosis. Unexpectedly, however, ectopic expression of the effector caspase inhibitor p35, resulted in a dramatic increase in the number of apoptotic germ cells. These results suggest that inhibition of effector caspases might promote an alternative germ cell-death pathway. Alternatively, it may suggest that effector caspases have a non-lethal function during early germ cell development and that p35 expression leads to developmental catastrophe and cell death. We are now performing a genetic screen for mutants that dominantly modify this developmental germ cell death. We identified a few genetic modifiers that either inhibit or increase this death. FUNCTION-BASED SYSTEMS LEVEL ANALYSIS OF THE CELL DEATH NETWORK BY SINGLE AND DOUBLE RNAi–MEDIATED PERTURBATIONS

E. Zalckvar, A. Kimchi

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

The programmed cell death network can be organized into three main functional modules driving different cell death phenotypes including apoptosis, autophagy and programmed necrosis. Here we developed a new approach for dissecting the network’s complexity, which is based on single and double sets of RNAi-mediated perturbations and combines two major read outs. The first quantifies the outcome of perturbations on the overall cell death performance by using high fidelity luciferase- based assays, and the second measures the perturbations’ effects on the protein connectivity within and between the different functional modules. The results prove that compensatory switches between functional modules contribute to the network’s robustness, and that inter-modular connectivity occurs at multiple positions via positive or negative interactions. This high throughput function-based analysis provides a powerful tool, that was not used before, for identifying new principles delineating the structure/function organization of molecular networks in mammalian systems. SHARED TRANSCRIPTIONAL CORRELATIONS IN SEED FORMATION AND IN PLANTS RESPONSE TO DROUGHT

M. Ashkenazi, M. Moshelion

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem, Rehovot, Israel

During plants life cycle several developmental stages involve the loss of considerable amount of water while keeping the tissue's vitality, such as the formation of seeds and pollen. Some of the genes expressed at these stages are known to be involved in response to other water stress conditions (e.g. drought stress, osmotic stress and salt stress). Our aim is to identify cliques of genes involved in the acquisition of desiccation tolerance (seeds and pollen) and in response to water stress. Expression measurements from 450 publicly available ATH1 arrays, representing Arabidopsis thaliana's response to various water deficit conditions (water stress or developmental stages, "states") were sorted into data sets. Each state is represented as a weighted, undirected graph resulting in networks of genes connected according to the degree of their expression correlation. Intersecting networks of two or more states (using Cytoscape) will produce a graph that includes only those genes that share correlation in all the states. This graph will reveal cliques (complete subgraphs) of genes whose expression is correlated in several states – suggesting a common regulation mechanism. Locating cliques enriched with water stress specific genes will enable us to assign functionality for the rest of the unknown genes in the clique and suggest them a role in plants response to drought. DETECTING CONSERVED GENE EXPRESSION PATTERNS BETWEEN D. MELANOGASTER AND A. THALIANA

O. Atias1, B. Chor2, D.A. Chamovitz1

1Department of Plant Sciences, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel, 2School of Computer Sciences, Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv, Israel

Comparison of microarray data from multiple species is an important tool in transcriptomics, which may become complicated when the data comes from different experimental conditions, chip technologies and analysis methods. We have compared two species, D. melanogaster and A. thaliana, using a network-based approach in search for gene modules, defined as groups of genes that have a shared function, that also share a conserved expression pattern. Detection of such modules points out regulatory pathways working under positive selection, teaching us about the evolution of gene expression regulation. We utilzed a collection of publicly available microarray experiments from both species to build co-expression networks, containing only edges that connect a pair of genes found to be significantly co- regulated in a large fraction of the experiments. Both single-species networks were built, as well as cross-species networks in which the definition of an edge relied on experiments from both species. Next, the networks were searched for clusters of highly interconnected regions, using the MCODE algorithm (Bader et al, 2003). These clusters, which appeared in the cross-species network at a rate much higher then that expected by random assignment of gene homologies, were regarded as putative gene modules. GO enrichment analysis showed that modules in the single- species networks were predominantly related to translation and processes of amino acid metabolism. However, the cross-species networks contained modules from other processes as well, such as DNA replication and electron transport, indicating that integrating data from the two species has increased the success rate of our method. To conclude, we have demonstrated how our method can combine microarray data from different species, preformed in a variety of experimental designs. We were able to detect putative gene modules that relate to basic cellular process and are suggested to have conserved regulatory pathways. NOISE RESISTANCE IN THE SPINDLE ASSEMBLY CHECKPOINT

A. Doncic1,2, E. Ben-Jacob3, N. Barkai1,4

1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2Department of Biomedical Engineering, The Iby and Aladar Fleischman Faculty, Tel Aviv University, Ramat Aviv, Israel, 3School of Physics and Astronomy, Beverly and Raymond Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv, Israel, 4Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel

Genetically identical cells vary in the amount of expressed proteins even when growing under the same conditions. It is not yet clear how cellular information processing copes with such stochastic fluctuations in protein levels. Here we examine the capacity of the spindle assembly checkpoint to buffer temporal fluctuations in the expression of Cdc20, a critical checkpoint target whose activity is inhibited to prevent premature cell cycle progression. Using mathematical modeling, we demonstrate that the checkpoint can buffer significant fluctuations in Cdc20 production rate. Critical to this buffering capacity is the use of sequestering-based mechanism for inhibiting Cdc20, as apposed to inhibition by enhancing protein degradation.We propose that the design of biological networks is limited by the need to overcome noise in gene expression. THE INTEGRATION OF A TRANSCRIPTOMIC AND PROTEOMIC APPROACHES FOR THE STUDY OF BIOLOGICAL NETWORKS IN THE INNER EAR

T. Elkan1, R. Hertzan1, A. Dror1, R. Elkon1, M. Irmler1, J. Beckers2, K.B. Avraham2

1Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel, 2Institute of Experimental Genetics, Neuherberg, Germany

Systems biology involves studying the interaction and interplay of many levels of biological information. We have combined transcriptomic and proteomic analyses of a comparison of early post-natal cochlear and vestibular sensory epithelia to identify networks of genes and proteins essential for the development and function of these inner ear organs. The analyses were performed on both wild type and Headbanger mutant mice. Expression profiling of vestibular and cochlear sensory epithelia using Affymetrix microarrays and proteomics analysis using the Q-TOF mass spectrometer with ITRAQ labeling (performed at the Smoler Proteomics Center, Department of Biology, Technion) has led to the identification of genes and protein networks. A network analysis was applied to find proteins of interest that are physically connected, pointing to some common function/pathway/complex. Two major sub-networks emerged from the integrated clusters, indicating multiple interactions between proteins expressed in the cochlear and vestibular systems. In addition, many novel genes/proteins that might play an important role in the inner ear were identified. Having established the baseline of transcript and protein profiles of the wild type sensory epithelia and in order to study the specific networks involved in stereocilia formation and maintenance, we analyzed the transcriptome and proteome of sensory epithelia from the Headbanger mutant. The Headbanger mutant mouse is a dominant ENU induced mouse mutant with a low frequency hearing loss and vestibular dysfunction caused by a mutation in the Myo7a gene, a model for human hereditary hearing loss. An analysis of its auditory and vestibular sensory epithelia revealed abnormal stereocilia formation and enlogation of the vestibular hair cells stereocilia. Due to the distinct phenotype of the Headbanger mutant mouse, we hope to use it as a model to study the more defined networks underling stereocilia formation and maintenance.

Research supported by the European Commission FP6 Integrated Project EUROHEAR. DYNAMIC PROTEOMICS IN INDIVIDUAL HUMAN CELLS IN RESPONSE TO DRUGS

N. Geva-Zatorsky1, A.A. Cohen1, E. Eden1, A. Sigal2, R. Milo3, N. Perzov1, T. Danon1, L. Cohen1, U. Alon1

1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 2Department of Systems Biology, Caltech, Pasadena, CA, USA, 3Department of Systems Biology, Harvard Medical School, Boston, MA, USA

A long term goal of biology is to quantitatively understand gene and protein networks of human cells and their responses to stimuli. Attaining this goal requires the ability to accurately measure protein levels and their dynamics in living cells. We have developed a method for dynamic proteomics that can randomly label endogenous proteins with a fluorescent tag in mammalian cells. The system is a library of annotated reporter cell lines (LARC), where in each cell a different endogenous protein is fluorescently tagged. This system allows real-time measurements of protein dynamics in individual living cells using a time-lapse microscopy strategy. We are studying the response-networks of this system to drug perturbations. One of our tagged proteins is topoisomerase I, a direct anti-cancer drug target. We are studying the dynamics of this protein and also of the entire library in response to the topoisomerase I inhibitor – Camptothecin. As a continuous to this study we also investigate the dynamic proteomic response to other anticancer drugs and to their combinations. A NOVEL PROBABILISTIC METHOD FOR COMPARING TRANSCRIPTION FACTORS BINDING SITE PROFILES

N. Habib1,2, T. Kaplan1,2, H. Margalit2, N. Friedman1

1School of Computer Science and Engineering, Hebrew University of Jerusalem, Israel, 2Department of Molecular Genetics and Biotechnology, Faculty of Medicine, Hebrew University of Jerusalem, Israel

Characterizing the DNA-binding specificities of transcription factors is a key problem in computational biology that has been addressed by multiple algorithms. These usually take as input sequences that are putatively bound by the same factor and output one or more DNA motifs. A common practice is to apply several such algorithms simultaneously to improve coverage at the price of redundancy. In interpreting such results two tasks are crucial: clustering of redundant motifs, and attributing the motifs to transcription factors by retrieval of similar motifs from previously characterized motif libraries. Both tasks inherently involve motif comparison. Here we present a novel method for comparing and merging motifs, based on Bayesian probabilistic principles. This method takes into account both the similarity in positional nucleotide distributions of the two motifs and their dissimilarity to the background distribution. We demonstrate the use of the new comparison method as a basis for motif clustering and retrieval procedures, and compare it to several commonly used alternatives. Our results show that the new method outperforms other available methods in accuracy and sensitivity. We incorporated the resulting motif clustering and retrieval procedures in a large-scale automated pipeline for analyzing DNA motifs. This pipeline integrates the results of various DNA motif discovery algorithms and automatically merges redundant motifs from multiple training sets into a coherent annotated library of motifs. Application of this pipeline to recent genome-wide transcription factor location data in S. cerevisiae successfully identified DNA motifs in a manner that is as good as semi-automated analysis reported in the literature. Moreover, we show how this analysis elucidates the mechanisms of condition-specific preferences of transcription factors. NOISE PROPAGATION AND SIGNALING SENSITIVITY IN BIOLOGICAL NETWORKS: A ROLE FOR POSITIVE FEEDBACK

G. Hornung1, N. Barkai1,2

1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel

Interactions between genes and proteins are crucial for efficient processing of internal or external signals, but this connectivity also amplifies stochastic fluctuations by propagating noise between components. Linear cascades were shown to exhibit interplay between the sensitivity to changes in input signals and the ability to buffer noise. We searched for gene circuits that can minimize noise propagation while maintaining signaling sensitivity. Negative feedback can buffer noise, but this buffering comes at the expense of an even greater reduction in signaling sensitivity. By systematically analyzing all three-gene circuits, we identify positive feedback as a central motif allowing for the buffering of propagated noise while maintaining sensitivity to long-term changes in input signals. We show analytically that noise reduction results from a longer averaging time in the presence of positive feedback, and discuss in detail a particular positive feedback circuit that is involved in the control of nutrient homeostasis. The design of biological networks requires a compromise between multiple constrains. Accordingly, we identify a new role for positive feedback in balancing between responsiveness and noise attenuation. DEVELOPMENT OF THE C. ELEGANS NEURAL NETWORK

R. Itzhack, Y. Louzoun

Department of Mathematics, Bar-Ilan University, Ramat Gan, Israel

A number of recent studies have focused on the statistical properties of networks systems. Researchers have concentrated particularly on a few properties that seem to be common to many networks: the small-world property, power-law degree distributions, and network transitivity. Currently proposed models do not seem to represent the properties of the Caenorhabditis elegans neural network. In order to find a suitable network model that can explain the stochastic process creating the neural networks in general, we suggests a new model of spatial network based on the physical distance between Neurons. We show that a simple model based on a constant synapse formation probability and the inhibition of short coherent cycles can explain all the properties of the C.Elegans neural network. We propose this model as a general framework to discuss Neural network generation even in more advanced networks. INVESTIGATING THE HIERARCHY OF INTERACTING DOMAIN PAIRS

Z. Itzhaki, H. Margalit

Department of Molecular Genetics and Biotechnology, Faculty of Medicine, Hebrew University of Jerusalem, Israel,

Recent databases based on solved protein complexes and biochemical experiments have listed pairs of protein domains that are used repeatedly in different protein contexts for mediating protein-protein interactions. Obviously, when multi-domain proteins interact there are multiple putative combinations of domain pairs that could mediate the interaction. However, thorough investigation of the data reveals that most of the protein interactions are mediated by a single domain-domain interaction, and that the interacting domain pairs vary in regard to their use as interaction mediators. There are domain pairs that are always used for mediating the interactions when they occur in interacting proteins, domain pairs that mediate the interactions of some proteins, and domain pairs that never mediate an interaction even when they occur in interacting proteins. Furthermore, when more than one domain pair can potentially mediate an interaction between two proteins, the interaction between a preferred domain pair often dominates an interaction between a less preferred one. Here we study this hierarchy of interacting domain pairs – we characterize the preferred domain pairs, provide a score that expresses the domain interaction preference, and use this score for predicting the domains mediating the interaction in reported interacting proteins. We also show that this score may be used to predict protein- protein interactions, based only on the domain content of the proteins. A SYSTEM LEVEL VIEW OF GENETIC INTERACTION NETWORKS

A. Jaimovich1,2, R. Rinott1, H. Margalit2, N. Friedman1

1School of Computer Science and Engineering, Hebrew University of Jerusalem, Israel, 2Department of Molecular Genetics and Biotechnology, Faculty of Medicine, Hebrew University of Jerusalem, Israel

Functional relations between proteins can be inferred from their physical interactions, but also from other indirect relations, such as epistatic relations. Two proteins are epistatically related if the phenotypic effect of their double knockout differs substantially from the expectation based on their single independent knockouts. Recently, several works made large-scale measurements of such relations in the budding yeast S serevisiae. It is a major challenge to systematically analyze the data from these large-scale assays in an attempt to extract new biological knowledge. In the first part of our work we describe the discovery of sub-structures in the network of epistatic relations (network motifs) that appear in this network more than expected at random. We analyze these connected patterns and try to understand their biological meaning. Interestingly, we discovered that various functional sub-networks have a different motif content, hinting perhaps into differences in the biological characteristics of these functional sub-networks. We show that many (but not all) of the network motifs can be explained by the existence of larger structures such as cliques and bipartite graphs. Finally we combine these findings along with standard clustering techniques to create a system level view of the network. This view consists of large modules, each involving many proteins. The modules are chosen in a way that creates ‘coherent’ interactions with other modules in the sense that proteins in one module have the same relation with most of the proteins in the other module. Importantly, these modules are not necessarily cliques in the original graph. Along with the structures we found in the previous section we show that this analysis enables automatic inference of biological insights. A UNIVERSAL MOLECULAR TIMEBOMB MECHANISM UNDERLIES TRINUCLEOTIDE DISEASES

S. Kaplan1,2, S. Itzkovitz1,3, E. Shapiro1,3

1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 3Department of Computer Science, Weizmann Institute of Science, Rehovot, Israel

Trinucleotide diseases are a broad family of hereditary diseases characterized genetically by an expanded DNA region consisting of a repeated three-letters code. Patients inheriting such an abnormally expanded DNA region experience sudden disease onset, at an age that decreases as the size of the expanded region increases, followed by inevitable and highly-predictable suffering and death. Despite more than a decade of research, the underlying mechanism of these diseases remains an enigma. Although the genes implicated with the various trinucleotide diseases are unrelated and the defects in these genes occur in different parts of the DNA coding for the gene, the diseases shared characteristics suggest that a common mechanism underlies their root cause. Here we suggests, for the first time, a time-bomb like mechanism that uniformly explains how the inherited DNA repeats genetically encode the time of onset and the rate of progression of trinucleotide diseases. The mechanism suggests that the disease manifests and progresses through the further expansion, during the patient's lifetime, of the inherited abnormally-expanded DNA region. The mechanism explains clinical data of many diseases in this family including previously unexplained onset-related phenomena. In addition, it predicts that a general therapy for these diseases could be found in a drug or a procedure that successfully interferes with the ongoing expansion of the disease trinucleotide repeat during the patient's lifetime. SPONTANEOUS RAPID EVOLUTION OF MODULARITY

N. Kashtan, E. Noor, U. Alon

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

Biological networks have an inherent simplicity: They are modular, with a design that can be separated into units that perform almost independently. Little is known about the evolutionary origin of these properties. Current models of biological evolution typically produce highly optimal non-modular networks. Here we suggest a possible explanation for the origin of modularity in biology. We use standard genetic algorithms to evolve networks. The new feature in our study is evolution under an environment (evolutionary goal) that changes in a modular fashion. That is, we repeatedly switch between several goals, each made of a different combination of sub- goals. We find that such 'modularly varying goals' lead to the spontaneous evolution of modular network structure. The resulting networks rapidly evolve to satisfy each of the different goals. Such switching between related goals may represent biological evolution in a changing environment that requires different combinations of a set of basic biological functions. We further find that evolution towards modularly varying goals dramatically speed up evolution compared to evolution towards a fixed goal. The speedup increases with the complexity of the goal: the harder the problem the larger the speedup. Modularly varying goals push populations away from local fitness maxima, and guide them towards evolvable and modular solutions. This study suggests that modularly varying environments promote modularity and significantly contribute to the speed of natural evolution. SIMPLE THERMODYNAMICS UNDERLIE NUCLEOCYTOPLASMIC

R.B. Kopito, M. Elbaum

Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel

Nucleocytoplasmic exchange of proteins and RNAs is mediated by receptors that usher their cargo through the nuclear pores. Peptide localization signals on the cargo determine the receptors with which it will interact. Those interactions are normally regulated by the small GTPase Ran. Hydrolysis of GTP provides the chemical energy required to create a bona fide thermodynamic pump that selectively and directionally accumulates its substrates across the nuclear envelope. A common perception is that cargo delivery is irreversible, e.g. a protein imported to the nucleus does not return to the cytoplasm except perhaps via a specific export receptor. Recently we have shown (Kopito RB & Elbaum M PNAS 2007), that cargo translocation across the nuclear pore occurs bidirectionally. Furthermore, we have demonstrated that steady state in cargo accumulation is a thermodynamic endpoint, which may be reached by a variety of kinetic paths. Thus the nuclear localization signal dictates the fate of a protein population rather than that of the individual molecule. Based on these results, we offer a simple biophysical model that predicts the observed behavior. Instead of a detailed description of all involved biochemical reactions, the model captures the most generic, fundamental features of the nucleocytoplasmic process. The transport system can be likened to an enzymatic reaction in the presence of two substrates competition. Steady state in the cargo transport system is reached only when the concentrations of receptor-cargo complex in the nucleus and the cytoplasm are equalized. This occurs when the nuclear cargo concentration is high enough to compete with the RanGTP. RanGTP indeed serves as a molecular switch, but only for recycling the transport receptor. it suggests that there is only a statistical coupling between the nucleocytoplasmic transport cycle and the Ran cycle. STRATEGY OF TRANSCRIPTION REGULATION IN THE BUDDING YEAST

S. Levy1, J. Ihmels1,2, M. Carmi1, A. Weinberger1, G. Friedlander1, N. Barkai1

1Departments of Molecular Genetics and Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel, 2Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California - San Francisco, CA, USA

Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirements. First, characters of the internal physiological state, such as growth rate, metabolite levels, or energy availability, could be feedback to tune gene expression. Second, internal needs could be inferred from the external environment, using evolutionary-tuned signaling pathways. Coordination of ribosomal biogenesis with the requirement for protein synthesis is of particular importance, since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis. To define the relative contribution of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression in yeast, we subjected S. cerevisiae cells to conditions which decoupled the actual vs. environmentally-expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in continuous cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer-term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmentally-mediated changes in cell growth presented a major selection force during yeast evolution. STRUCTURAL SIMILARITY ENHANCES INTERACTION PROPENSITY OF PROTEINS

D. Lukatsky1,2, B. Shakhnovich2, J. Mintseris3, K. Zeldovich2, E. Shakhnovich2

1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA, 3Harvard Medical School, Boston, MA, USA

Several independent analyses of accumulating high-throughput and specific data on protein-protein interactions revealed a general statistical bias for homodimeric complexes. It was also shown experimentally that the sequence similarity is a major factor in enhancing the propensity of proteins to aggregate. The physicochemical or evolutionary reasons for these striking observations remain unexplained. We study statistical properties of interacting protein-like surfaces and predict two strong, related effects: (i) Statistically enhanced self-attraction of proteins; (ii) Statistically enhanced attraction of proteins with similar structures. The effects originate in the fact that the probability to find a pattern self-match between two identical, even randomly organized interacting protein surfaces is always higher compared with the probability for a pattern match between two different, promiscuous protein surfaces. This theoretical finding explains statistical prevalence of homodimers in protein-protein interaction networks reported earlier. Further, our findings are confirmed by the analysis of curated database of protein complexes that showed highly statistically significant overrepresentation of dimers formed by structurally similar proteins with highly divergent sequences (“superfamily heterodimers”). We predict that significant fraction of heterodimers evolved from homodimers with the negative design evolutionary pressure applied against promiscuous homodimer formation. This is achieved through the formation of highly specific contacts formed by charged residues as demonstrated both in model and real superfamily heterodimers. NEGATIVE AUTO-REGULATION OF A TRANSCRIPTION FACTOR CAN INCREASE THE DYNAMIC RANGE OF ITS DOWNSTREAM GENES

A. Bren1, D. Madar1, E. Dekel1, S. Kaplan1,2, U. Alon1

1Departments of Molecular Cell Biology and Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel, 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Many transcription factors show negative auto-regulation, where they repress their own promoter. Since this is a recurring regulatory pattern, it is of interest to understand its functional roles. Negative auto-regulation has been shown to increase the speed of transcription responses, and to stabilize expression of the transcription factor against noise. Here we demonstrate an additional function of this network motif: negative auto-regulation of a transcription factor can increase the range of input signals over which its downstream genes respond. This function is demonstrated in the arabinose system of Escherichia coli, using high resolution measurements of promoter activities using fluorescent reporter strains. Negative auto-regulation increases the dynamic range in this system by about an order of magnitude compared to mutants without auto-regulation. The increased dynamic range provided by negative auto-regulation can be tested in other systems, and, together with its other functions, can help explain its widespread appearance in biological regulatory networks. DECIPHERING FUNCTION – FORM MOTIFS BY SYSTEM LEVEL ANALYSIS OF GENE EXPRESSION

A. Madi1,2, Y. Friedman1, D. Roth2, T. Regev1, S. Bransburg-Zabary1,2, E. Ben-Jacob1

1School of Physics and Astronomy, Tel Aviv University, Ramat Aviv, Israel, 2Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel

DNA chips are a valuable and powerful tool that allows measuring simultaneously genome wide response of thousands of genes. Many advanced methods have been developed over the years that usually involve analyses of the gene expression level by means of clustering algorithms. Here we present a new approach motivated by the idea that valuable information is embedded in the similarities (correlations) between the expressions profiles of different genes. Consequently, we employ the functional holography method in which the normalized matrices of correlations between gene expressions are analyzed. The approach is demonstrated by analyzing database of Bacillus subtilis gene expression response to sub-lethal levels of 37 different antibiotics. Using an unsupervised clustering (dendrogram algorithm) of the gene expression correlation matrix we found that the genes of each operon (that is involved in the response) form a distinct cluster and sub-clusters in correspondence to their sub-units in the operon. The functional holography method also includes dimension reduction of the correlation matrix using the Principal Component Analysis - projection of the genes on the 3-D space of the three principal vectors. Doing so we found that the genes locations in the reduced space recover (for operons whose internal structure is known) the known form motifs of the operon such as gene order, separation by non coding segments and translational start and stop sites. Thus, the analysis enables to decipher (for operons with partial knowledge) intra-operon form motifs from the functional ones (genes response). The approach also enables to expose inter operon relationships such as operons that have functional similarity, operons that share functional regulation factors and more. In particular we will demonstrate detection of activators and inhibitors for the competence and sporulation pathways. AN AUTONOMOUS SYSTEM FOR IDENTIFYING AND GOVERNING A CELL'S STATE IN YEAST

L. Nissim, T. Beatus, R. Bar-Ziv

Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel

We present an approach for an autonomous system that detects a particular state of interest in a living cell and can govern cell fate accordingly. Cell states could be better identified by the expression pattern of several genes than of a single one. Therefore, autonomous identification can be achieved by a system that measures the expression of these several genes and integrates their activities into a single output. We have constructed a system that diagnoses a unique state in yeast, in which two independent pathways, methionine anabolism and galactose catabolism, are active. Our design is based on modifications of the yeast two-hybrid system. We show that cells could autonomously report on their state, identify the state of interest, and inhibit their growth accordingly. The system's sensitivity is adjustable to detect states with limited dynamic range of inputs. The system's output depends only on the activity of input pathways, not on their identity; hence it is straightforward to diagnose any pair of inputs. A simple model is presented that accounts for the data and provides predictive power. We propose that such systems could handle real-life states-of-interest such as identification of aberrant versus normal growth. DISENTANGLING SIGNALING GRADIENTS GENERATED BY EQUIVALENT SOURCES

N. Rappaport1, M.J. Concalves De Sa2, M. Piel3, A.W. Murray2, N. Barkai1

1Departments of Molecular Genetics and Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel, 2MCB, Harvard University, Cambridge, MA, USA, 3Biologie du Cycle Cellulaire et de la Motilite, Institut Curie, Paris, France

Direction sensing has an important role in many biological contexts, such as axonal guidance, cell polarity and chemotaxis. Normally, sensing is obtained by secretion of a chemical from a localized source and detection of its gradient by a receiver cell, which is followed by the proper biological response. Uniform degradation of the chemical is canonically believed to facilitate the gradient detection. In yeast, detection of mating factor gradients secreted from opposite cell types is essential for mating, and is followed by a gene expression response, cell cycle arrest, polarized growth towards the source and eventually cell fusion. Thus, this system can serve as a model system for studying short range gradient detection. We examine strategies for gradient sensing in the yeast mating system. We show that local degradation of the secreted molecule can facilitate efficient gradient sensing and is especially helpful under conditions of confusing signals at short range or high diffusion coefficient. Indeed, experimental results support our prediction, and indicate that uniform degradation alone cannot be the mechanism for gradient detection in the yeast mating system. DISSECTION OF A COMPLEX GENETIC NETWORK DETERMINING NATURAL GENETIC VARIABILITY

G.H. Romano, Y. Gurevich, M. Kupiec

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Natural selection is driven by changes in the nucleotide sequence of genomes. These mutations increase the genotypic and phenotypic diversity of organisms and determine their fitness by affecting their ability to survive and multiply in a variety of ecologic niches and under different, often changing, environmental conditions. Genetic variability is therefore a crucial component of evolution, yet the mechanisms that shape and affect genetic variability remain largely unknown. This is mainly due to the fact that the fitness of an individual is determined by several levels of complexity: Each phenotypic trait influences and is influenced by many other characteristics of an individual, and most traits are determined by many quantitative trait loci (QTLs) and their combinations. Thus, the variability in the phenotypic expression of any particular trait is affected by many genes, in complex ways that remain basically uncharacterized. We present a new global strategy to isolate, for the first time, the network of QTLs determining the genetic variation of a single phenotypic trait. Our strategy enables the isolation and mapping of a complete genetic network underlying a specific trait, rather than the mapping of its individual QTLs. In this study we identify a genetic network, composed of QTLs in fifteen different genomic regions, that determines the relative fitness of yeast cells under alkali stress. One of the QTLs identified is affected by the presence or absence of a mobile genetic element, demonstrating the influence of these genetic units in shaping phenotype. Our strategy opens the way for further analysis of the basic molecular mechanisms underlying genetic diversity. DNA ABERRATIONS IN COLON CANCER: THE ROLE IN TUMOR INITIATION AND PROGRESSION

M. Sheffer1, M. Bacolod2, F. Barany2, P.B. Paty3, D.A. Notterman4, E. Domany1

1Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel, 2Department of Microbiology, Weill Medical College of Cornell University, New York, NY, USA, 3Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA, 4Department of Molecular Biology, Princeton University, NJ, USA

The development of colorectal cancer takes decades and requires the accumulation of mutations of key regulatory genes. Some of these alterations may be due to changes in genome copy number and structure. We are interested in mapping the regions that exhibit the most frequent copy number alterations in colon cancer; in particular, in those copy number changes that are reflected in the transcriptional level of the affected genes. Based on a data set of 337 Affymetrix U133A RNA chips and 122 Affymetrix Xba2 DNA SNP chips, comprising different stages of the disease as well as normal tissue, we developed a statistical method that searches for CINons - regions whose DNA copy number is frequently amplified (amplicon) or deleted (deleton), based on both copy numbers and genotype calls. We then filtered only those regions that contain genes with significant correlation between their DNA copy number and expression level, and constructed a map of these CINons in colon cancer. Clustering analysis of this map divided the colon cancer samples into two groups. The first group, characterized by low degree of DNA changes, is mostly comprised of adenomas (non-cancerous tumors) and MIN (micro-satellite instability) tumors. Some of these low degree changes may play a role in early tumorigenesis. The second group of samples, characterized by high degree of DNA changes, is mostly comprised of carcinomas and metastases. Annotation analysis of the genes that are found within these CINons reveals enrichment of pathways that are known to be related to cancer progression. SINGLE CELL ANALYSIS OF SIZE CHECKPOINT OF BUDDING YEAST

I. Soifer1, A. Doncic1,3, M. Carmi1, N. Barkai1,2

1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel, 3Department of Biomedical Engineering, Tel Aviv University, Ramat Aviv, Israel

Most cell populations are characterized by a relatively constant cell size. When perturbed by changing nutrient conditions or inhibiting cell division, this characteristic cell size is restored as fast as in one cell division. The size control in budding yeast operates by means of size checkpoint in G1 which is prolonged in small cells. Systematic genetic screens that measured the average cell size of a whole population implicated various pathways in setting the cell size. However, the global view of the size control is missing We are using a high-throughput microscopy and automated image analysis software for cell tracking and analysis of the cell cycle of budding yeast. To this end we developed software that allows us to simultaneously follow and measure thousands of yeast cells per experiment. Using this tool we show that the timing of the initiation of S phase in the yeast cell cycle has a considerable variability which is high in the daughter cells and low in the mother cells. However, the variability of size at which the cells enter S phase is much smaller. This finding indicates that the cell cycle of budding yeast is under tight size control. GENETIC INTERACTIONS BETWEEN GENES INVOLVED IN TELOMERE LENGTH REGULATION IN SACCHAROMYCES CEREVISIAE

L. Ungar, M. Kupiec

Department of Molecular Biology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

Telomeres are the nucleoprotein complexes that protect chromosome ends from degradation and allow their complete replication. In most organisms, telomere DNA consists of an array of short TG-rich repeats. Saccharomyces cerevisiae telomeres consist of a 250- to 400-bp array of the heterogeneous repeat TG1-3. Previously our lab conducted a large scale screen of all the non-essential genes in Saccharomyces cerevisiae, using a large collection of strains, each deleted for a different gene. We found 171 TLM (Telomere Length Maintenance) genes that cause telomere length alterations when deleted. 121 of the mutants showed a short telomeric phenotype and 50 exhibited long telomeres. Combining our results and those obtained in a similar screen conducted in another lab (Gatbontone et al) we assembled a panel of 98 genes that when deleted show a long telomeric phenotype. Using the SGA methodology, we are creating a large set of double-mutants. Using the information gathered about the phenotype of the double-mutants, bio-informatics, and proper modeling, we will create an informative blueprint of telomere length regulation in yeast. ROBUSTNESS OF SACCHAROMYCES CERVISIAE TO DELETION MUTATIONS AND RESPONSE TO ENVIRONMENTAL STIMULI THROUGH THE LENS OF HIGH-THROUGHPUT METABOLIC PROFILING

I. Venger1,2, I. Rogachev2, S. Mintz2, D. Ackerman3, N. Raban3, Y. Pilpel1, A. Aharoni2

1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel, 3Ruppin High School, Ben Gurion Educational Complex, Emek Hefer, Israel

Metabolomics is the study of the concentrations of all the small molecules present within a cell in a particular condition. It is currently estimated that about 550 metabolites are produced by the yeast Saccharomyces cerevisiae. To date only a few studies have employed large scale metabolic profiling to address questions of functional genomics in S. cerevisiae. We have set up a system that utilizes a gas chromatography – mass spectrometry (GC-MS) method to analyze the yeast metabolome. We apply metabolic profiling of S. cerevisiae to address two major problems that perplex the field of functional genomics. The first stems from the fact that a large amount of deletion mutations do not result in an observable decrease in the fitness of an organism. The second relates to the association between changes in the environment and cellular response to them. First, we explore the metabolic changes that occur within a cell in response to the deletion of metabolic genes that have a paralog elsewhere in the genome. We observe that for some, but not all paralogous gene pairs, a strain with deletion in one of the genes exhibits a metabolic phenotype different than the other deletion strain. Understanding the nature of these differences would help us uncover the biochemical foundations of robustness to gene loss. In the second part of the study, we analyze the response of S. cerevisiae metabolome to an environmental stress; specifically, exposure to 12% ethanol. We observe multiple metabolomic changes; some metabolites increase in their levels (e.g. sorbitol and trehalose), while others show a marked decrease (e.g. serine and threonine). Both studies show that metabolomics is a powerful tool for studying the cellular phenotype in a large-scale, quantifiable fashion. It allows us to gain a new insight into the intricacies of cellular response to genetic changes and environmental stimuli. A NETWORK-BASED ANALYSIS OF PROTEIN COMPLEX EVOLUTION

N. Yosef1, M. Kupiec2, E. Ruppin1,3, R. Sharan1

1School of Computer Science ,Tel-Aviv University, Ramat Aviv, Israel, 2Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel, 3School of Medicine, Tel-Aviv University, Ramat Aviv, Israel

The recent availability of protein interaction data for several species allows now to study protein complexes in an evolutionary context. In this work we develop a novel computational framework for reconstructing the evolutionary history of protein complexes from a network perspective. Our method is based on generalizing evolutionary measures for single proteins to the level of subnetworks, considering both the potential preservation of protein sequences and their interaction patterns. Specifically, we show how to compute sets of orthologous complexes across species and derive evolutionary rate and age measures for a protein complex. With these measures in hand, we investigate how different attributes of a protein complex impact its evolution. We find that the evolution rate of a protein complex highly correlates with its level of connectivity in a network of protein-protein interactions, its functional specificity (according to the GO process annotation of its members) and its essentiality. These findings are consistent with those previously reported for single proteins. Furthermore, we find a high level of agreement between the evolutionary rate of proteins and that of the complexes they form, strongly supporting the view that protein complexes tend to undergo evolution as units, concomitantly to changes at the sequence level. Finally, we turn to explore the role of homomeric gene duplication in the evolution of complexes. The hypothesis that these events are key to the development of modularity and complex formation in protein-protein interaction networks was so far verified only in an indirect manner, e.g., by showing that paralogous dimers are likely to evolve from the duplication of homodimers. Explicitly measuring the dominance of paralogous homodimers in the formation of orthology relations between complexes, we find that ~24% of the sets of orthologous complexes originate from conserved cores of homodimers which underwent duplication. The integrated computational approach presented here is likely to lead to many further new insights concerning complexes' evolution as molecular interaction databases continue to expand in their size and species coverage. A NOVEL APPROACH FOR GENOME-WIDE MAPPING OF SPLICING FACTORS BINDING SITES

M. Akerman, M. Stark, H. David, Y.G. Assaraf, Y. Mandel-Gutfreund

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Alternative splicing is a post-transcriptional process that enhances genetic expression by leading to the production of several protein products from a same gene. Alternative splicing is regulated by a variety of splicing factors (SF) that can produce positive or negative effects on the splicing reaction by differentially interacting with splicing factor binding sites (SFBS) along the exons and introns. In general, SFs bind SFBS by assembling into large complexes, therefore SFBS are often found in clusters possessing relatively high degree of evolutionary conservation. Here we present a novel computational tool for predicting SFBS of known splicing factors which considers both the clustering propensity and the evolutionary conservation of the cis- regulatory elements. Our method was trained and tested using experimentally validated sequences and displays a low false positive rate (~1%). In addition, we show that the method can be used to detect enrichment of SFBS in a population of cassette exons flanking introns as well as interesting discrepancies in the regulation of highly specific and broadly expressed SFs. Finally we tested our tool on a set of splicing defective genes derived from a drug-treated leukemia cell line and predicted a significant enrichment of hnRNP H/F binding sites flanking exon junctions which was not observed in the non effected genes. These result reinforce that our method is both sensitive and selective and can be generally applied for genome-wide mapping of other splicing factor binding sites BUILT-IN LOOPS FINE-TUNE DOMAIN-DOMAIN RECOGNITION

E. Akiva, Z. Itzhaki, H. Margalit

Department of Molecular Genetics and Biotechnology, Faculty of Medicine, Hebrew University of Jerusalem, Israel

Recent compilations of domain-domain interactions based on solved structures suggest that there are distinct domain pairs that are used repeatedly in different protein contexts to mediate protein-protein interactions. However, not all protein pairs with the corresponding domains that can potentially mediate interaction do interact. This may suggest that protein-protein interaction modularity is achieved by a hierarchy of features. Domain pairs are at the top of this hierarchy, and below this level there are additional sequential and structural elements within the domain that define more delicate levels of this modularity. To prove this hypothesis we analyzed domains that are known to mediate homodimerization by self-interactions. Since these domains appear also in monomers, the comparison of the monomeric and dimeric instances of a domain should uncover discriminative features, below the domain level. Indeed, we discovered distinct loops within the domains that play critical roles in determining the interactions. There are enabling loops which are found in homodimers but are absent from the monomers and mediate self-interaction, and there are disabling loops which are present in the monomers but absent from the homodimers and prevent the interactions. The presence of the enabling and disabling loops in homodimers and monomers, respectively, is highly preserved in evolution. This suggests that along with the preservation of structural elements that enable interaction, evolution maintains elements intended to prevent unwanted interactions. The enabling and disabling loops discovered in this study provide fine-tuning of domain-domain interactions with direct implications for prediction of protein-protein interactions (achieving 79% accuracy). Our results provide a proof of concept for the existence of more refined levels of modularity below the domain level. INDEL BASED GENOTYPING - EXPANDING THE HORIZON OF GENETIC ASSOCIATION STUDIES

P. Akiva1,2,3, S. Pollock2, A. Vaknin2, T. Zekharya2, L. Bazak2, S. Edelheit2, L. Dassa2, A. Golubev2, E.Y. Levanon3, A. Wool2, G. Rechavi1, Y. Cohen2

1Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer and Sackler Medical School, Tel Aviv University, Ramat Aviv, Israel, 2Compugen Ltd., Tel Aviv, Israel, 3Department of Genetics, Harvard Medical School, Boston, MA, USA

Recent reports indicate that Insertions and Deletions (INDELs) play a significant role in human polymorphism. Here, we developed a novel platform to identify, catalog and genotype fine-scale human INDELs that is practical for genetic association studies. Based on DNA and expressed sequences we mapped all INDELs varying in length from 15bp to 500bp. Each INDEL was evaluated for its potential capacity to exert phenotypic effects and was linked in an integrated database to disease, drug response and pathway data. Genotyping involves PCR, sequencing and computational analysis of resultant chromatograms. Innovative algorithmic tools enable the deciphering of heterozygous chromatograms. We applied our platform to search for susceptibility loci for type II diabetes and discovered a novel 15bp insertion within the gene PFKP (Phosphofructokinase, platelets). We make a case that INDEL-based genetic association studies compliment current methods based on Single Nucleotide Polymorphisms, and thus offers a more complete view of genome variability AN INTEGRATIVE STUDY OF BACTERIAL INTEIN-LIKE AUTO- CATALYTIC DOMAINS IN THEIR NATIVE BACTERIA

B. Dassa, S. Pietrokovski

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Inteins and related protein domains post-translationally modify their protein molecules by auto-catalytic protein-splicing, self-cleavage and ligation reactions. During protein-splicing an intervening protein domain (intein) cleaves itself out of the precursor host protein and efficiently ligates its two flanks with a new peptide bond. In animal hedgehog developmental proteins, an intein-like domain processes its precursor protein by cleaving off the N-terminal half and attaching a cholesterol molecule to it. Although these post-translational activities are part of different biological processes, their biochemistry is similar and carried out by related families: inteins, Hedgehog, and Bacterial Intein-Like (BIL) domains. We study the sequence- to-function relation of BIL domains, integrating computational and experimental approaches. Genomic analyses revealed new BIL families in over 80 diverse bacterial species, including pathogens of humans, animals and plants. BILs are usually modularly organized at the junction between conserved and hyper-variable regions of bacterial proteins that undergo rapid evolutionary changes, such as adhesins, secreted or surface-exposed proteins. We experimentally demonstrated the protein-splicing and self-cleavage activities of BILs. Remarkably, the protein-splicing mechanism of BILs appears to be novel and distinct from that of inteins. Additionally, we demonstrated the in vivo transcription, translation, secretion and processing of a new type of a BIL- containing gene in the predator B. bacteriovorus protein, and showed its differential processing throughout the dimorphic life-cycle of the bacteria. BILs may also ligate various nucleophilic molecules (including amino acids, peptides and proteins) to their N-terminal flank. We propose BILs as a new type of bacterial post-translational mechanism for generating protein variability, particularly in extracellular proteins. In some cases BILs activity joins micro-evolutionary processes to generate this diversity. Auto-catalytic processing by BILs can thus play a role in bacterial adhesion, immunogenicity and adaptation. Studying BILs may reveal new ways of protein maturation and control by cis- and trans-ligation of proteins and other moieties. PREDICTING DELETERIOUS MUTATIONS ON THE RIBOSOMAL RNA USING SUPPORT VECTOR MACHINES

H. David-Eden, Y. Mandel-Gutfreund

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

The ribosome is a complex macromolecular machine that catalyzes protein synthesis in all organisms. A large proportion of clinically useful antibiotics exert their antimicrobial effects by inhibiting protein synthesis on the ribosome. In the last years, several molecular structures of the ribosome have been solved in high resolution. These structures as well as biochemical and genetic studies provide valuable information to understand ribosome function at the atomic level. In an attempt to identify new functional sites on the ribosome that can serve as a potential targets for new antibacterial drugs, we trained an SVMs (Support Vector Machines) algorithm on structural and functional parameters of 76 mutations with mild (negatives) and deleterious (positives) phenotypes. Among the features, we included new parameters which we have recently applied to model the ribosome structure. In addition, we included classical structural features such as solvent accessible surface area, crystallographic B-factor, and evolutionary conservation of the corresponding nucleotides. Importantly, evolutionary conservation, which is a classical parameter used to identify functionality, is high for both the deleterious mutations, and for a large fraction of the mild mutations subset, therefore not sufficient information to assess the phenotypic effect of a single point mutation. Nonetheless, our results show that we can distinguish weak from deleterious mutations based on structural and functional information from the mutations and their local environment (accuracy = 87%). In summary, we present a computational knowledge-based approach to identify nucleotides that are critical to ribosome functionality. FROM GENE SEQUENCE TO PROTEIN FUNCTION ON THE GENOMIC LEVEL: 3D METAL-BINDING SITE PREDICTIONS FROM TRANSLATED DNA SEQUENCES

R. Levy, M. Edelman, V. Sobolev

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel

Homology modeling is an accepted bioinformatics tool that increasingly is being used to study a wide range of structural problems. Currently, the community considers a model of a protein acceptable if the overall RMSD between X-ray and modeled structure is within 2A. We recently demonstrated the ability to effectively predict metal binding-sites from apo structures resolved by X-ray crystallography using the "CHED algorithm" (Babor et al, 2007, PROTEINS 10.1002/prot.21587, on line). We now ask: Can structures obtained by modeling translated gene sequences be effectively used for metal binding-site prediction? This issue is especially relevant in light of current attempts to go from sequence to function via structural homology modeling. We first examined the extent by which modeling alone reduces the ability to predict metal binding sites. This was found to be minor (< 5%). Next, we determined the current general overlap between structural and linear database space; i.e., the fraction of PDB structures that can productively serve as structural templates for TrEMBL sequences. This was found to be significant (~ 42%). An outline of our procedure to locate potential metal binding sites from translated gene sequences is as follows: Input a Swiss-Prot/TrEMBL sequence ("Target"); seek a homologous PDB sequence ("Template") using PsiBlast; structurally model the Target amino acid side chains using the Template as backbone; output the predicted metal binding site using the CHED algorithm. A statistical analysis of our approach using structurally resolved protein sequences as both targets and templates showed that selectivity (correct predictions/total predictions) remains quite high (85-90%) using modeled PDB templates, irrespective of the presence of a metal in the Template or the level of sequence homology to the Target over a very broad range (18-99%). REVEALING THE tRNA-BINDING AND RECOGNITION SITES OF AMINO-ACYL tRNA SYNTHETASES BY MEANS OF CORRELATED MUTATIONS ANALYSIS

M. Frenkel-Morgenstern, D. Tworowski, M.G. Safro

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

The aminoacyl-tRNA synthetases (aaRSs) family comprises twenty enzymes that covalently attach amino acids to the corresponding nucleic acid adaptor molecules, tRNA. We addressed the question of tRNA binding by aaRSs, and tRNA-recognition site adaptation or stabilization using the correlated mutation analysis inside the aaRS molecules. Eight of aaRS-tRNA complexes with known structures were subjected to the analysis. For each aaRS, we compared a relative number of correlated mutations for a group of exposed residues with those for a group of the recognition site residues with or without charged ones. We found that a frequency of correlated mutations in the tRNA-recognition site is significantly higher in average than for the group of exposed residues and the protein itself. Moreover, a group of charged residues in the recognition site has a higher rate of correlated mutations for each aaRS. It is of interest that a level of correlated mutations for all charged residues, inherent in the protein is comparable to those of the recognition site. Thus, correlated mutations analysis being in excellent agreement with the hypothesis, predicted before that non- specific electrostatic interactions are the driving forces for primary stickiness of aaRSs and tRNA. This property of correlated mutations inside aaRSs and their recognition sites may be defined, in fact, as a new effective method of mapping the tRNA-binding and recognition sites in tRNA-aaRSs complexes. This also sheds light on the physical nature of correlated mutations as compensatory mutations intended to preserve protein functional regions such as tRNA-recognition sites in the aaRS. tRNA-DEPENDENT PATHWAY OF CYSTEINE BIOSYNTHESIS IS ACCOMPANIED BY INCREASE OF CYSTEINE CONTENT IN METHANOGENIC ARCHAEA

L. Klipcan, M. Frenkel-Morgenstern, M. Safro

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

The genetic code is established by a 20 aminoacyl-tRNA synthetases (aaRSs) that specifically attaching amino acids to their cognate transfer RNAs. However, since rich variety of genomes of methanogenic Archaea were sequenced, it was found that a given organisms lack the canonical class-I CysRS as well as components of cysteine biosynthetic pathway. Recently, it was reported that another distinct aaRS, called phosphoseryl-tRNA synthetase (SepRS), ligates phosphoserine to tRNACys. Then phosphoseryl-tRNACys is converted to canonical Cys-tRNACys by another enzyme SeptRNA:Cys-tRNA synthase (SepCysC). Therefore, these reactions can be dubbed as “tRNA- dependent”. We examined the cysteine content among Archaea proteomes, and observed their clustering to two separate groups: first demonstrates relatively low cysteine content (~0.6 % of whole proteome) and the second contains twice as much cysteins as compare to the first one (~1.2%). Proteomes with the low cysteine content are widespread among the different Archaea species, however all organisms within the cluster contain canonical bacterial-like CysRS-I only. Oppositely, cysteine rich proteomes belongs to Euryarchaeota and all but one are methanogenic organisms. By this means all cysteine rich proteomes display correlation between the tRNA- depended pathway and raise in cysteine content. This suggests a linkage between amino acid biosynthetic pathway and its relative incorporation in protein sequences. We also demonstrated that earliest branch of prokaryotes contains relatively high cysteine content and utilize phosphoserine as precursor of cysteine biosynthesis via tRNA-depended or special non-tRNA dependent archaeal pathway. Moreover, we observed that methanogens create special clusters of proteins extremely rich with cysteins. These archaeal proteins are associated with primitive metabolic reactions as electron transfer and nucleic acid binding. Our findings suggest revision of role of cysteine in early life forms. IDENTIFICATION AND GENOMIC CHARACTERIZATION OF DNA REPLICATION TIMING MUTANTS

A. Koren1, N. Barkai1,2

1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel

Eukaryotic cells replicate their DNA according to a defined temporal program. The protein factors that establish the DNA replication timing program are, nonetheless, largely unknown. We have utilized a flow-cytometry-based assay to screen for Saccharomyces cerevisiae deletion mutants exhibiting abberant S-phase profiles, and have identified genes involved in both replication origin firing and replication fork progression as determinants of the proper execution of the replication program. Using a microarray-based method for assaying replication timing genome-wide, we characterized the molecular abnormalities resulting from deletion of these genes. We show that the most frequent perturbations of S-phase progression result from defects in the firing of particular replication origins, throughout the genome. However, in mrc1ִ cells, an elongated S-phase is rather associated with an increased number of origins firing, as well accelerated timing and/or elevated efficiency of origin activation. In many but not all replicons, this enhanced replication activity is correlated with a strong inhibition of replication fork progression. Thus, the elongation defect is a consequence of enhanced origin firing, possibly due to excessive torsional stress created by converging replication forks. An abnormal number of active replication origins is also associated with genomic instability. Our results suggest that the DNA replication timing program is optimized for maximal replication progression rate while maintaining the stability of the genome. COMPARATIVE TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS DURING DIFFERENTIATION OF THE PARASITIC PROTEOSAN LEISHMANIA

T. Lahav1, D. Sivam2, D. Rosenzweig1, N. Holland1, H. Volpin3, P. Myler2, D. Zilberstein1

1Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel, 2Seattle Biomedical Research Institute, WA, USA, 3Keygene N.V., Wageningen, The Netherlands

Leishmania donovani is an intracellular protozoan parasite that causes kala-azar in humans. During infection the extracellular insect forms (promastigotes) undergo rapid differentiation to intracellular amastigotes that proliferates inside phagolysosomes of mammalian macrophages. We aimed to compare mRNA and protein abundance during L. donovani differentiation using a host free system that simulates the differentiation process. mRNA abundance was determined using high-density oligonucleotide array of all ORFs in the L. infntum genome. The microarray indicated that 60% of the genomic expression is changed significantly during differentiation. The results were compared with the recent proteomic data gathered from the same cells: 32% of the genes showed positive correlation between mRNA and protein abundance, 22% showed negative correlation and the rest displayed low correlation. Interestingly, most (71%) of the genes in the positive correlation group were down regulated during differentiation, 19% were up regulated and 4% were constitutively expressed. Whereas major metabolic pathways enzyme abundance changed during differentiation, their corresponding mRNAs remained unchanged, suggesting translation and posttranslational regulation of these genes. FACTORS GOVERNING THE AFFINITY AND SPECIFICITY IN THE COHESIN-DOCKERIN COMPLEX

D. Reshef1, M. Slutzky2, Y. Barak2, E.A. Bayer2, O. Schueler-Furman1

1Department of Molecular Genetics and Biotechnology, Hebrew University of Jerusalem, Israel, 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

The cellulosome is a multi-protein complex used by bacteria for the degradation of cellulose. It is composed of a scaffold, containing multiple repeats of a cohesin domain. Each cohesin binds a dockerin which is covalently bound to a cellulase. This tree-like structure enables highly efficient degradation of a heterogenous cellulose polymer. In general, the cellulosome is an ideal framework for major biotechnological applications. The cohesin-dockerin interaction is of high affinity and remarkable specificity: cross-species cohesin and dockerins do not bind, indicating the evolutionary pressure on the interaction. We used Rosetta to create models of different Clostridium thermocellum and Clostridium cellulolyticum cohesin-dockerin complexes to determine key residues that are thought to be responsible for the affinity and specificity of this interaction. These analyses reveal a complex network of inter- and intra-monomer contacts that mediate overall specificity and affinity. We designed mutations that would create a specificity switch, i.e. replace an existing interaction with a new interaction. Preliminary experimental validation indicates that a new interaction has indeed been created, but that the original interaction is not abolished yet. Current efforts are underway to complete the specificity switch, as well as altering the affinity of the interaction. DNA – THE QUANTITY IS NOT ALL THAT COUNTS

T. Shay1, W.L. Lambiv2, A. Murat2, A. Misra3, B. Feuerstein3, R. Stupp4, M.E. Hegi5, E. Domany1

1Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel, 2Laboratory of Tumor Biology, Genetics and Neurosurgery, University Hospital Lausanne (CHUV), Switzerland, 3Department of Neurology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ, USA, 4Multidisciplinary Oncology Center, University Hospital Lausanne (CHUV), Switzerland, 5National Center of Competence in Research (NCCR) Molecular Oncology, ISREC, Epalinges sur Lausanne, Switzerland

Many cancers contain cells that possess an abnormal number of chromosomes. There are several datasets containing both expression data and copy number data for the same tumors available in public repositories. It was already shown that gain of a chromosome is reflected in over expression of genes on that chromosome, while loss of a chromosome results in downregulation of corresponding genes. However, not all genes on the chromosome are affected. Our aim is to isolate the effect of DNA copy number on expression, and characterize the differences between genes whose expression level is altered by the copy number and those that are not. We focused on Glioblastoma, the most common and aggressive brain tumor, with its typical gain of chromosomes 7, 19 and 20, and loss of chromosomes 10 and 13. The effects of basal transcription level, promoter type, tissue specificity and other factors on expression were investigated. Though not fully understood, these studies shed some light on why some transcript levels are affected by copy number, while others are not. A comparative analysis of copy number effect on expression in several types of cancer, as well as Down syndrome, is displayed and analyzed. GENECARDS: ONE STOP SITE FOR HUMAN GENE RESEARCH

G. Stelzer1, A. Harel1, I. Dalah1, N. Rosen1, M. Shmoish1, T. Iny Stein1, A. Sirota1, A. Madi1, Y. Sole1, M. Safran2, D. Lancet1

1Departments of Molecular Genetics (Bioinformatics Unit), Weizmann Institute of Science, Rehovot, Israel, 2Department of Biological Services (Bioinformatics Unit), Weizmann Institute of Science, Rehovot, Israel

The individual scientist, who seeks knowledge for the research of a gene of interest, can become overwhelmed by the deluge of data from worldwide genome projects. The laborious task of sifting through thousands of records can be reduced by the use of integrated flexible and user-friendly searchable databases. For over a decade, GeneCards (www.genecards.org) has withstood this challenge, providing a one-stop gene-centric site with comprehensive information about human genes, automatically mined and integrated from over 70 sources, including NCBI, ENSEMBL, UniProt, Invitrogen iPath, HapMap, and UCSC, as well as from in-house transcriptome results (http://genecards.weizmann.ac.il/genenote). The extracted data is organized into a "card" format for each gene, with sections that encompass a variety of topics (e.g. genomic location, gene function, transcription, disorders, literature, and pathways). An important GeneCards feature is information on gene-related commercial products, e.g. antibodies and functional assays. Recently, the GeneCards database suite has been enriched with set-centric components. These include: 1) GeneALaCart, which provides batch query support, whereby the user submits a gene list (e.g. from a microarray experiment) along with the desired GeneCards annotation fields, and receives tabulated output that enables facile overview. 2) GeneDecks, which receives as input a single gene or a group of genes, and outputs a set of potential functional gene paralogs. To this end, GeneDecks employs concepts of combinatorial annotation and quantitative enrichment analyses. Such gene-set-related information, together with the single-gene data, results in a unique wealth of compound annotation entries, which enables innovative scientific scrutiny. NUCLEOSOME POSITIONING FROM TILING MICROARRAY DATA

M. Yassour, T. Kaplan, A. Jaimovich, N. Friedman

School of Computer Science and Engineering, Hebrew University of Jerusalem, Israel

The packaging of DNA around nucleosomes in eukaryotic cells, plays a crucial role in transcriptional regulation, e.g. by altering the accessibility of short transcriptional regulatory elements. To better understand transcription regulation, it is therefore important to identify the position of nucleosomes in 5-10bp resolution. Toward this end, several recent works measured nucleosomal positions in a high-throughput manner using dense tiling arrays. Here we present a fully automated algorithm to analyze such data. Using a probabilistic graphical model, we suggest to improve the resolution of the nucleosome calls beyond that of the microarray platform used. We show how such a model can be compiled into a simple HMM, allowing for a fast inference of the nucleosome positions, without any loss of accuracy. We applied our model to nucleosomal data from mid-log yeast cells reported by Yuan et al. (Science, 2005), and compared our predictions to those of the original paper, to a more recent method that uses five times denser tiling arrays (Lee et al., Nat. Genet. 2007), and to a curation of literature-based positions. Our results suggest that by applying our algorithm to the same data of Yuan et al., we were able to trace 13% more nucleosomes, and increase the overall accuracy in about 20%. We believe that such an improvement opens the way for a better understanding of the regulatory mechanisms controlling gene expression, and how they are encoded in the DNA. MLV INFECTION CONFERS DNA REPLICATION STRESS AND LEADS TO PREFERENTIAL INTEGRATION OF THE VIRAL DNA INTO COMMON FRAGILE SITES

A.C. Bester, B. Kerem

Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel

Common fragile sites (CFSs) are chromosomal loci exhibiting perturbed chromatin condensation which appear as constrictions, gaps or breaks on metaphase chromosomes from cells exposed to partial inhibition of DNA replication. CFSs correlate with genomic instability in tumors such as deletions, translocations, amplifications and with integration sites of oncogenic DNA viruses such as HPV16, HPV18, HBV, EBV and AAV. In a gene therapy trial of 10 patients with X-linked severe combined immunodeficiency, using a murine leukemia virus (MLV)-derived vector, two patients developed leukemia due to an activating integration of the vector near the LMO2 gene. We show that the LMO2 gene is located within FRA11E, a CFS on human chromosome 11. Furthermore, we found that CFSs are preferential integration sites of the MLV-derived vector. This led us to hypothesize that the MLV infection confers replication stress, which perturb replication elongation leading to CFS expression. To test this hypothesis we infected HeLa cells with an MLV vector and analyzed replication elongation rate on single DNA molecules using DNA combing. The analysis revealed that the replication rate was 40% slower in the infected cells then in non-infected control cells (P<4.5E-12). We further analyzed the formation of double strand breaks (DSBs) following MLV infection using antibodies against the phosphorylated form of histone H2AX (gama-HeAX), a marker of DSBs. The analysis showed a significant (P<0.003) increase in number of gama-H2AX foci following the infection. These results show that MLV infection causes replication perturbation leading to DSBs specifically at CFSs. Failure to successfully repair these breaks might leads to open chromatin conformation into which the MLV vector preferentially integrates. Preferential integration into CFSs might lead to high frequency of oncogene activatation within these regions. HYPERMETHYLATION AND EXPRESSION OF RASSF1A IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

H. Betzer1, S. Shukla1, I. Naumov1, B. Stark2, G. Avrahami2, D. Luria1,2, Y. Kodman2, S. Avigad1,2, I. Yaniv2

1Department of Molecular Oncology, Felsenstein Medical Research Center, Tel Aviv University, Ramat Aviv, Israel, 2Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah Tikva and Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel

Epigenetic modifications including promoter hypermethylation and histone acetylation are emerging as one of the factors in controlling the expression of tumor suppressor genes. One such gene is the RAS-associated domain family 1 (RASSF1A), a member of RAS effectors that regulate cell proliferation and apoptosis. This gene is frequently methylated in many adult cancers and childhood solid tumors, but little is known about its role in childhood acute lymphoblastic leukemia (ALL). The aim of this research was to study RASSF1A methylation and expression and evaluate mutations in ras oncogene in childhood ALL and correlate it with clinical outcome. Bone marrow samples at diagnosis were obtained from 147 patients diagnosed and treated at the Schneider Children's Medical Center of Israel. The cohort consisted of 101 and 46 B-lineage and T-cell ALL, respectively. The median age at diagnosis was 6 years (range 0.3-18.8 years). Nineteen (13%) of the patients responded poorly to steroids at day 8. Relapse occurred in 41 (28%) patients. Median follow up was 80.5 months (range 5-253 months). DNA was extracted from BM at diagnosis and treated by bisulfite prior to methylation specific PCR (MSP) analysis of RASSF1A and directly sequenced for N, H and K ras mutations. RNA was extracted for the evaluation of RASSF1A expression. Methylation of RASSF1A was identified in 30% of the samples. A border line significant correlation was found between methylation and relapse free survival in the standard risk group of patients. RASSF1A expression analysis correlated with clinical risk groups: high and low expression in the standard and high risk groups, respectively. 17 mutations were detected (11%) in KRAS, NRAS and HRAS. Methylation in the standard risk group might have a role as a prognostic marker for this group for identifying a sub-group with a worse prognosis. G9a-MEDIATED DE NOVO METHYLATION IS NECESSARY AND SUFFICIENT TO PREVENT REPROGRAMMING OF EMBRYONICALLY SILENCED GENES

S. Epsztejn-Litman1, N. Feldman1, A. Gerson1, J. Ueda2, Y. Shinkai2, H. Cedar1, Y. Bergman1

1Departments of Experimental Medicine and Cellular Biochemistry, Hebrew University Medical School, Ein Kerem, Jerusalem, Israel, 2Experimental Research Center for Infectious Disease, Institute for Virus Research, Kyoto University, Sakyo-Ku, Kyoto, Japan

Normal development appears to take place through a unidirectional process characterized by a step-wise decrease in cell potency, and it is presumably this phenomenon that is mainly responsible for the difficulty in reprogramming differentiated somatic cells in vivo. The pleuri-potency determining gene, Oct-3/4 undergoes post implantation silencing in a process that is largely mediated by the histone methyltransferase (HMT) G9a. Microarray analysis now shows that this enzyme actually plays a major role in the inactivation of multiple early embryonic genes by bringing about methylated-histone H3K9 heterochromatinization, as well as de novo DNA methylation. Genetic studies in differentiating ES cells demonstrate that a point mutation in the G9a SET domain prevents the heterochromatinization reaction, but still allows de novo methylation, while biochemical and functional studies indicate that G9a itself is capable of bringing about de novo methylation through its ankyrin (ANK) domain, by recruiting Dnmt3a and 3b in a manner that is independent of HMT activity. These modifications appear to be programmed for carrying out two separate biological functions, with histone methylation serving as a block to target-gene reactivation in the absence of transcriptional repressors, while DNA methylation prevents reprogramming to the undifferentiated state. ALLELIC MARK AND GENE CHOICE AT THE IgkappaV LOCUS

S. Fraenkel, H. Cedar, Y. Bergman

Hebrew University - Hadassah Medical School, Jerusalem, Israel

The genes of the B-lymphocyte antigen receptors are assembled in a stochastic manner from several gene families in a specialized recombination process termed V(D)J recombination. In the Igkappa light chain locus, these gene families are comprised of the variable (V), joining (J) and constant (C) gene segment families. Amongst them, the variable family exceeds all others in the number of its candidate gene segments. The accessibility of the Ig segments for rearrangement is determined by the locus epigenetic state, with acetylated histone H3 characteristic of an active chromatin structure. Here, we report that Vkappa gene families are differentially acetylated at the pre-B cell stage prior to Igkappa rearrangement. Moreover, the level of acetylation appears to correlate with the Vkappa gene usage. The Vkappa region is also subject to allelic exclusion, a phenomenon defined by the expression of only one allele by each B-cell. Studies on the Jkappa and Ckappa regions revealed that allelic exclusion is established at the level of rearrangement, and that a multitude of positive and negative epigenetic marks gained during B-cell development turns one allele into the preferred substrate for the recombination machinery. Interestingly, we found that five Vkappa families are H3-acetylated in an allelic manner, but the acetylated gene segment switches alleles when different Vkappa families are considered. Further, the allelic mark at the pre-B cell stage may determine whether the Vkappa gene segment would subsequently be used in rearrangement. Switching of the allele preference is in agreement with the model of limited transcription factors. EPIGENETIC REGULATION OF SOMATIC RECOMBINATION AT THE IMMUNOGLOBULIN LAMDA LIGHT CHAIN

M.R. Gouzman, Y. Bergman

Hadassah Medical School, Hebrew University of Jerusalem, Israel

V(D)J recombination is a major event in B cell development, generating the vast repertoire of antibody molecules. The recombination process can operate on the three Ig loci, the heavy chain and the two light chains, kappa and lamda. It was previously shown that the Igkappa locus undergoes a series of epigenetic changes to become accessible for rearrangement, and most importantly, that these changes occur in a monoallelic fashion thus serving as the basis for allelic exclusion. Epigenetic regulation of V(D)J recombination of the Iglamda locus has not been studied in detail so far. We studied the changes in chromatin structure across the Iglamda locus during B cell development using ChIP assays, examining both active and repressive chromatin modifications. The active H3-acetylation levels were found to increase with development, whereas the repressive H3K9me2 levels decreased. Altogether, the Iglamda locus becomes more accessible during development, facilitating V(D)J recombination and gene expression. Allelic differentiation assays based on polymorphisms revealed that histone modifications at the Iglamda locus are monoallelic at the pre-B cell stage. These include the active H3Ac and H3K4me2/3 marks, as well as the repressive H3K9me2. Moreover, we show that the epigenetic state of the alleles correlates with the subsequent allele choice. EPIGENETIC MECHANISM OF FMR1 INACTIVATION IN FRAGILE X SYNDROME

M. Hecht, A. Tabib, I. Keshet, H. Cedar

Department of Cellular Biochemistry and Human Genetics, Hebrew University Medical School, Jerusalem, Israel

The primary defect in Fragile X syndrome is a triplet repeat expansion in the 5’ UTR of the FMR1 gene on chromosome X. This amplification probably takes place during oogenesis or in the very early embryo and is made progressively worse from generation to generation in a process called anticipation. Once the repeat region attains a critical size, it induces de novo methylation of the repeat region as well as the nearby CpG island promoter, and it is this embryonic event that brings about the epigenetic repression of FMR1 and results in mental retardation. The mechanism for this de novo methylation has not yet been deciphered. In order to gain insight into the nature of this process, we have used cell fusion technology to recapitulate the events that occur during early embryogenesis. These experiments show that the Fragile X- FMR1 5’ region undergoes inactivation post implantation in a Dicer-dependent targeted process that involves local SUV39H-mediated tri-methylation of histone H3(K9), exactly mimicking molecular events that take part in the formation of constitutive heterochromatin in vivo. THE EVOLUTION OF TRANSPOSABLE ELEMENTS FOLLOWING GENOME DOUBLING

K. Kashkush, K. Vadim, L. Snir, Z. Kraitshtein

Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Combining two or more different genomes in the same nucleus by inter-specific or inter-generic hybridization followed by chromosome doubling can lead to the formation of new allopolyploid species. Recent studies have shown that allopolyploidy is associated with rapid genetic and epigenetic changes. The mechanisms by which two or more different genomes achieve a harmonic coexistence in the same nucleus and those causing rapid genomic changes (genetic and epigenetic) are largely unknown. The biological impact of TE activation on the host genome structure and expression as well the evolution of transposable elements (TEs) following allopolyploidization is poorly understood. TEs are usually silent but can be activated by biotic and abiotic stresses. In addition, interspecific hybridization and allopolyploidy can cause a “genomic shock” and thus activate TEs. In this study, the methylation patterns of TEs and their flanking sequences were undertaken in the first generations of newly synthesized wheat allotetraploid and its diploid parents, using a novel technique called Transposon- Methylation- Display (TMD). TMD was successfully applied on various high copy TE elements. We showed that TE methylation alters due to genome doubling. In many instances the alteration of TE methylation was correlated with TE transcriptional activation, where demethylted TEs found to be transcriptionally active in the newly formed polyploid. Barbara McClintok’s prediction, nearly twenty years ago (McClintock, 1984), that interspecific hybridization can cause a “genomic shock” and thus activate TEs which could thereby restructure the genome is gaining support. STRESS ACTIVATED MAPK REGULATE TELOMERIC HETEROCHROMATIN IN S. CEREVISIAE

Y. Mazor, M. Kupiec

Department of Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Variegated expression of genes contributes to phenotypic variation within populations of genetically identical cells. Such variation plays a role in development, host pathogen interaction and can play a role in adaptation to harsh environments. The expression state of genes placed near telomeres show a variegated pattern of inheritance due to heterochromatin formation, a phenomena that is called telomere position effect (TPE). We show that in yeast, TPE is controlled by the locus MAT, which dictates developmental decision in response to environmental changes. From the genes controlled by MAT we show that the stress activated MAPK, HOG1, is the major effector of this heterochromatin regulation pathway. We examine a possible link between telomeric DNA and rDNA stability which is mediated by this stress regulated pathway. Our results show that some of the same components that regulate gene expression states in euchromatin regions regulate heterochromatic expression states and that stress can play a part in turning genes placed in heterochromatic regions on or off. CHROMATIN REMODELING CONTROLS PHASE TRANSITION THROUGH PLANT EVOLUTION

A. Mosquna, R. Izhaki, R. Yaari, A. Katz, N. Ohad

Department of Plant Sciences, Tel-Aviv University, Ramat Aviv, Israel

During the plant life cycle, particular gene expression programs are established requiring a cell memory mechanism for their activation and maintenance. In Arabidopsis, the polycomb group protein complex (PcG) takes part in regulating gene expression programs. The PcG complex contains three main protein families: WD, single zinc finger and SET domain proteins. Mutation in each of the Arabidopsis PcG proteins triggers precocious abnormal developmental programs of the gametophyte and/or of the sporophyte. For example, mutation in FIE coding for a WD40 protein, leads to autonomous endosperm development, a tissue witch nourish the embryo, without fertilization. Using the moss Physcomitrella patens as a model system representing early terrestrial plants, we have examined PcG function along plant evolution. We identified a putative Physcomitrella PpFIE PcG gene and elucidated its function by the analysis of knock out PpFIE moss plants and plants bearing GUS fusion to the endogenous PpFIE gene, generated by homologues recombination. PpFIE knockout plants display arrest at a gametophytic stage, forming buds that continue to proliferate into a callus-like tissue, instead of differentiating gametophores bearing gametes. Transgenic PpFIE-GUS moss show specific protein accumulation pattern in the gametophore meristems. This pattern correlates in time and place with the manifestation of PpFIE-KO mutant phenotype. Applying reciprocal complementation assays in which the moss PpFIE gene was introduced into Arabidopsis fie mutant plants and vice versa, demonstrated that FIE has maintained its function through evolution. The combined approach presented above reveals the central role of chromatin remodeling, mediated by the PcG complex, in controlling the transition from vegetative to reproductive stages during plant evolution. INTERPLAY BETWEEN ATM AND ATR IN THE REGULATION OF COMMON FRAGILE SITE STABILITY

E. Ozeri-Galai, M. Schwartz, A. Rahat, B. Kerem

Department of Genetics, The Silberman Life Sciences Institute, Hebrew University of Jerusalem, Israel

Common fragile sites are specific genomic loci that form constrictions and gaps on metaphase chromosomes under conditions that slow, but do not arrest, DNA replication. These sites have been shown to have a role in various chromosomal rearrangements in tumors. Different DNA damage response proteins regulate fragile site stability, including Ataxia-Telangiectasia and Rad3-Related (ATR) and its effector Chk1. Here we investigated the role of ataxia-telangiectasia mutated (ATM), the main transducer of double strand break (DSB) signal, in this regulation. We demonstrate that replication stress conditions which induce fragile site expression lead to DNA fragmentation and recruitment of phosphorylated ATM to nuclear foci at DSBs. We further show that ATM plays a role in maintaining fragile site stability, which is revealed only in the absence of ATR. Following conditions which induce fragile site expression both ATR and ATM phosphorylate Chk1, suggesting that both proteins regulate fragile site expression probably via their effect on Chk1 activation. Our findings provide new insights into the interplay between ATR and ATM pathways in response to partial replication inhibition and in the regulation of fragile site stability. THE IMPRINTING MECHANISM AT THE PRADER-WILLI/ANGELMAN SYNDROMES DOMAIN USING A TRANSGENIC SYSTEM

S. Rabinovitz, R. Shemer, A. Razin

Department of Cellular Biochemistry and Human Genetics, Hebrew University - Hadassah Medical School, Jerusalem, Israel

A number of imprinted genes are clustered within the 2 Mb imprinted domain on human chromosome 15q11-q13 and its orthologue on mouse chromosome 7c. Loss of this 2 Mb domain on the paternal or the maternal allele results in two neurogenetic disorders, Prader-Willi Syndrome or Angelman Syndrome, respectively. Imprinting of the genes in this region appears to be coordinated by an imprinting control center (IC), which itself consists of two regulatory elements – PWS-IC and AS-IC. PWS-IC plays a role in the regional activation of all the paternally expressed genes, and is maternally imprinted as a result of repression conferred by AS-IC. In an effort to decipher the mechanism by which the regional imprinting control works we have used transgenic mice and transfected cells in culture. To this aim several transgenic constructs have been constructed: 1. The NDN gene, one of the imprinted genes in the cluster. 2. The NDN gene adjacent to the PWS-IC 3. The NDN gene adjacent to the PWS-IC and the AS-IC. Our results reveal that NDN, when included PWS-IC and AS-IC, was imprinted being unmethylated upon paternal transmission and methylated after maternal transmission. Yet NDN in a transgene that did not include the PWS-IC was, as expected, methylated upon both paternal and maternal transmission. This result indicates that the activation and transcription of the NDN gene depends exclusively on the status of imprinting at PWS-IC. To examine whether the activation of the NDN by the PWS-IC is specific, depending on its sequence, we tested a transgenic line that contains the PWS-IC and the AS-IC transgene ligated to the human non-imprinted gene ApoAI. Surprisingly, the ApoAI gene was unmethylated upon paternal transmission and silenced upon maternal transmission, indicating that activation of the genes by the PWS-IC on the paternal allele is sequence independent. THE Dm ELEMENT, A NOVEL cis REGULATORY ELEMENT IN KAPPA LIGHT CHAIN REARRANGEMENT AND ALLELIC EXCLUSION

C. Rosenbluh, A. Kirilov, Y. Bergman

Department of Experimental Medicine and Cancer Research, Hebrew University Medical School, Jerusalem, Israel

Somatic rearrangement in the immunoglobulin heavy and light chains plays a central role in generating the vast variety of antibodies necessary for normal immunological response. In order to guarantee that each cell will generate only one specific antibody it is crucial that only one allele will be rearranged. This phenomenon is known as “Allelic Exclusion”. Allelic exclusion is made possible through differential epigenetic status of the two alleles. While one allele, which will eventually be a preferred target for rearrangement, is in an open conformation, early replicating, and centrally located in the nucleus, the second allele is in a closed conformation, is late replicating and is recruited to heterochromatin. In addition, immediately prior to rearrangement, the chosen allele undergoes DNA demethylation. We here present data identifying a novel cis regulatory element in the Kappa light chain locus that we designated the Demethylation (Dm) Element, which is central in regulating DNA demethylation. Using knock out mice we demonstrate that this element is indeed central in enabling rearrangement, and pinpoint the developmental stage at which the DNA demethylation takes place. We also present data suggesting that this Dm element contains a Pax5 binding element, and demonstrate that indeed it is crucial in Pax5 recruitment to the Kappa region. Based on this new data we suggest a model for the monoallelic DNA demethylation at the Kappa light chain locus in mice. IMPAIRED REPLICATION STRESS RESPONSE IN CELLS CARRYING CERNUNNOS/XLF MUTATIONS

M. Schwartz, A. Rahat, B. Kerem

Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel

Non-Homologous End Joining (NHEJ) is a major Double Strand Break (DSB) repair pathway in mammalian cells. In humans, mutations in genes encoding different NHEJ components can lead to Severe Combined Immune Deficiency (SCID). Cernunnos/XLF is a recently identified factor of the NHEJ pathway that acts as part of the Ligase IV/XRCC4 complex- the complex that performs the ligation step in this pathway. Mutations in the gene encoding XLF/Cernunnos in humans lead to Immunodeficiency with microcephaly. Here we investigated the role of Cernunnos/XLF in the cellular response to replication stress using a cell line from a patient harboring a mutation in the gene encoding Cernunnos/XLF. We demonstrate that Cernunnos/XLF mutated cells are sensitive to replication stress conditions. Cernunnos/XLF exhibit impaired DSB repair following mild replication stress as demonstrated by H2AX phosphorylation analysis and prolonged arrest in G2/M. Moreover, Cernunnos/XLF mutated cells display increased chromosomal instability at common fragile sites, specific genomic loci which are sensitive to replication stress. Replication analysis revealed that deficiency in Cernunnos/XLF does not affect the replication process it self. Our data demonstrates a role for Cernunnos/XLF in the repair of DSBs and the maintenance of genomic stability under replication stress conditions. METHYLATION ANALYSIS OF SPECIFIC micro-RNAs IN EWING SARCOMA

S. Shukla1, I. Naumov1, M. Hameiri-Grossman1, I.J. Cohen1,2, S. Ash2, I. Yaniv1,2, S. Avigad1,2

1Department of Molecular Oncology, Felsenstein Medical Research Center, Tel Aviv University, Ramat Aviv, Israel, 2Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah Tikva and Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel

Micro-RNAs (miRNAs) are small non-coding RNAs of 18 to 25 nucleotides that regulate protein expression. While there is accumulating information and evidence of the involvement of miRNAs in various cancers, not much is known about the mechanisms which control the miRNA biogenesis. Epigenetic mechanisms including DNA methylation and histone modification have been implicated in many cancers through their regulation of gene expression. We initiated a study into the changes in DNA methylation pattern as a regulatory mechanism for miRNA expression in Ewing sarcoma (ES) which is the second most frequent bone tumor in adolescents and young adults. An in silico analysis of few of these microRNAs identified those situated close to/ embedded in CG islands. Experimental verification of changes in methylation patterns was carried out both by Methylation Specific PCR and by treatment with demethylating agent 5-Aza -2'-deoxycytidine. Two of the micro-RNAs - mir 34b and mir 34c located on 11q23.1 demonstrated altered pattern of methylation in 25 ES tumors and 4 ES cell lines in comparison with healthy control. While both miRNAs showed only an unmethylated band in healthy PBL, methylated bands were detected in tumors (48% for 34c and 24% for 34b) and cell lines (3/4 for 34c and 2/4 for 34b). The effect of methylation on the expression of these miRNAs was highlighted by a six fold higher expression of mir 34b in cell line MHHES1 where this miRNA is unmethylated as compared to that in cell line SKES1 where it is hemi-methylated. Treatment of SKES1 with 5-Aza -2'-deoxycytidine correlated with 50% increased expression of 34b, thereby verifying methylation in this cell line. Our study implicates methylation as one of the regulatory mechanisms in a distinct class of miRNAs in ES. Anticancer strategies using demethylating agents should be explored. MOLECULAR MECHANISMS UNDERLYING DROUGHT RESISTANCE: ZYGOPHYLLUM DUMOSUM - A PERENNIAL DESERT PLANT - AS A MODEL SYSTEM

G. Granot, N. Sikron, S. Talwara, G. Grafi

Department of Dryland Biotechnologies, Institute for Desert Research, Ben-Gurion University of the Negev, Sde Boker Campus, Beer-Sheva, Israel

Zygophyllum dumosum Boiss. is a perennial Saharo-Arabian phytogeographical element and a dominant shrub on the rocky limestone hill slopes of the Negev desert. The plant is active during the winter, and semideciduous during the dry summer, that is, it sheds its glabrous leaflets, while leaving the petiole, which can survive for up to three years, green and rather active during the dry season. Being resistant to extreme perennial drought, Z. dumosum appears to provide an intriguing model plant for studying epigenetic mechanisms underlying drought resistance. We collected plant material during the wet (March to May) and the dry seasons (June to August) and analyzed posttranslational modifications of core histones, changes in protein profiles, as well as in fatty acid composition. We found that dimethylation of histone H3 at lysine 4 (H3K4me2) – a modification associated with active gene expression - is high during the wet season (March-April) but gradually decreased on the transition to the dry season (May to August). Unexpectedly, dimethylation of H3 at lysine 9 – a modification associated with gene silencing - could not be detected in petioles during both the wet and the dry seasons. We also observed changes in protein profiles and fatty acids composition during the transition from the wet to the dry season. Our results imply that Z. dumosum responds and copes with drought by unique means of epigenetic reprogramming of gene expression. SIMULTANEOUS ANALYSIS OF TRANSLESION DNA SYNTHESIS AND HOMOLOGOUS RECOMBINATION REPAIR IN MAMMALIAN CELLS

S. Adar, Z. Livneh

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

DNA damage tolerance mechanisms act to cope with unrepaired DNA lesions during and after replication. There are two known mechanisms for gap filling. In translesion DNA synthesis (TLS), specialized DNA polymerases are capable of inserting nucleotides across from the damage thus allowing filling in of the missing strand. This mechanism bears an inherent risk of mutation due to the miscoding nature of many DNA lesions. The second known mechanism is homologous recombination repair (HRR), in which the intact sister chromatid serves as a donor for the missing information. This mechanism is essentially error free. In mammalian cells, TLS is highly robust and has been extensively studied. On the other hand, it is not known whether lesions can be tolerated by HRR. For this purpose, we have developed an episomal, plasmid-based assay for the detection of gap filling through homologous recombination. The assay is based on a gapped plasmid carrying a site-specific lesion in the single-stranded region, and a homologous duplex DNA donor. The poster will describe experiments designed to determine whether both TLS and HRR operate in mammalian cells to fill in gaps opposite DNA lesions. GENE-CONVERSION MEDIATED DELETION (GCMD) INDUCED BY A DSB

N. Agmon, M. Kupiec

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

The prevalence of repeated sequences in most genomes from yeast to humans represent a potentially dangerous situation. This is due to the fact that lesions such as double strand breaks (DSBs) occur frequently during cell growth, and the repair of even a single DSB can result in gene-conversion, deletion or tandem duplication depending on the mechanism and the sequence chosen for the recombinational repair. We are interested in examining how does the cell cope with a single DSB and multiple donors for repair. We constructed a yeast strain that harbors a single induced HO cut-site with several alternative homologous sequences for repair: intra- chromosomal (direct repeat), inter-chromosomal (ectopic) and/or allelic. Our results indicate that the intrachromosomal homologous sequence is preferred as the donor for repair, resulting in either gene conversion or deletion of the intervening sequences. We demonstrate the occurrence of a novel repair end-product which combines ectopic gene conversion and deletion. In addition, we show that the formation of our deletion products is RAD51-dependent, decreasing 400-fold in a rad51ִ mutant. Thus, we introduce a new system that can shed light on the cells' attempt to make the repair as efficient and risk-free as possible, balancing between potentially competing DSB repair pathways. TO EAT OR NOT TO EAT? TROPHIC BIOLOGY OF STYLOPHORA PISTILLATA LARVAE - A STABLE ISOTOPE APPROACH

A. Alamaru1,2, R. Yam3, A. Shemesh3, Y. Loya1

1Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel, 2The Interuniversity Institute for Marine Sciences of Eilat, Israel, 3Department of Environmental Sciences and Energy Research, Weizmann Institute of Science, Rehovot, Israel

Recruitment success of planktonic larvae is essential for the persistence of coral reefs. Feeding strategies may affect recruitment success and potentially determine species distribution. The aim of the study was to ascertain the feeding strategies of Stylophora pistillata larvae (planulae), implementing stable isotopes, a powerful method used in dietary studies. Planulae and fragments of parental colonies were collected from two and twenty meters at Eilat (Gulf of Aqaba) during 2007. Carbon and nitrogen stable isotopes composition and C/N ratios of the planulae were compared with those of parental colonies. Results showed that delta 13C in planulae was significantly lower than parental colonies but that C/N ratios were two times higher regardless of depth. The additional carbon in the C/N ratio in the planulae was depleted in 13C. Following lipid extraction, I found no significant difference in delta 13C and in C/N values between planulae and parental colonies. This indicated that the differences in delta 13C originate in the lipid content of the planulae and not from an isotopic fractionation during embryological development. In order to determine if released planulae feed heterotrophically, I conducted controlled feeding experiments using dissolved and particulate organic matter, phytoplankton and zooplankton. I compared the isotopic ratios between planulae and the food sources. To evaluate the contribution of photosynthates translocated from the symbiotic zooxanthellae to the planulae, experiments were carried out under light and dark conditions. Despite the presence of an oral opening the planulae did not feed. Moreover, following 2 weeks “starvation” in the dark, planulae started utilizing their lipid reservoirs, stressing the importance of photosynthesis to the planulae. My findings stress the importance of the physiological state of parental colonies in producing viable planulae, capable of going through metamorphosis and successful recruitment to a coral reef. STRUCTURAL CHARACTERIZATION OF A TYPE III COHESIN MODULE FROM THE MULTI-ENZYME CELLULOSOME COMPLEX OF RUMINOCOCCUS FLAVEFACIENS

O. Alber1, I. Noach1, R. Lamed2, L. Shimon3, F. Frolow2, E.A. Bayer1

1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel, 3Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

Ruminococcus flavefaciens is a predominant fiber-degrading bacterium that inhabits the rumen of herbivores. R. flavefaciens strain 17 produces a highly organized multi- enzyme complex, termed the cellulosome that plays a key role in the degradation of plant cell walls. The main factor that dictates cellulosome assembly and attachment to the cell wall is the interaction between two complementary modular families, i.e., the cohesin and the dockerin. ScaE is one of the critical components of the R. flavefaciens cellulosome, owing to its anchoring role in cellulosome assembly. The attachment of the entire cellulosome to the surface of the bacterial cell wall is mediated via a typical gram-positive anchoring motif for sortase-mediated covalent coupling to peptidoglycan layer. In the present work, the selenomethionine derivative of the ScaE cohesin (Rf-ScaECoh) has been crystallized, and the X-ray crystal structure has been solved to a resolution of 1.96 ?, using Single-wavelength Anomalous Diffraction (SAD) technique. The cohesin module folds into a nine-stranded beta-sandwich with “jelly roll” topology, typically observed for cohesins. Despite the great similarity in overall topology, the presence in Rf-ScaECoh of an additional structural element represents a dramatic divergence from other known type-I and type-II cohesin structures. In this context, an elaborate 13-residue alpha-helix is located between beta- strands 8 and 9. The alpha-helix is enveloped by an extensive N-terminal loop, unseen in any other known cohesin, which embraces the helix thereby enhancing its stability. Differences in the composition of conserved residues comprising beta-strands 5, 6 and 3 may account for the distinct specificity that Rf-ScaECoh exhibits for its target dockerins. A putative dockerin-binding site on the cohesin surface is proposed. STRUCTURE AND MECHANISM OF QSOX, EXTRACELLULAR SULFHYDRYL OXIDASE PAR EXELLENCE

A. Alon, D. Fass

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

Oxidative folding occurs mainly in the secretory pathway, assisted by specific proteins in vivo. Sulfhydryl oxidases are enzymes that couple the de novo generation of disulfide bonds with the reduction of a terminal electron acceptor, such as molecular oxygen. Two families of eukaryotic sulfhydryl oxidases using FAD as a cofactor have been described so far: ERV/ALR and Ero1. The QSOX enzymes are a subfamily of ERV/ALR, with one or more N-terminal thioredoxin (TRX) domains fused to a sulfhydryl oxidase domain. QSOX enzymes are found in high eukaryotes, mostly in multicellular organisms, where they operate late in the secretory pathway or even outside the cell. Based on our structural studies of single-domain Ero1 and ERV/ALR enzymes by X-ray crystallography, a general mechanism for the function of sulfhydryl oxidases has been described. However, the first structure of a QSOX protein will specifically further our understanding of how the sulfhydryl oxidase and TRX-like modules of this protein family interact during catalysis of disulfide bond formation and transfer. We have successfully expressed a human QSOX protein recombinantly in bacteria and optimized a purification procedure. The enzyme is highly active both towards small-molecule substrates such as DTT and towards RNase A, a model protein substrate. Surprisingly, human QSOX appears as a monomer under all procedures tested, although all other proteins in the ERV/ALR family described to date are dimeric. This observation is in contrast to the previously suggested mechanism of catalysis by QSOX, which makes clear the need for structure-based studies of the catalytic mechanism of this enzyme family. DEPRESSION- AND ANXIETY-LIKE BEHAVIORS IN THE DIURNAL FAT SAND RAT INDUCED BY SHORT DAYLIGHT CYCLE OR MELATONIN INJECTIONS

T. Ashkenazi1, N. Kronfeld-Schor1, H. Einat2

1Department of Zoology, Tel-Aviv University, Ramat Aviv, Israel, 2College of Pharmacy, University of Minnesota, Duluth, MN, USA

Seasonal affective disorder (SAD) is characterized by winter depression episodes and spring/summer remissions. Affective disorders SAD in particular, have repeatable been suggested to be related to melatonin and circadian rhythms. These relations may differ significantly between nocturnal and diurnal mammals. Therefore our goals were: to test the effects of short photoperiod (SD) on affective-like behavior of a diurnal rodent [the fat sand rat (Psammomys Obesus)], and to test the role of melatonin on these effects. After 3-week acclimation to SD (5/19 light/dark) or long photoperiod (LD, 12/12 light/dark) animals were divided to two subgroups that received I.P injections of either 100µg melatonin or vehicle solution. Injections were administered twice daily, 5 and 8.5 hours after light onset, to mimic the effects of SD cycle in the animals housed under LD. Depression-like behaviors were evaluated in a modified forced swim test (FST), sweet solution test, plus maze test, and resident intruder tests. Results demonstrate that SD or melatonin administrations result in depression- and anxiety-like phenotype, including faster emergence of despair-like behavior in the FST and reduced saccharine consumption, reduced time in the open arms of the plus maze and reduced aggression in the resident intruder tests. The results support a significant involvement of melatonin in the observed behavioral changes, suggesting that the underlying biological mechanism is related to circadian rhythms. We therefore suggest that the diurnal fat sand rat may be an appropriate animal model to explore the effects of photoperiod on emotional behavior, the biological basis of SAD, and the interactions between circadian rhythms and affect. Such a model can contribute to the attempts to develop novel treatments for the disorder. Supported by The National Institute for Psychobiology in Israel Founded by the Charles E. Smith Family. MOLECULAR DYNAMICS SIMULATIONS OF PALMITATE BINDING TO THE HUMAN Tim44 C-TERMINAL DOMAIN

Y. Avneon1, M. Gutman1, E. Nachliel1, Y. Tsfadia

1Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

The entry of proteins into the mitochondria’s matrix space is mediated by a specific system that consists of two trans-membranal channels, TOM and TIM, one for the outer and one for the inner mitochondrial membranes. One component of this system, Tim44, is the object of our research. Tim44 is functioning at the junction between the membrane and the mitochondrial matrix, and is capable to adsorb to a phospholipid membrane containing acidic residues such as cardiolipin. The site where the protein recognizes the negative lipid is our subject. Azem et al. showed that Tim44 binding to negatively charged vesicles was electrostaticly based, but was abolished only by very high concentrations of salt (M=0.5). These observations suggest involvement of hydrophobic interactions. The structure of the C- terminal domain of Tim44, either human (PDB 2cw9) or from yeast (PDB 2FXT) exhibits a hydrophobic caveola large enough to accommodate a medium size molecule. To evaluate whether this locus is the membrane attachment site, we carried out Molecular Dynamics simulation of the human protein, surrounded by water and ions, to gain electroneutrality and ionic strength of physiological solution. In the first step of the research we represented the acidic phospholipid by a simpler model - palmitate anion. In contrast with our expectations, it was noticed that the positive charges, located on the alpha-helix connecting the C-terminal domain of Tim44 to its N-terminal domain, is the most probable attachment site for reacting with charged membrane.To validate this assumption, in vitro experiments were conducted at Dr. Azem lab, Tel-Aviv University. The N-terminal section of the Yeast's Tim44 gene was truncated and expressed. Its binding to cardiolipin enriched DPPC membranes was measured. The results indicate that removal of the N-terminal alpha-helix, of the C-domain, abolishes the capacity of the modified protein to be attached to membrane. A GENOME SCALE LOOKUP FOR HORIZONTAL GENE TRANSFER

D. Bar

Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel

Horizontal gene transfer (HGT) is one of the main mechanisms that contribute to microbial diversification, but its role in higher eukaryote evolution has yet to be studied. Here we present a genome wide scan for HGT events between house mice and men, two species we have reason to believe are prone to HGT. We compared small sequences from the mouse genome with the rat and human genomes, finding sequences that show higher similarity to man then to the phylogenetically closer rat and compared that to the same analysis done on the rabbit genome, where no HGT events are expected. Many potential HGT sequences were found. Focusing on mouse chromosome 19 lead to six potential mouse-human HGT events, compared to no mouse-rabbit events. Of these, three sequences were transcribed to RNA, one of them being part of the FRAT2 gene, a regulating member of the WNT signaling pathway, known to be near several integration sites of mouse mammary tumor virus. We believe these finding suggest that HGT might play an important role in eukaryote evolution. ANTIFREEZE PROTEIN SCAFFOLD FOR THE DESIGN OF SURFACE BINDING BIOMOLECULES

M. Bar1, T. Sherf2, I. Braslavsky3, D. Fass1

1Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel, 2Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel, 3Department of Physics and Astronomy, Ohio University, Athens, OH, USA

Protein scaffolds are increasingly being adapted for selection of novel activities or binding specificities. The Tenebrio molitor thermal hysteresis protein (TmTHP), also known as antifreeze protein, is a potential scaffold for generation of non-natural proteins that bind to two-dimensional (2D) surfaces of useful inorganic materials, such as semiconductors. TmTHP is a small, repetitive, highly disulfide-bonded beta- helix with a periodic 2D face naturally evolved to bind ice. These properties make TmTHP beneficial relative to other proteins or peptides, potentially increasing the specificity of surface recognition. We developed an efficient protocol for expression and purification of TmTHP and showed that it is active and stable. To explore the suitability of TmTHP for re-engineering, we constructed a series of mutants with global replacement of the ice binding face. Each mutant contains an exposed array of a particular amino acid, providing a repetitive arrangement of altered functional groups on the surface. Structural studies (circular dichroism, NMR) show that most of the TmTHP mutants are folded despite the comprehensive and severe changes in sequence and chemical properties. We are developing a phage display system for selection of TmTHP variants with specific surface binding properties. We engineered phage displaying TmTHP and showed that TmTHP is folded on its surface. We designed a TmTHP gene library of ~107 unique variants with diversified amino acids on the ice binding surface and intend to custom-evolve novel 2D surface-binding proteins by selection from the TmTHP library. COHESIN-DOCKERIN MICROARRAY: DIVERSE SPECIFICITIES BETWEEN TWO COMPLEMENTARY FAMILIES OF INTERACTING PROTEIN MODULES

S. Ouanounou, Y. Barak, R. Haimovitz, E.A. Bayer

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

The cellulosome is an intricate multi-enzyme machine, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular architecture of a cellulosome is the consequence of the types and specificities of the interacting modules – the cohesins and dockerins – borne by the different cellulosomal subunits. The high-affinity cohesin-dockerin interaction dictates the overall architecture of a given microbial cellulosome complex. In this study, we describe the use of a microarray system for determination of cohesin-dockerin specificity. For this purpose, matching recombinant fusion protein constructs were prepared that contained one of the interacting modules. The cohesins were joined to an appropriate cellulose-binding module (CBM) and the relatively small dockerin modules were fused to a thermostable xylanase that served to enhance expression and proper folding. In both cases, it is believed that the resultant fusion protein emulated the natural setting for the given module. The CBM-fused cohesins were immobilized on cellulose-coated glass slides, to which xylanase-fused dockerin samples were applied. In effect the microarray system described herein allows a global comparison among the interactions between various members of two complementary families of interacting protein modules. Knowledge of the specificity characteristics of native and mutated members of the cohesin and dockerin families provides insight into the architecture of a given parent cellulosome and allows the selection of suitable cohesin-dockein pairs for biotechnological and nanotechnological application. Using this approach, extensive cross-species interaction among type-II cohesins and dockerins is shown here for the first time, as opposed to the commonly observed specificity characteristics among the different type-I and type-II cohesin-dockerin pairs. Selective intraspecies binding of an archaeal dockerin to two complementary cohesins is also demonstrated. CAROTENOID IMPROVE ENDOTHELIAL CELL FUNCTION

Y. Beck1, H. Potashnik1, K. Hirsch1, I. Hazan-Hallevy1, E. Paran2

1Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Hypertension Unit, Department of Clinical Biochemistry, Soroka University Hospital, Beer-Sheva, Israel

Hypertension is one of the clinical conditions where endothelial dysfunction has been confirmed. Endothelial damage may account for the increased vascular resistance in hypertensive patients, and contributes to clinical consequences of this condition such as atherosclerosis. Dietary supplementation of fruits and vegetables has been linked to a rise in plasma vitamin antioxidant levels and blood pressure values reduction in human. Due to their high consumption, tomatoes and their products represent a main dietary source of vitamins and carotenoids with antioxidant properties. In a previous study we have demonstrated that tomato-extract (oleoresin) has blood pressure- lowering effects in individuals with essential hypertension, but the mechanism remains to be clarified. Thus, the aim of the present study was to examine the effect of lycopene, lutein and oleoresin on endothelial cells (EC) function. Furthermore in order to mimic the effect of diet rich in carotenoids we determined the effect of combination of low physiological concentrations of these carotenoids. Lycopene, lutein and oleoresin inhibit the adhesion of neutrophils to two cytokine-induced endothelial cells: ECV-304 and EA.hy926 cells. The adhesion of neutrophils to EC was synergistically inhibited by the combinations of oleoresin with lutein. All the carotenoids tested reduced ET-1 secretion (ELISA Kit) and increased NO release (Griess reagent) from EA.hy926 cells. In these cells the level of eNOS expression measured by western blot did not change, however increase in the phosphorylation of ser1177, which was reported to induce eNOS activity, was observed. The possibility that the increase in NO production is the mechanism behind the inhibitory effect of carotenoids on the adhesion of leukocytes to EC is under investigation. In conclusion our results suggest that tomato-extract and lutein may improve and even prevent endothelial dysfunction by their ability to increase NO and reduce ET-1 secretion and the adhesion of leukocytes to EC. THE GUT BACTERIAL COMMUNITY OF THE MEDITERRANEAN FRUIT FLY (CERATITIS CAPITATA) AND ITS FUNCTIONS DURING THE DIFFERENT LIFE STAGES OF THE HOST

A. Behar1, E. Jurkevitch2, B. Yuval3

1Department of Entomology, Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, 2Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, 3Department of Entomology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel

The Mediterranean fruit fly is a holometabolous insect with two ecologically different life stages: the larval and the adult stage, separated by a static pupal stage. We examine the hypothesis that the fly-associated bacterial community changes adaptively during the insect's life cycle, and plays different roles during fly development according to the specific ecological needs in each stage. We found that members of the Enterobacteriaceae constituted the dominant bacterial populations of the medfly’s gut. Using direct and cultured-based approaches we demonstrated that diazotrophic and pectinolytic Enterobacteriaceae are vertically transmitted from parents to offspring. During oviposition, these bacteria are transferred to the fruit, establish and proliferate within it, causing its decay. After bacterial-mediated hydrolysis, pectin may constitute an additional carbon source of readily metabolizable sugars for the larvae growing within the fruit. Moreover, we assume that while causing fruit decay, the pectinolytic process macerates the fruit cell walls, hence assisting larval movements within the fruits, and emergence from the fruit to pupate in the soil. We discovered that in adult medflies diazotrophic bacteria actively fix nitrogen within the fly's gut, potentially supplying a significant proportion of the nitrogen used by the adult fly. Our results also indicate that nitrogen fixation occurs in medfly larvae and thus may supply nitrogenous compounds essential for larval growth. Furthermore, we suspect that the pectinolytic function expressed within the fruit may also provide an ample supply of readily metabolizable carbohydrates to the bacterial microbiota and fuel the energy-demanding nitrogen-fixation process. We propose that larvae get an "enterobacterial package" of pectinolytic and diazotrophic bacteria that establish during the fly's life, contribute to the fly’s nitrogen and carbon metabolism, affecting its development and ultimately, fitness. DIRECTED EVOLUTION OF A MUTANT BETA-XYLOSIDASE TOWARDS IMPROVED OLIGOSACCHARIDES SYNTHESIS ACTIVITY

A. Ben-David1, G. Shoham2, Y. Shoham1

1Department of Biotechnology and Food Engineering, Institute of Catalysis Science and Technology, Technion–Israel Institute of Technology, Haifa, Israel, 2Department of Inorganic Chemistry, Laboratory for Structural Chemistry and Biology, Hebrew University of Jerusalem, Israel

Oligosaccharides play pivotal roles in many biological systems, from defined structural functions to specific molecular recognition elements. Despite the enormous importance of oligosaccharides, efficient synthetic methods for their large-scale production are limited. Most of the difficulties arise from the need to perform protecting group manipulations in order to obtain well-defined products. For that reason, enzymatic synthesis of oligosaccharides provides an attractive alternative to the classic chemical methods. Several years ago Withers et al. introduced a new type of mutant glycosidases, coined glycosynthases, for oligosaccharide synthesis. In these enzymes, which are mostly retaining glycosidases, the acidic catalytic nucleophile is replaced by a smaller non-nucleophilic residue, resulting in practically inactive hydrolase. However, in the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes transfer the activated sugars to suitable acceptors. Since the synthesis of oligosaccharides is not the original activity for which glycosidases were evolved, reactions catalyzed by glycosynthases are slow relative to wild type glycosidase activities, requiring large quantities of enzyme and/or extended incubation. In order to improve the activity of glycosynthases we have developed a novel screening assay. The assay is based on the reaction mechanism and follows the byproduct of the reaction – hydrofluoric acid. This on-agar assay is high throughput and allows examination of thousands of variants a day. In addition, the assay is general to all glycosynthases and is simple and affordable. To test the feasibility of the assay we used a nucleophile mutant of a Geobacillus stearothermophilus beta-xylosidase (XynB2E335G) as a model enzyme. Library of XynB2E335G variants was produced using error prone PCR. Preliminary screening of the library for the transfer of alpha-D-xylopyranoside fluoride donor to various acceptors resulted in the isolation of several variants exhibiting 1.2- to 3-fold improved glycosynthetic activity. STRUCTURAL GENOMICS OF POSSIBLE VIRULENCE FACTORS FROM THE PROPHAGES IN PATHOGENIC BACTERIA

M. Ben-David1, S. Albeck2, O. Dym2, Y. Peleg2, T. Unger2, A. Branzburg2, J.L. Sussman1

1Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel, 2Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot, Israel

Prophages are transferable DNA segments that integrate into the bacterial chromosome. These mobile DNA elements encode various important functions, including toxins, virulence factors and antibiotic resistance that confer new phenotypes to the host bacteria. The function of many of these proteins still remains unknown. This study describes first attempts of the use of structural genomics initiatives, in order to provide insights into the function of unknown prophage proteins from pathogenic bacteria. This initiative includes the setting-up of a protein crystallographic pipeline from protein-targets selection to structure determination, combining testing and establishment of high-throughput methods. After establishment of the target selection strategy, two lists of protein-targets were generated, list-A (5 targets) and list-B (40 targets). In both lists, the prophage-encoded targets are with higher then average likelihood to express and crystallize. This was achieved via the use of selection criteria based on bioinformatics and biophysical protein properties that were applied as part of the target selection strategy. In list-A, in addition, the protein-targets are related to the bacteria virulence, according to in-vivo studies. Part A of the study, shows the parallel cloning, expression, purification and crystallization of list-A targets. In this part we were able to show high success rates in soluble expression (60%) and crystallization (40%). In part B, the same parallelization, as in part A, was carried out. Here, we found success rates of 50% in soluble expression (crystallization process is still in progress). Comparison of our results with other structural genomics initiatives confirmed successful design of the target selection strategy. Two protein-targets from list-A produced crystals, both of which diffract to poor resolution (>7A). Attempts to optimize the crystals results in improvement of the diffraction resolution from 7A to 3.7A. However, this resolution is still too low for atomic resolution structure determination, and additional crystal optimization is needed. MOLECULAR DYNAMICS SIMULATIONS FOR THE BINDING INTERACTIONS OF CHICKEN AND TOAD LIVER FABP WITH SMALL FATTY ACID MICELLES AT THE ALTERNATIVE-PORTAL REGION

L. Ben Avraham, E. Nachliel, M. Gutman, Y. Tsfadia

Department of Biochemistry, Tel-Aviv University, Ramat Aviv, Israel

Fatty acid binding proteins (FABP) are intra cellular proteins, widely spread in organisms, which serve as intra-cellular carriers of free fatty acid (FA), or related compounds in enterocytes, adipocytes and other tissues, reducing the level of free fatty acid to a non-harming concentrations, Previous studies have shown that there is more than one possible mechanism for the entry of the ligand into the protein. In this study, we report for the first time, results of a molecular dynamic simulation indicating that the proteins react with mini-micelles, composed of 3-4 palmitate anions, which might be built up when the concentration of the FA is high. The simulations have shown that the binding interactions occur at the alternative portal site of the protein. The interactions were seen either with spontaneously formed micelle during the simulation, or with a pre-existed micelle. The analysis of the binding interactions shows that the main driving forces for the penetration are the van der Waals interactions. HEPARANASE IS CRUCIAL FOR IN-VIVO DENDRITIC CELL TRANSMIGRATION

S. Benhamron1,2, E. Zecharia2, I. Vlodavsky3, D. Mevorach1

1Laboratory for Cellular and Molecular Immunology, Hadassah-Hebrew University of Jerusalem, Israel, 2Laboratory for Oncology, Hadassah-Hebrew University of Jerusalem, Israel, 3Cancer and Vascular Biology Research Center, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

After antigen capture and exposure to danger stimuli, maturing dendritic cells (DCs) migrate to regional lymph nodes, where antigenic peptides are presented to T lymphocytes. Chemokines play an important role in the migration of DCs. Particularly; CCR7 is up regulated in mature dendritic cells and is required for migration to draining lymph node. However, the expression of CCR7 is not sufficient for migration of DCs into lymphatic. Heparanase is an endo-beta-D-glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM). Heparanase activity has been traditionally correlated with the metastatic potential of tumor-derived cells facilitating cell invasion. Heparanase activity is so associated with autoimmunity and inflammation like neutrophils, macrophages, and lymphocytes. In our previous work (Benhamron S. et al. J. of immunology. 2006 176:6417-6424), we have showed the presence and the migratory role of heparanase in human DCs. We have now established a model of migration in vivo : mature bone-marrow (BM) derived dendritic cells generated from the heparanase deficient mice (Hpa-/-) and from wild type mice were labeled with 2.5 or 3 microM 5- and 6-carboxyfluorescein diacetate sucinimidyl (CFSE) and injected into footpad of wild type mice. After 24 hours the popliteal lymph nodes are removed and analyzed by FACS analysis for presence of BM derived DCs into lymph nodes. We show that the migration in vivo of mature BM derived DCs from the heparanase deficient mice are reduced in 70 % compared to the migration of mature BM derived DCs from the wild type mice. SYNAPTIC ZINC SIGNALING, MEDIATED BY A ZINC SENSING RECEPTOR, IN THE CA3 HIPPOCAMPAL REGION

L. Besser1,3, U. Chorin1,3, I. Sekler2, M. Hershfinkel1

1Department of Morphology, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Physiology, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 3Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Synaptic Zn2+ is found at high concentrations in presynaptic glutamatergic vesicles in the CA3 region of the hippocampus. Sequestration of Zn2+ into the synaptic vesicles is mediated by the zinc transporter 3, ZnT3. During neuronal activity, synaptic Zn2+ is released and allosterically modulates the activity of ion channels, most notably the NMDA and GABA, thereby reshaping synaptic transmission. But a signaling role, via a specific receptor on the post synaptic neurons, has not been identified. Using fluorescent Ca2+ imaging of acute slices, we show that addition of extracellular Zn2+ (200µM) or electrical stimulation of the mossy fibers triggers a Ca2+ rise in the postsynaptic CA3 neurons. Application of the extracellular Zn2+ triggers a metabotropic Ca2+ rise mediated by a Gq coupled receptor and the IP3 pathway. The Ca2+ response following electrical stimulation of the mossy fibers in the ZnT3 KO mice, lacking synaptic Zn2+, was significantly reduced compared with the WT, indicating that synaptic Zn2+ activates postsynaptic metabotropic Ca2+ release. Finally, application of extracellular Zn2+ was followed by phosphorylation of qבERK1/2 and CAMKII in the postsynaptic neurons, that was blocked by the G inhibitor indicating that it was triggered by the Zn2+- dependent metabotropic response. Our results provide the first evidence for a specific Zn2+ sensing receptor, ZnR, that is activated by synaptically released Zn2+, on the postsynaptic neurons in the CA3 region. The Zn2+- dependent metabotropic Ca2+ and kinase signaling mediated by the ZnR may play a role in regulating synaptic plasticity. INITIATION OF THE HUMAN mtDNA REPLICATION: CONTROL OF THE L-STRAND PRIMING AT THE OL

R. Bezalel-Buch, I. Kapeller, J. Shlomai

The Kuvin Center for the Study of Infectious and Tropical Diseases, Hebrew University of Jerusalem, Israel

The mitochondrial genome (mtDNA) is a double-strand circular DNA molecule containing two separate replication origins for its light (L) strand (OL) and heavy (H) strand (OH). DNA synthesis from these replication origins is asynchronous and unidirectional. mtDNA replication initiates with H-strand synthesis. L-strand synthesis initiates at the OL using the displaced parental H-strand as a template. OL consists of a 30 bp-sequence, located in a non-coding region. The sequence has a strong potential to form a stem&loop structure. It is presumed that generation of a hairpin structure could be triggered at the OL by the displacement of the H-strand and its exposure as a single stranded conformation. However, while there is a lot of information available regarding the mechanism of OH recognition and the events that lead to the initiation of H-strand synthesis, our understanding of the molecular events that result in the priming of L-strand synthesis is quite poor. Here we have addressed the long-standing question of OL recognition of by the mtDNA replication apparatus, by searching for a putative human mtDNA OL-origin binding protein (hmtOL-BP), which specifically recognizes the OL and directs the L-strand replication initiation machinery onto this site. For this purpose we have used lysates of mitochondria, purified from human placenta. Mitochondrial extracts were fractionated, using protein chromatography methods, and hmtOL-BP has been detected and partially purified, by the electrophoretic mobility shift analysis (EMSA), using OL sequences ligands, as an assay. Our analyses revealed a high affinity interaction between a ~20kDa hmtOL-BP and a 30 bp sequence, derived from the stem-loop OL structure, of the mtDNA H- strand, which is the template strand for L-strand synthesis, but not with sequences derived from the mtDNA L-strand of OL. Both the stem and the loop structure were found to be essential for the recognition of OL by the hmtOL-BP. BONE RIDGE DEVELOPMENT IS REGULATED BY TENDONS AND MUSCLES

E. Blitz1, J. Kahn1, D.A. Breitel1, P. Maire2, E. Zelzer1

1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2Departement de Genetique et Developpement, Institut Cochin, Universite Paris Descartes, Paris, France

The skeleton provides an anchoring point for muscles, attached via tendons, which exert a pulling force. Bone ridges are projections of the bone, which serve as anchoring points for muscles, inserted into the bone via tendons. We are currently studying the development of the deltoid tuberosity, an example of such a bone ridge, as a model for studying the bone-tendon-muscle unit. The development of bone ridges is poorly understood. In order to understand the mechanism regulating tuberosity development, we analyzed its growth in mice embryos. We show that tuberosity growth from the humerus begins at E13.5 and continues to grow and ossify till birth. In addition, the tuberosity cell population contains proliferating and differentiating chondrocytes, resembling growth plate organization. It has been well established that the musculature plays a major role in postnatal bone homeostasis and development. By utilizing mice with mutations resulting in absent limb musculature or paralysis, we studied the involvement of muscles in bone ridge development. In these mice we observed initiation of the tuberosity, however this growth was arrested by E14.5. This growth cessation was characterized by a 13 fold reduction in percentage of cell proliferation, but normal cell differentiation in paralyzed mice. These results demonstrate that deltoid tuberosity initiation is a bi-phase process of initiation and growth: Its initiation is musculature independent, but its growth depends on muscle contraction. Analysis of the involvement of tendons in tuberosity development using the TGF-beta RII KO with absent tendon formation, revealed blocked initiation and tuberosity growth. Furthermore in scleraxis-null mice, shown to have defective tendons, tuberosity initiation and growth was disturbed. These results indicate that deltoid tuberosity initiation is mediated by tendons. Moreover, scleraxis-null mice are genetic evidence for scleraxis signaling through cell-nonautonomous mechanisms to coordinate the timing and growth of tuberosity. MORPHO-NEUROLOGICAL PARALLELS IN THE RAT MODEL OF STROKE

M. Boyko

Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Acute brain ischemia (stroke) is one of the leading causes of death and long-term disability in adult humans. Numerous experimental models have been developed for investigating the mechanisms of ischemic brain injury and for searching the potential therapeutic targets. Among the currently widely used animal models of stroke is the rat model of focal brain ischemia caused by a middle cerebral artery occlusion (MCAO). We used this model for (i) comparison of neurological changes with the size and dynamics of ischemic brain injury, and (ii) elaboration of the optimal Neurological Severity Score (NSS). The previously proposed (NSSs) included a limited number of tests (from 4 to 8) and were mostly used for validation of brain ischemia. We suggested that using the more NSS tests could provide a better evaluation of neurological deficit and ischemic brain injury as well. For this purpose, we developed NSS which include 42 tests. Focal brain ischemia (transient and permanent) were induced in male Sprague-Dawley rats weighing 300-350 g. Permanent MCAO caused a considerable brain damage, from 20 to 30%, whereas 24 h following a 2-hour transient MCAO, a mean injury size was 15%. After one week, the size of brain injury decreased by approximately 5% in both types of ischemia. Morphological changes did not significantly correlated with NSSs of 4-8 tests, whereas they strongly correlated with the NSS which included a battery of 42 tests. Thus, the proposed NSS may be helpful for a non-invasive evaluation of ischemic brain injury in rats. THE BETA AMYLOID AND ISLET AMYLOID POLYPEPTIDES AS A MODEL TO STUDY ASSEMBLY OF AMYLOID OLIGOMERS

Y. Bram, S. Gilead, A. Frydman-Marom, M. Levi, I. Yanai, E. Gazit

Department of Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Peptide and proteins convert under some conditions from their soluble native forms into highly ordered aggregates termed amyloids. Such transitions can give rise to pathological conditions ranging from neurodegenerative disorders to systematic amyloidosis. In the past decade there was very substantial efforts directed toward identifying, isolating, and characterizing the oligomeric species, both because of their likely role in the mechanism of fibril formation and because of their implication as the toxic species. Several studies had shown that in some cases protofibrils can be on- pathway to fibrils. In other cases, they appear to be off-pathway. Regardless of the precise role played by protofibrils in the overall process of fibril formation, the elucidation of their mechanism of formation and of their structures is extremely important, not least because these species could be the primary toxic agents involved in amyloid associated disorders. In my study I am analyzing the terms that contribute to self assembly of the oligomer species, by usage of different aromatic molecules we were able to conclude that aromatic interaction are essential for oligomer assembly, In order to fully understand the principles of inhibition mechanism we used bioinformatics tools, we were able to point out a set of role that a molecule most posses in order to inhibit oligomer formation. By using Pep-Spot technique we were able to determine the sequences that are important for oligomer assembly. We are the first group that have succeeded in preparing SDS stable IAPP oligomers in vitro, we believe that this IAPP oligomers characterized by a new structural epitope will provide an unprecedented possibility to elucidate the pathology associated with IAPP oligomers and to clinically address deficits of patients with type 2 diabetes by specific treatment. UNRAVELING TRANSCRIPTION REGULATION BY THE Ldb-BASED TRANSCRIPTION COMPLEX

R. Bronstein, D. Segal

Department of Biotechnology, Tel Aviv University, Ramat Aviv, Israel

The intricate regulation of gene expression in multi-cellular organisms depends on multi-protein transcription complexes. Transcription complexes are now viewed as composing core elements, which are relatively ubiquitous proteins, and a variety of context-dependent cofactors. The diverse roles of each transcription complex depend on the unique combinations of its cofactors. The ‘Ldb-based’ transcription complex is a typical example for such a high order complex. The core of this complex is a dimer of a ubiquitous nuclear adaptor called LIM-domain binding protein, LDB. Ldb-based complexes were found to regulate development in species ranging from C. elegans to man. When malfunctioning they can cause several types of cancers primarily leukemia. Understanding how transcription complexes regulate the multitude of different processes in higher organisms is crucial for understanding normal development and diseases. The long term goal of this project is to identify the genes regulated by the Ldb-based transcription complex. This will enable us to comprehend and unravel the processes it regulates and its mode of action during development. To that end, we used micro-array technology to monitor genome wide differences in transcription caused by mutations in several components of the Ldb-based transcription complex. As a first stage for verification of the array results we are using a genetic screen. So far several downstream targets that came up in the array results were found to genetically interact with mutations disrupting the function of the Ldb- based transcription complex. MOLECULAR CO-EVOLUTION AND THE EMERGENCE OF LUCA

O. Carny, E. Gazit

Department of Biotechnology and Microbiology, Tel Aviv University, Ramat Aviv, Israel

One of the basic questions regarding the development of the last universal common ancestor (LUCA) is how did biological organization evolve from an abiotic supply of small organic molecules? The most prevalent paradigm, regarding the primordial soup, points at RNA molecules as chief candidates for the development of a self- replicating system. However, this ‘RNA world’ hypothesis suffers from a main Achilles heel – the instability of the RNA molecules in the primordial earth conditions. It was recently demonstrated by our group that peptide molecules as simple as dipeptides can self-assemble into well-ordered tubular, fibrilar, and closed- cage structures. In addition, we have confirmed the ability of such assemblies to bind nucleotide bases. In light of these examples, regarding the self-assembly propensity of short peptides and their ability to bind and recognize RNA, we examine the possible role of self-assembled peptide structures in RNA stabilization and early chemical catalysis. These features lay the grounds for molecular co-evolution of short peptide and RNA sequences in the emergence of LUCA. THE EFFECT OF CELLULOSOMAL ASSEMBLY ON THE SYNERGISTIC CELLULOSE-DEGRADING ACTIVITY OF THERMOBIFIDA FUSCA CELLULASES

J. Caspi1, D. Irwin2, R. Lamed3, D.B. Wilson2, E.A. Bayer1

1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA, 3Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

The organization of cellulases (cellulose-degrading enzymes) into a multi-enzyme complex (cellulosome) is considered to be the most organized system among all cellulase systems of polysaccharide-degrading microbial species. Cellulosomes are composed of different cellulose degrading enzymes assembled on a non-catalytic scaffolding protein (scaffoldin) through the affinity of their docking module (dockerin) to the scaffoldin’s anchoring modules (cohesins). Over the years, various research groups have investigated the relative efficiency of the cellulosomal system in synergistic degradation of recalcitrant insoluble polysaccharides, compared to other cellulase systems. The assembly of designer cellulosomes, based on selected combinations of cellulases is a means by which a non-cellulosomal system can be compared to a cellulosomal system composed of the same enzymes. Moreover, systematic construction of artificial cellulosomes can lead to new biotechnological applications, especially in the field of environmental pollution and bio-fuel production. This work is the first attempt to convert a complete, free cellulase system of a single bacterium into the cellulosomal mode of action. For this purpose, we are exchanging the ancillary modules (divergent dockerins for the resident cellulose- binding modules) of the different cellulases, produced by the aerobic bacterium Thermobifida fusca, to allow their incorporation into defined “designer cellulosomes”. Different combinations of chimaeric cellulases were tested for crystalline cellulose hydrolysis, and in some cases were shown to be more efficient than the parallel wild-type combinations. We are now very close to completing the full conversion of the T. fusca free cellulase system to work as a designer cellulosome. THE UNIQUE SYMMETRY AND DESIGN OF ARCHAEAL FLAGELLA

S. Cohen-Krausz, S. Trachtenberg

Faculty of Medicine, Hebrew University of Jerusalem, Israel

Archaeabacteria, characteristically inhabit extreme environments and comprise one of life's domains, alongside Eubacteria and Eukarya. Bacteria that belong to this group swim by rotating flagellar filaments that are phenomenologically and functionally similar to those of Eubacteria. However, biochemical, genetic and structural evidence has pointed to significant differences and greater similarity to eubacterial type IV pili. We have now determined the three-dimensional symmetry and structure of flagellar filaments for two distantly related archaeabacteria that thrive in very different environments. We have studied the halophile Halobacterium salinarum, strains R1M1and M175 [Cohen-Krausz & Trachtenberg J.Molec.Biol, 321:383, 2002; Trachtenberg et al, J.Molec.Biol, 346:665, 2005] and the acidothermophile Sulfolobus shibatae B12 [Cohen-Krausz & Trachtenberg, J.Molec.Biol, in press]. Both structures were studied using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Three-dimensional reconstructions of the flagellar filaments were generated from cryo-negatively stained filaments. This included analysis of their helical symmetry subsequent single particle reconstruction. These two structures are similar, with basic three-start helical symmetry and a conserved core domain that lacks a central channel. They differ from eubacterial flagellar filaments, which have a larger diameter, a symmetry based on five and six- start helical symmetry and a central channel, which is important for their assembly. Work is in progress on alkalihalophiles, which support this scheme of design. This implicates that the archaeal flagellar filament has a different mode of assembly and is not an environmental adaptation of eubacterial flagella but rather appears to be convergence to a similar structural solution for motility. IDENTIFICATION AND ISOLATION OF PEPTIDE BIOMARKERS FOR CANCER DIAGNOSIS IN BODY FLUIDS

M. Cohen-Kupervaser1, R. Lichtenstein2, A. Porgador1

1The Shraga Segal Microbiology and Immunology Department, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel

In the last few decades, the 5-years survival rates of many cancers had hardly improved. This led to the realization that early diagnosis is crucial for the treatment of cancer. There is a great need for simple, accurate and cost-effective screening tests that will enable early diagnosis of cancer. Despite decades of effort, single biomarkers have not been found that can reach the levels of specificity and sensitivity required for routine clinical use for the detection or monitoring of many cancers. This is due to the molecular heterogeneity of tumors from patient to patient, its location, size, histology, grade and stage. Since malignant cells exhibit specific changes in protein expression and alterations in proteolytic activities, peptides are capable of reflecting these pathological changes and are educible by dedicated analytical technologies. Some of these peptides spill from the tumor into the blood stream and become a part of the blood peptidome. By analyzing signatures of typical peptides in a blood sample, it may be possible to diagnose and monitor different kinds of cancers. Here we introduce a new platform for the peptidome analysis of serum samples from pancreas cancer, stomach cancer and healthy individuals. We developed a reverse phase liquid chromatography (RPLC) approach for high-throughput sample purification, followed by different mass spectrometry techniques. These methods were applied onto serum samples from pancreas and stomach cancer patients at various stages, before and after surgical removal of the tumor. Due to the high cost of mass spectrometry operation and the differences in the results obtained by different MS machines, we are now developing a new ELISA based array using a phage-display library, that will over- come the need of mass spectrometry measurements and will facilitate the detection of a specific set of peptides for cancer diagnosis. TOLL-LIKE RECEPTOR SIGNALING PLAYS A CRITICAL ROLE IN AUTOIMMUNE CENTRAL NERVOUS SYSTEM DISEASE

S.J. Cohen1, I.R. Cohen2, G. Nussbaum1

1Institute of Dental Sciences, Hebrew University - Hadassah School of Dental Medicine, Jerusalem, Israel, 2Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

Multiple Sclerosis (MS) is a disease of the central nervous system that involves autoimmune inflammation leading to de-myelinization. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model for MS. Both innate and adaptive immunity are required for EAE to develop. T-cells specific for myelin antigens can transfer EAE, however the development of pathogenic cells require signals from the innate immune system. These signals are produced by. antigen presenting cells (APCs) in response to an array of environmental stimuli, many of which activate through members of the Toll-Like Receptor (TLR) gene family. The majority of TLR signaling proceeds through an intracellular adaptor protein called MyD88. To study the involvement of TLR2 and TLR4, and the TLR signaling molecule MyD88 in the development of autoimmune CNS disease using gene knock-out mice. Active EAE was induced by immunization with a peptide derived from myelin oligodendrocyte protein (MOG35-55) emulsified in adjuvant. Adoptive transfer EAE was induced by transfer of encephalitogenic anti-MOG35-55 T cells derived from wild-type (WT) mice to irradiated recipients. No difference was found between TLR4 deficient mice and WT mice in the active model of EAE, however TLR2 deficient mice developed lower EAE disease scores. In contrast, the MyD88 deficient mice were completely resistant to active EAE. WT anti-MOG35-55 T cells adoptively transferred the same level of EAE to WT and TLR2 deficient mice, however MyD88 deficient mice were again completely resistant. We found that encephalitogenic T-cells proliferate and migrate to the CNS of MyD88 deficient mice following adoptive transfer. We found that resistance was due to a shift in the cytokine profile of anti-MOG T-cells rather than deletion of pathogenic cells. Inhibition of signaling through MyD88 may generate this shift which would be of therapeutic benefit in autoimmune disease. CRYSTALLIZATION AND STRUCTURE DETERMINATION OF THE PHYCOBILISOME COMPLEX

L. David, N. Adir

Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel

The photosynthetic process is initiated by the absorption of light energy by protein complexes called light harvesting antennas. In cyanobacteria and red algae the major antenna is called the Phycobilisome (PBS). The PBS is an extremely large complex, with a molecular weight of 3-7MDa which is made up of pigmented proteins known as phycobiliproteins (PBPs) and unpigmented proteins known as linker proteins. Our goal is to obtain an atomic resolution structure of the entire PBS complex from the cyanobacterium Thermosynechococcus vulcanus using x-ray crystallography. Intact PBS was isolated in high phosphate buffer by sucrose gradient ultracentrifugation. Small blue crystals shaped like half moons were obtained in stabilization buffer in two to four weeks. Material obtained from the dissolution of extensively washed crystals was analyzed by fluorescence, SDS-PAGE and mass spectrometry. The results of these experiments indicate that the crystals contain intact, functional PBS complex. Dynamic light scattering indicates a molecular weight of at least 2.8MDa. Preliminary diffraction experiments have indicated that the present crystals diffract poorly. In the process of PBS isolation, a sample of phycocyanin (PC, one of the PBP components of the PBS) containing linker proteins was also obtained. Large blue crystals were obtained and two data sets were collected on beam line ID14-1 at ESRF, Grenoble. The structure was determined by MR to a resolution of 1.8 A in the space group C2, with three PC monomers in the asymmetric unit arranged as half a hexamer, which is different from all previously determined PC structures. Within the PC disks, additional unstructured electron density could be identified, in the position thought to be occupied by the linker. This indicates that the linker may be flexible within the PC disks. STUDIES ON A DOCKERIN-CONTAINING CLOSTRIDIUM THERMOCELLUM SUBTILASE AND ITS RELATIONSHIP TO THE CELLULOSOME COMPLEX

A. Demishtein1, Y. Barak1, R. Lamed2, M. Morrison3, E.A. Bayer1

1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel, 3Department of Animal Sciences, The Ohio State University, Columbus, OH, USA

Several anaerobic bacteria responsible for plant cell wall polysaccharide degradation in Nature produce a multiprotein complex called cellulosome, which coordinates glycoside hydrolase assembly, bacterial adhesion to cellulosic substrates and polysaccharide hydrolysis. Cellulosomal proteins possess a dockerin module, which mediates their species-specific attachment to the scaffoldin subunit via high-affinity interaction with cohesin modules. Until recently, only glycoside hydrolases and other carbohydrate active enzymes were known to reside within the cellulosome. With the publication of the full genome of the Clostridium thermocellum ATCC 27405, other types of enzymes and proteins were also identified to carry a dockerin module. Among these, serine proteinase inhibitors (serpins) were shown to reside within the C. thermocellum cellulosome. Another type of enzyme, a serine proteinase (subtilisin), has also been identified from the genome that contains two tandem dockerins. However, it is unknown whether this enzyme in fact functions in the context of the cellulosome. Interestingly, both of its dockerins are atypical in their recognition determinants, compared to the consensus type-I dockerins of this bacterium. We show herein that although this subtilisin resides in C. thermocellum, its dockerin interacts with the cohesins of another bacterium: C. cellulolyticum, even though the two bacteria occupy different niches. Surprisingly, examination of the C. thermocellum ATCC 27405 genome revealed a stop codon in the middle of the subtilisin gene, which was absent in the same gene of a different C. thermocellum strain (YS), the CDS of which contains a complete serine protease. The role of this component, though not known, invites speculation: For example, the serpins might be involved in regulation of proteolytic events during bacterial growth, whereas the subtilisin might have a protective, antibacterial role, since it failed to bind the C. thermocellum cohesin. Indeed, a new type of cohesin, to which the atypical subtilisin dockerin might bind, awaits discovery. THE ORGANOTELLURIUM COMPOUND ,AMMONIUM TRICHLORO(DIOXOETHYLENE-O-O’)TELLURATE, REACTS WITH HOMOCYSTEINE TO FORM HOMOCYSTINE AND DEREASES HOMOCYSTEINE LEVELS IN HYPERHOMOCYSTEINEMIC MICE

Y. Dikshtein1, E. Okun1, A. Carmely1, H. Saida1, G. Frei4, B.A. Sela2, L. Varshavsky1, A. Ofir3, E. Levy3, M. Albeck1, B. Sredni1

1CAIR Institute, The Safdie AIDS and Immunology Research Center, Bar-Ilan University, Ramat Gan, Israel, 2Institute of Chemical Pathology, Chaim Sheba Medical Center, Tel-Hashomer, Israel, 3Department of Chemistry, Faculty of Exact Sciences, Bar-Ilan University, Ramat Gan, Israel, 4Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

Ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) is an organotellurium compound with pleiotropic functions that has been associated with antitumoral, immunomodulatory and neuroprotective activities. Tellurium compounds with a +4 oxidation state, such as AS101, react uniquely with thiols, forming disulfide molecules. In light of this, we tested whether AS101 can react with the amino acid homocysteine both in vitro and in vivo. AS101 conferred protection against homocysteine-induced apoptosis of HL-60 cells. The protective mechanism of AS101 against homocysteine toxicity was directly mediated by its chemical reactivity, whereby AS101 reacted with homocysteine to form homocystine, the less toxic disulfide form of homocysteine. Moreover, AS101 was shown here to reduce the levels of total homocysteine in an in vivo model of hyperhomocysteinemia. As a result, AS101 also prevented sperm cells from undergoing homocysteine-induced DNA fragmentation. Taken together, our results suggest that the organotellurium compound AS101 may be of clinical value in reducing total circulatory homocysteine levels. REVERSE MODULATION OF ZnT-1 BY ATRIAL AND VENTRICULAR TACHYPACING IN THE ISOLATED RAT HEART

S. Dror, M. Mor, O. Beharier, A. Moran, A. Katz, Y. Etzion

Department of Physiology, Ben Gurion University of the Negev, Beer-Sheva, Israel

Background: ZnT-1 has been shown to inhibit the L-type calcium channels and facilitate Raf-1 serine/threonine kinase in the ERK MAPK signaling pathway. Recently, we demonstrated that acute rapid pacing (RP) of the rat atrium in-vivo as well as human AF are associated with increased expression of atrial ZnT-1, presumably as a protective response against calcium overload. In the present study, we compared the expression of ZnT-1 in the left atria versus the left ventricle in response to RP stimulus Methods: Isolated-perfused rat heart was maintained under constant perfusion pressure of 100mmHg. Arial pacing at 200 ms cycle (sham, n=3) 60 ms cycle (n=3) or minimal 1:1 atrial capture (min-1:1, n=3) was applied to the right atrium at double diastolic threshold. Capture was assessed in the left atria. Similarly, ventricular pacing at 200 ms (sham, n=2), 100 ms cycle (n=2) and 60 ms cycle (n=3) was applied to the left ventricle and capture was assessed in the right ventricle. In all experiments pacing was applied for 3h or until persistent tachyarrhythmia has evolved. Following 3h ZnT-1 levels were assessed and normalized to GAPDH (ZnT-1/GAPDH). Results: Atrial tachypacing augmented ZnT-1/GAPDH levels following either the 60 ms cycle pacing (148±14 % of sham, P=0.07) or min-1:1 pacing (180.4±4% of sham, p<0.01). Atrial tachypacing did not induce persistent tachyarrhythmia in any of the preparation. In contrast, rapid ventricular pacing at 100 ms cycle or 60 ms cycle, invariably induced VT\VF following a mean duration of 118.5±3 min and 32±6 min, respectively. Consequently ventricular ZnT-1/GAPDH levels was changed to 92.5±19% (ns) and 53.6±6 % (p<0.05) of sham levels, respectively. Conclusions: As demonstrated in-vivo, atria tachypacing increases atrial ZnT-1 levels in the isolated rat heart. In contrast, ventricular tachypacing\tachyrrhythmia down-regulates ventricular ZnT-1 levels. These results indicate that calcium overload per-se can not fully account for the modulation of cardiac ZnT-1. These findings may mark an important dissimilarity in the adaptive response to electrical tachypacing between the atrium and the ventricles. THE DUAL ROLE OF PDE3A IN OOCYTE MATURATION

I. Edry, D. Galiani, N. Dekel

Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

Meiotic arrest in mammalian oocytes is maintained by a constant transfer of cAMP from the somatic follicle cells. Following the mid-cycle surge of luteinizing hormone (LH), an increase in cAMP concentrations in the granulosa cells is followed by its rapid decrease inside the oocyte. This decrease, achieved by a concurrent cessation of the cAMP transfer and the hydrolyzing activity of the oocyte-specific phosphodiesterase type 3A (PDE3A), serves as the primary trigger for resumption of meiosis. Previously, nitric oxide (NO) and its inducible synthesizing enzyme iNOS were shown to participate in the events leading to oocyte maturation as well. We hypothesize that PDE3A is upregulated prior to meiosis reinitiation and aim at exploring the mechanism that controls its activity. We show that incubation of denuded oocytes with a permeable derivative of cAMP, dibutyryl cAMP (dbcAMP), results in a significant activation of PDE3A. Employment of a specific inhibitor of cAMP-dependent protein kinase (PKA), H-89, depicted a PKA dependency of the observed PDE3A activation. Further, we demonstrate that a brief exposure of follicle enclosed oocytes (FEOs) to LH induces a transient elevation of intraoocyte cAMP concentration, which is immediately followed by an activation of PDE3A above basal levels. A more complex regulation of PDE3A activity is indicated by new arising data; the LH-induced activation of PDE3A is blocked in the presence of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), and is mimicked in the presence of the iNOS inhibitor, aminoguanidine (AG). Based on our findings, we conclude that the oocyte-specific PDE3A plays a dual role. By exhibiting basal activity of cAMP hydrolysis under the inhibiting influence of follicular NO levels, it maintains meiotic arrest. Alternatively, at a state of phosphorylation dependent hyper activation, induced by a transient elevation of cAMP levels in the oocyte, PDE3A mediates LH action in resumption of meiosis. PHAGE THERAPY OF CORAL DISEASE

R. Efrony, E. Rosenberg

Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Coral reefs are one of the most diverse and important ecosystems on earth. Unfortunately, during the last several decades, reefs have undergone considerable destruction, at least in part as a result of emerging and re-emerging diseases. At present there are no known procedures for preventing or treating infectious diseases of corals. In this study, the use of phage therapy of coral disease has been investigated. Lytic bacteriophage (phage BA3) was isolated for the bacterial pathogen, Thalosomonas loyaeana that causes the white plague-like disease of Favia favus on the Eilat coral reef. Phage BA3 is characterized as dsDNA lytic phage belonging to the Podoviridea family. The genome of phage BA3 contains ~37 Kbp (41% G+C content) with 47 ORFs. By using this pathogen-specific phage in controlled aquaria experiments, it was demonstrated that the disease could be controlled by addition of the phage. The data indicate that initially the phages bind to the pathogen in seawater and are then brought to the coral surface where they multiply and lyse the pathogen. The phages remained associated with the coral and could prevent subsequent infections. Additionally, it was shown that addition of the phages one day after the infection prevented the disease, whereas applying the phage 2 or 3 days after infection failed to prevent the disease. The white plague-like disease is transmitted through the water. Using phages in controlled experiments prevented the transmission of the disease from a sick coral to a healthy one. A subsequent infection of the healthy phage-treated coral did not cause the disease, even though no additional phages were added. Corals treated with phage retained the phages for weeks after they were inoculated. The data presented suggest, for the first time, that phage therapy has the potential to control the spread of infectious coral diseases. INTERACTION BETWEEN PSEUDOMONAS AERUGINOSA AND VIBRIO CHOLERAE IN PLANKTONIC AND BIOFILM CULTURES

S. Elias, E. Banin

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

Microbial life in the environment is mostly characterized by multiplicity, controlled by a variety, often changing conditions. The challenge obviously is to address the microbial community scenarios as they appear in the environment. Bacteria colonize diverse ecological habitats where they can exhibit different lifestyles - free-living or biofilm. Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Biofilms constitute a protected mode of growth that allows survival in a hostile environment and increased resistance to killing (e.g. chemical disinfection, immune system and antimicrobial therapy). In nature most bacteria are in constant competition and interaction with other microorganisms, and are under conditions that are dramatically different than studying pure cultures in the laboratory. This raises the need to start and examine mixed biofilms and the role multispecies cell-cell interactions. In this project we use a community composed of two bacterial species, Pseudomonas aeruginosa and Vibrio cholerae, to explore experimentally how these two organisms interact in planktonic and in biofilm cultures. We focus on the role of iron as an essential and limiting factor in this interaction. Growth curve experiments show lysis of V. cholerae by P. aeruginosa, yet P. aeruginosa strain defective in siderophores (iron-binding compounds; pyoverdine and pyochelin) synthesis show reduced V. cholerae lysis. We hypothesize that P. aeruginosa can use the V. cholerae siderophore, vibriobactin, to acquire iron. In support of this hypothesis P. aeruginosa siderophore mutant can utilize condition medium from V. cholerae to support its growth in iron limiting conditions. In search for the Pseudomonas vibrobactin uptake system we searched the genome and located an ORF (i.e. PA4156) with 48% homology to the vibriobactin receptor. We demonstrate this gene is indeed induced in the presence of V. cholerae condition medium and under low iron conditions. STRUCTURE AND FUNCTION ANALYSIS OF DNAJC19 AND MAGMAS, HOMOLOGUES OF THE YEAST Pam18 (Tim14) AND Pam16 (Tim16) MITOCHONDRIAL PROTEIN - IMPORT MOTOR COMPONENTS

S. Elsner, D. Simian, A. Azem

Department of Biochemistry, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Recently, Pam18/Tim14 and Pam/16Tim16, two novel proteins involved in mitochondrial protein import, have been identified in the yeast Saccharomyces cerevisiae (REF). Tim14 is a member of the J protein family whereas Tim16 is ascribed to the J-like protein family. Both proteins were found to be essential components of the mitochondrial Tim23 preprotein translocase (also regarded as the PAM complex). Homologues of yeast Pam18/Tim14 and Pam16/Tim16 were also identified in humans (DNAJC19 and Magmas respectively). DNAJC19 and Magmas are of great interest since they are associated with several human disorders. A novel autosomal recessive disorder called "DCMA syndrome" has already been associated with a mutation in the DNAJC19 protein, while Magmas is suspected to be involved in increased rates of anaerobic metabolism, resistance to apoptosis and altered growth-factor sensitivity, characteristic of cancer cells. The goals of this work are i) To carry out a structural analysis of human Tim14(Pam18)/Tim16(Pam14). ii) To determine whether the function of Tim14(Pam18)/Tim16(Pam14) is evolutionarily conserved between yeast and human. To this end, we have overexpressed the human Tim proteins in bacteria, purified them and studied their ability to interact with their yeast homologues, using crosslinking with a bifunctional reagent, CD spectroscopy and gel filtration. The in vitro results indicate that the interaction between human and yeast Tim proteins is conserved. Furthermore, we are currently trying to determine whether the human homologues are able to replace the function of their yeast counterparts, by conducting in vivo complementation experiments. We hope to create a yeast system for investigating the function of these two human disease-causing proteins. Kv CHANNELS ION INDEPENDENT EFFECT ON FREQUENCY AND CHARACTERISTICS OF FUSION EVENTS

L. Feinshreiber1, R. Friedrich2, U. Matti3, J. Rettig3, U. Ashery2, I. Lotan1

1Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv, Israel, 2Department of Neurobiochemistry, Life Science Institute, Tel Aviv University, Ramat-Aviv, Israel, 3Physiologisches Institut, Universitaet des Saarlandes, Homburg/Saar, Germany

Kv2.1 channel is commonly expresses in the soma and dendrites of neurons, where it could influence the release of neuropeptides and neurotrophins and in neuroendocrine cells, where it could influence hormone release. The traditional role of this channel is to inhibit neurotransmitter release by influencing the membrane potential and thereby inhibiting voltage-gated calcium channels activation. Recently (Singer-Lahat et al., 2007) , we suggested an unexpected role for Kv2.1 of facilitating neuropeptide release from rat PC12 cells expressing recombinant fluorescent atrial natriuretic factor polypeptide. The enhancement was independent of the channel ion conducting function and occurred through a direct interaction with syntaxin 1A, a member of the SNARE proteins which are component of the vesicle release machinery. These data implied a complex role for this channel in the regulation of exocytosis, and raised the need for understanding the mechanism underlying this enhancement effect. Here, we further investigated the enhancement effect on release from bovine chromaffin cells using amperometry, which allows direct measurements of single secretion events. We show that over expression of Kv2.1 wild-type or a non-conducting mutant channel enhances release (~ two fold) of catecholamines triggered by external application of high potassium solution, compared with control cells. Detailed single spike analysis reveals that the enhancement can be attributed mostly to a higher fusion events frequency, indicating a possible involvement of Kv2.1 in vesicle docking and/or priming. In addition, significant changes in single spike parameters are obtained, suggesting a role for Kv2.1 in regulating the vesicle fusion efficiency. This study substantiates the non-conducting function of Kv2.1 channel in regulating vesicle exocytosis and provides new mechanistic insights into this novel machinery participating in the regulation of secretion. CRYSTALLIZATION AND MOLECULAR-REPLACEMENT STUDIES OF HUMAN CYTOPLASMATIC PHENYLALANYL-tRNA SYNTHETASE

I. Finarov, N. Moor, M. Safro

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

A cytoplasmatic human phenylalanyl-tRNA synthetase (hPheRS) is one of the biggest and most complex enzymes among aminoacyl-tRNA synthetases. HPheRS is a hetero-tetramer consisting of two alpha and two beta subunits, overall 2194 amino acids. Overexpressed protein has been crystallized in three different crystal forms. The crystals that belong to space group C2 diffracts to ~3A resolution and have unit cell parameters of a=362.8 b=213.15 c=215.2 alpha=gamma=90 beta=125.15. The experiment data was collected at ESRF (Grenoble, Freance). Two other crystal forms reveal P422 and P21 space groups and unit cell parameters (a=b=110.9 c=290.25 alpha=beta=gamma=90) and (a=178.69 b=112.6 c=378 alpha=gamma=90 beta=93.8) respectively. Both of them produce diffraction spots beyond 4.0A resolutions. A molecular replacement (MR) method was implemented to obtain initial phases and first model of protein structure. Structures of T. thermophilus and Pyrococcus Horikoshii PheRSs were combined to obtain the most complete preliminary model. For MR search performed by Phaser program a polyserine model was constructed. The search was made up of two steps: determination of intramolecular organization using alpha-beta heterodimer as a search model, and localization of four hPheRS copies in asymmetric unit using the whole heterotetramer. Matthews parameter suggests presence of 3-6 molecules in asymmetric unit. From examination of self rotation function map, showing presence of four-fold non-crystallographic axis, we derive existence of four copies of hPheRS in asymmetric unit. This is consistent with Matthews parameter. Four molecules with the total molecular weight ~1MD within asymmetric unit have been localized. The Further improvement of the model was performed using model building function in Phenix program. Human cytoplasmatic PheRS structure refinement is in progress now. THE ‘NEW MIGHTY’ (NUMT) HYPOTHESIS OF AGING AND LONGEVITY

K. Muradian1, V. Fraifeld2

1Department of Physiology, Institute of Gerontology, Kiev, Ukraine, 2The Shraga Segal Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Insertions of mitochondrial DNA into the nuclear genome, NUMTs (pronounced ‘new-might’), are predominantly dispersed in the non-coding regions of the chromosomes as pseudogenes. Although NUMTs represent a valuable resource for phylogenetic analyses, their ontogenetic functions and especially relevance to aging and longevity remain completely unknown. Here we discuss for the first time our findings shedding light on the relationship between NUMTs and maximum life span (MLS) and their possible role in aging. The analysis of currently available data on the number and size of NUMTs in 12 species (human, chimpanzee, mouse, rat, vole, cattle, cat, chicken, mosquito, flour beetle, D. melanogaster and C. elegans) revealed a highly significant positive correlation between MLS and the NUMTs number (n = 12; r = 0.944; p < 0.000004). The correlation remained significant for the mammalian species as well (n = 7; r = 0.947; p < 0.001). Also, MLS correlated positively with the NUMTs total size or percentage per the nuclear genome. The molecular mechanisms of how the NUMTs may influence the aging and longevity are currently unknown. Regardless of the exact nature of the phenomenon, the larger NUMTs pool could be associated with an enhanced affinity and ‘dialog’ between the nuclear and mitochondrial genomes, resulting in viability advantages. An alternative but not mutually exclusive explanation of the higher NUMTs number in the longer-lived species could be the elevated capacity of their genomes to preserve genetic information necessary in certain critical situations. Definitely, further research is warranted to support the suggested ‘new mighty’ hypothesis of aging, as well as to explain the correlations and cause-and-effect links, if any, between NUMTs, aging and longevity. TOWARDS CLONING OF ZETA, A NOVEL TOMATO MUTANT IMPAIRED IN CAROTENOID BIOSYNTHESIS

C. Gafni-Amsalem1, S. Glasner1, D. Zamir2, J. Hirschberg1

1Department of Genetics, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem, Israel

Carotenoid pigments serve crucial roles in plants as components of the photosynthetic apparatus and as protectors against oxidative damages. As such, carotenoids are indispensable for plant growth. In fruits and flowers they are secondary metabolites contributing colors to attract animals. We are studying the genetic and environmental regulation of carotenoid biosynthesis in plants. To this end we are characterizing novel mutations in tomato that affect pigmentation of fruits or flowers. We have identified a new recessive mutation in tomato (S. lycopersicum cv M82) named ZETA. Fruit of ZETA have are orange instead of red and flowers are paler than the wild type. Both fruit and flowers accumulate zeta-carotene. Young leaves are virescent but eventually synthesize beta-carotene and xanthophylls and turn green. However, in the dark, leaves of ZETA accumulate zeta-carotene. It was determined that zeta-carotene desaturase (ZDS) and carotene isomerase (CRTISO) are not impaired in ZETA. We hypothesize that a mutation in ZETA affects an accessory mechanism essential for ZDS-specific activity. To identify the mutation ZETA gene we are using map-based cloning and candidate-gene approaches. We have screened F2 plants of a cross between ZETA (CV M82) and the wild tomato S. pimpinellifolium, and found that the mutation maps between the markers C2_At1g48300 and CT80 (Tomato-EXPEN 2000 map) on chromosome #12. We are currently carrying out "chromosome walking" using a Bacterial Artificial Chromosome (BAC) genomic library. Sequences of BAC-ends are used as markers for screening F2 plants of a cross between ZETA and the wild tomato S. pennellii. THE EFFECTS OF PRE-STORAGE 1-MCP TREATMENT ON PEAR AT DIFFERENT RIPENING STAGES

D. Gamrasni1, R. Ben Arie2, M. Goldway1

1Department of Molecular Biology, MIGAL - Galilee Technology Center, Kiryat Shmona, Israel, 2Department of Postharvest, Fruit Storage Research Laboratory, Kiryat Shmona, Israel

1-MCP (1-methylcyclopropene), an inhibitor of ethylene action, delays climacteric fruit ripening according to its stage of maturity and 1-MCP concentration. 'Spadona', the main pear cultivar cultivated in Israel, is a climacteric fruit producing large amounts of ethylene during ripening. The aim of this work was to investigate the effects of 1-MCP on 'Spadona' pears at different ripening stages, determined by ethylene production level: H- no ethylene production at harvest; C1 - beginning of the climacteric rise (after 12 days at 20 C); C2 - mid-climacteric rise (after 19 days). Following 1-MCP treatment, the pears were stored at -0.5 C in 1.5% O2 + 5% CO2 for 6 months and then transferred to shelf-life (20 C, 70% R.H.) for 2 weeks. At removal from storage ethylene production was inhibited by 1-MCP to similar levels following application at stages H and C1. Application at stage C2 inhibited ethylene production far less. The firmness of pears treated with 1-MCP at stages H and C1 did not change during 2 weeks' shelf life. However, there was no effect of 1-MCP treatment on softening of pears treated at C2. Superficial scald developed during shelf-life at 20 C on all untreated control fruit, independent of the stage of maturity when stored. 1-MCP totally inhibited the development of the disorder at all three stages. The expression of the ERS1 gene, which declined in control fruit with advancing maturity, also became less sensitive to 1-MCP. In contrast, ACO expression increased with advancing maturity, but 1-MCP inhibition still declined. Conclusions: Pear sensitivity to 1-MCP is high at pre-climacteric and early climacteric stages, but declines during the climacteric rise. At all stages 1-MCP treatment controls superficial scald on stored fruit. STATUS EPILEPTICUS AND MYOCARDIAL DAMAGE FOLLOWING SARIN EXPOSURE IN THE RAT

G. Yacov, L. Raveh, T. Kadar, S. Chapman, A. Rosner, I. Rabinovitz, R. Sahar, R. Brandeis, B.A. Weissman, E. Gilat

Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, Israel

Exposure to organophospates (OP) initiates a cascade of toxic symptoms, such as salivation, fasciculations, tremors, convulsions, respiratory distress and death. These toxic manifestations are due to hyperactivity of the cholinergic system as a result of inhibition of ChE, and the subsequent increase in the concentration of the neurotransmitter ACh. The Immediate therapeutic regimen with atropine sulfate and an oxime, such as TMB4, although increasing survival, does not ameliorate nerve agent-induced seizure activity. This study focused on the correlation between cardiac damage and convulsive activity following sarin exposure. Cardiac damage following sarin exposure was determined by blood Troponin T assay and histological evaluation. Measurement of brain peripheral benzodiazepine receptor density served as a measure for brain damage. Exposure to 1.2LD50 sarin with TA1 treatment (TMB-4, 7.5 mg/kg and Atropine, 5 mg/kg, 1 min post exposure) resulted in a significantly (p<0.003) higher clinical score than identical exposure with TAB1 or TA1+MID1 (Benactyzine or Midazolam 1 mg/kg) 1 min treatment. Marked elevation in troponin-T level characterized exposure to 1.2LD50 sarin with TA1 or TA1+MID60 treatment (0.83±0.11 and 0.48±0.18 ng/ml respectively). When animals were subjected to an immediate anticonvulsant, such as midazolam or a central anticholinergic drug such as benactyzine, the rate of occurrence of abnormal Troponin-T levels was significantly (p<0.014-p<0.018) lower (0.05±0.03, 0.03±0.03 ng/ml, respectively). Histological evaluation of cardiac damage significantly (p<0.002) corroborated these results. The order of PBR density in brain tissue 7 days following exposure to 1.2LD50 sarin and treated with TA1 or TA1+MID60 was 2-3 fold higher than those with TAB1 or TA1+MID1 (p<0.0001). Correlations between the 4 parameters ranged between R=0.69-0.83, p<0.01. In conclusion, Benactyzyne and midazolam if given immediately following sarin exposure provide neuroprotection as well as cardioprotection. Further studies are necessary to reveal the mechanism responsible for the induction of cardiac damage following sarin exposure. TWO ACTIVE FORMS OF ZYMOMONAS MOBILIS LEVANSUCRASE: A UNIQUE MICROFIBRIL STRUCTURE OF THE ENZYME PROMOTES THE SYNTHESIS OF HIGH MOLECULAR WEIGHT LEVAN

D. Goldman, N. Lavid, A. Schwartz, D. Danino, Y. Shoham

Department of Biotechnology and Food Engineering, Institute of Catalysis Science and Technology, Technion–Israel Institute of Technology, Haifa, Israel

Many microorganisms synthesize polysaccharides based on fructan for carbon storage, protection and adhesion. These beta-D-fructofuranose polymers (inulin or levan) can consist of up to 100,000 fructose units. The beta-(2-6)-fructofuranose linked levan is synthesized directly from sucrose by levansucrase, which is able to catalyze both sucrose hydrolysis (to glucose and fructose) and the polymerization of fructose to levan. We have recently found that Zymomonas mobilis levansucrase exist in two forms depending mainly on the pH and the ionic strength of the solution. At pH above 7.0 and low ionic strength the protein is mainly a dimer in solution. At pH below 6.0 the protein tends to form long microfibrils that form a turbid solution and can precipitate out of solution. These two forms are readily interchangeable simply by changing the pH. Surprisingly, the manner in which the enzyme is arranged strongly effects its product specificity, kinetic properties and stability. At pH above 7.0 the activity of the dimer enzyme is mainly sucrose hydrolysis and the generation of short oligosaccharides (degree of polymerization 2 to 4). At pH below 6.0 the enzyme forms microfibrils, and its main product is high molecular weight levan (degree of polymerization greater than 20,000). Negative stain transmission electron microscopy images reveal that the enzyme forms a very ordered structure of long fibrils which appear to be made of repeating rings. Each ring consists of six to eight enzyme monomers. This form of enzyme corresponded to a molecular weight greater than 1,000,000 Da based on gel permeation chromatography. The microfibrils formation kinetics increases at high temperature and high ionic strength, suggesting that hydrophobic interactions are the main driving force of the protein self assembly. A NOVEL FUNCTION OF RED WINE POLYPHENOLS IN HUMANS: PREVENTION OF ABSORPTION OF CYTOTOXIC LIPID PEROXIDATION PRODUCTS

S. Gorelik1,3, M. Ligumsky2, R. Kohen1, J. Kanner3

1Department of Pharmaceutics, School of Pharmacy, Hebrew University of Jerusalem, Israel, 2Gastrointestinal Unit, Division of Internal Medicine, Hadassah Medical Center, Jerusalem, Israel, 3Department of Food Science, ARO, Volcani Center, Bet Dagan, Israel

Current evidence supports a contribution of polyphenols to the prevention of cardiovascular disease, but their mechanisms of action are not understood. We investigated the impact of red wine polyphenols on postprandial cytotoxic lipid peroxidation products (MDA) levels in human. In a randomized, crossover study, the effect of red wine polyphenols on postprandial levels of plasma and urine MDA was investigated. Three meals of 250g turkey cutlets: supplemented by water (A); soaked in red wine after heating and 200ml of red wine (B); or soaked in red wine prior to heating and 200ml of red wine (C), were administered to ten healthy volunteers. Subjects' baseline plasma levels of MDA were 50±20nM. Following turkey meat cutlets meal plasma MDA levels increased by 160nM (P<0.0001), following (B) there was 75% reduction in the absorption of MDA (P<0.0001). However, following (C), the elevation of plasma MDA was totally prevented (P<0.0001). Similar results were obtained for MDA accumulation in urine. Our study suggests that red wine polyphenols exert a beneficial effect by the novel new function, absorption inhibition of the lipotoxin MDA. These findings explain the potentially harmful effects of oxidized fats found in foods, and the important benefit of dietary polyphenols in the meal. Locating the biological site of action of polyphenols in the gastrointestinal tract may lead to a revision in our understanding of how antioxidants work in vivo and may help to elucidate the mechanism involved in the "French paradox" phenomena and the protective effect of the Mediterranean or the Japanese diet. HUNTINGTIN INTERACTING PROTEIN 1 (HIP1) IS INVOLVED IN EARLY STEPS OF CLATHRIN MEDIATED ENDOCYTOSIS

I. Gottfried1, O. Yizhar1, M. Ehrlich2, U. Ashery1

1Department of Neurobiochemistry, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel, 2Department of Cell Biology and Immunology, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Huntingtin-interacting protein 1 (HIP1) is related to clathrin mediated endocytosis (CME), However, its exact role and the exact stage in CME are still a debate. It was shown to colocalize with several endocytic proteins but its effect on internalization of cargoes and its involvement in early steps of CME are unclear. To study the involvement of HIP1 in early steps of CME we followed fluorescently tagged proteins, using TIRF and confocal microscopy. We show that HIP1 particles are collocalized with clathrin on the plasma membrane and appear and disappear concomitant with clathrin particles following treatments that influence pits formation (such as butanol and sucrose). In addition, similar to clathrin, HIP1 colocalizes with transferrin receptors on the plasma membrane but not often with those in internalized vesicles, suggesting it might detach from the vesicle close to clathrin uncoating. Nevertheless, examination of the two proteins behavior in untreated cells reveals some differences; HIP1 particles stay in the vicinity of the plasma membrane for longer times and are less mobile than clathrin particles suggesting interaction with other components of the endocytotic machinery or with lipids. We also show that a fragment of HIP1 (HIP1(218-604)) is mislocalized and creates large cellular structures that contain clathrin, AP2 and EEA1. These structures are comprised from smaller units that attach and detach from each other dynamically. We found some HIP1(218-604) in smaller clusters close to the plasma membrane. These HIP1(218- 604) clusters behave in a way that resembles clathrin. These results suggest that HIP1 is found on coated pits, but not vesicles and therefore plays a role in the early stages of CME. MICE DEFICIENT FOR THE SPERMATID-SPECIFIC PROTEIN, NURIT, DISPLAY NORMAL FERTILITY

Y. Gubbay, E. Feige, B. Motro

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

The differentiation of round spermatids into motile spermatozoa, is a highly complicated reconstruction and physiological process. During this transition, the nucleus becomes compacted, an acrosome and a tail are formed, the cytoplasm is eliminated, and the spermatozoon acquires movement and fertilization capacity. Accordingly, many spermatid-specific proteins are needed for coordination and execution of this complex process. Previously, we have cloned and characterized a novel evolutionary conserved spermatid-specific leucine-zipper protein, designated Nurit. Immunogold electron microscopy localized the protein to a unique spermatid organelle called the flower-like structure, which is believed to be involved in spermatid reorganization and cytoplasmic elimination. To elucidate the functional roles of Nurit, we have used gene targeting in embryonic stem cells to generate mice lacking the nurit gene. The mutants are viable and do not display any gross anatomical, morphological or behavioral abnormalities. The fertilization abilities of the mutants were not affected, and the number of pups was similar. The mutants' epididymal sperm count was normal, and no significant difference was observed in sperm motility assays using Computer Assisted Sperm Analysis. Moreover, light and electron microscopy did not reveal any detectable defects. In fertility terms, the most prominent difference between animal colony conditions and life in the wild is in the constant temperature maintained in the animal colony. We thus examined the influence of heat shock in wt and nurit-deficient mice. However, testis morphology and fertility following heat shock, did not differ significantly in the mutants. Therefore, our current data suggests that Nurit protein is dispensable for sperm differentiation and fertility, and the functions of this conserved protein are still obscure. CLASSICAL WOUND-HEALING CASCADES TRIGGER UROCHORDATE WHOLE BODY REGENERATION

O. Haber, N. Guttmann-Raviv, R. Reshef

Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel

Regeneration in adult chordates is confined to a few model cases and terminates in restoration of restricted tissues and organs. Here, we study the unique phenomenon of whole body regeneration (WBR) in the colonial urochordate Botrylloides leachi. This regeneration produce a new functional organism from a small isolated piece of the vascular system. We have previously shown that retinoic acid (RA) regulates diverse developmental aspects in WBR. However, the initial signals that trigger this process remain unknown. In the present study we tested the hypothesis that the WBR process initiates by using wound-healing molecular cascades. Therefore, we decided to investigate the early events and the signalling cascades that take place rapidly after an injury of blood vessels and ampullae in B.leachi colony, leading to WBR. It is known that physical injury induces the release of ATP from cells. These extracellular nucleotides stimulate cells by activating the P2 purinergic receptors (P2Y) present on the cell surface and by activation of the Mitogen-activated protein kinases (MAPK) cascade. We found that wounding the network of blood vessels and ampullae, in B.leachi colony, results in release of ATP into the surrounding medium. We demonstrate that the inhibition of P2Y with the specific antagonist abrogates the migration of ampullae and prevents the initiation of WBR. We also show that the wounded tissue induces rapid activation of the extracellular signal-regulated kinase (ERK) pathway. This activation is attenuated by addition of a chemical inhibitor of ERK. This inhibition results in a complete arrest of the WBR process. These results suggest that early events in WBR resemble the ones that take place during wound healing processes in higher vertebrates. Therefore, it is plausible that some regeneration abilities that are expressed in early choradates potentially exist in higher verterbrates but during the course of evolution were altered or repressed. REGULATION OF HEME SYNTHESIS AND PROTEASOMAL ACTIVITY BY COPPER IN WILSON DISEASE

R. Hait-Darshan, Z. Malik

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

Wilson disease (WD) is a genetic disorder characterized by mutations in ATP7B gene regulating Cu++ cellular transporting and resulting Cu++ accumulation in a variety of tissues, particularly in the liver and brain. However, the biochemical mechanism causing the pathology in WD is unknown. In vivo studies have shown a correlation between WD and porphyrin synthesis implicating that enhanced cellular Cu++ causes malfunctions in porphyrin regulating. The purpose of the present study was to assess the multiple effects of Cu++ on regulation of heme synthesis which in turn affects proteasomal degradation. Exposure of hepatoma cells HepG2 to Cu++ induced reduction in heme synthesis, upregulation of heme oxygenase1 and enhanced proteasomal activity. The heme pathway enzymes PBGD and ALAD were inhibited by Cu++ similarly to the effects of Pb++ in HepG2 cells. Cellular accumulation of Cu++ induced ER disruption and mitochondrial swelling. Under these conditions Ub conjugated proteins were gradually accumulated, while treatment with the Cu++ chelator BCS reversed this effect. The present results shed light on the toxic mechanisms of copper in WD which are well correlated with the clinical manifestations. In addition, it could facilitate development of new and improved drugs for Wilson disease patients. DISULFIDE BOND FORMATION IN THE CYTOPLASM? VIRAL SULFHYDRYL OXIDASES

M. Hakim, D. Fass

Department of Structural Biology, Weizmann Institue of Science, Rehovot, Israel

Structural disulfide bonds are abundant in extracellular proteins and in proteins localized to internal membrane-bounded compartments of the cell. In contrast, the cytosol of prokaryotes and eukaryotes is maintained as a reductive environment that facilitates the breaking of disulfides. Remarkably, a pathway dedicated to the catalysis of disulfide bond formation in the cytosol has been identified. Poxviruses and related large double-stranded DNA viruses encode a sulfhydryl oxidase that catalyzes the formation of disulfide bonds de novo in the cytoplasm of infected cells, as well as a series of oxidoreductases that transfer these disulfides to virus capsid proteins assembling in the vicinity. The poxviral sulfhydryl oxidase belongs to the ERV/ALR family, members of which catalyze oxidation of cysteines in the endoplasmic reticulum and mitochondria. The viral enzyme exhibits some prominent deviations from its eukaryotic homologs. By exploring these differences structurally, biochemically and biophysically, we may better understand how disulfides are formed and retained in an environment that has evolved to generally prevent cysteine oxidation. Here I present the first crystal structure of a viral sulfhydryl oxidase and biophysical evidence for membrane affinity of the enzyme. Our studies support a model in which the poxvirus cytoplasmic disulfide bond formation pathway is restricted to specialized membrane-associated microenvironments that protect virus capsid proteins from host cell reducing agents during oxidative folding and virus assembly. THE BENEFICIAL EFFECT OF AS101 IN A RAT MODEL OF TYPE 2 DIABETES: ASSOCIATION WITH UPREGULATION OF SIRT1 PROTEIN EXPRESSION AND ACTIVITY

M. Halperin, Y. Kalechman, H. Cohen, B. Sredni

The Mina and Everard Goodman Faculty of Life Sciences, Safdie Institute for AIDS and Immunology Research, Bar-Ilan University, Ramat-Gan, Israel

Type 2 diabetes is characterized by chronic deregulations in lipid and carbohydrate metabolism. The disease manifests itself in individuals who loose the ability to produce sufficient quantities of insulin to maintain normoglycemia due to insulin resistance. The histone deacetylase SIRT1 regulates lipid metabolism through its various target genes in several organs, and is involved in increasing insulin sensitivity. Ammonium trichloro (dioxoethylene-o,o’) tellurate (AS101) is a potent immunomodulator, with a wealth of potential therapeutic applications. In this study we show that treatment of normal rats with AS101 upregulates Sirt1 protein expression and activity. Increased activity was manifested by deacetylation of Sirt1 target genes, p53 and PGC1alpha. These effects of AS101 were associated with decreased serum insulin like growth factor-1 (IGF-1) levels. Although addition of AS101 to in vitro cultures of HEK293 cells did not affect Sirt1 protein expression directly, replacement of the standard serum culture by sera from AS101 treated rats (containing decreased IGF-1 levels) increased Sirt1 expression in these cells, implying a role of IGF-1 inhibition in SIRT1 upregulation by AS101. These results promoted us to investigate its therapeutic role in type 2 diabetes model, induced by high fat diet and low dose of streptozocin. These rats are characterized by hyperglicemia, glucose tolerance and insulin resistance that precede overt Diabetes. Treatment with AS101 before manifestation of hyperglycemis prevented disease symptoms while treatment after disease induction resulted in partial beneficial effects. These included increased insulin sensitivity, decreased blood glucose levels, effective glucose clearance, prevention of fatty liver appearance and normal distribution of insulin-producing beta cells in the pancreas. These were associated with upregulation of liver Sirt1 protein expression. Disturbed fat metabolism that precede overt type 2 Diabetes may allow to use approaches that apply pharmacological means that may regulate metabolic disorders by increasing Sirt1 levels thus interfering with the disease progression. A SCREEN OF MEDICINAL PLANTS FOR NEW BIOLOGICALLY ACTIVE COMPOUNDS TARGETED TO THE CYTOSKELETON

M. Halpert1, M. Abu-Abied1, Z. Kerem2, A. Perl1, E. Sadot1

1Institute of Plant Sciences, Volcani Center, Bet-Dagan, Israel, 2Institute of Biochemistry Food Science and Nutrition, Faculty of Agriculture, Food and Environmental Quality Science, Hebrew University of Jerusalem, Rehovot, Israel

We have established a system in which extracts from Israeli medicinal plants are screened for the presence of biologically active compounds targeted to the cytoskeleton. The activity found in one of the extracts, #21, is described here. When extract #21 was subjected to Hela cervical cancer cells the area occupied by the cells on the substrate was doubled and the formation of stress fibers was enhanced accompanied by the formation of focal cell-substrate contacts. The effect was observed already after 5 minutes and even when the initial extract was diluted 1:5000 suggesting a highly active compound. The option that the increase in actin stress fibers is the outcome of microtubule disruption was tested but no microtubule disruption by the extract, neither in HeLa cells nor in rat fibroblasts was observed. In order to correlate the formation of actin stress fibers and focal contacts with rate of cell motility a wound healing assay was performed. In this assay a scratch is formed in a confluent culture, and the time required for the cells to migrate and re-occupy the empty space is monitored. After 20 hours the control cells entirely filled the scratch whereas, when subjecting the extract, HeLa cells migration was inhibited and the scratch remained visible. It was then tested whether the extract is potent in cell cycle arrest using fluorescence activated cell sorter (FACS) analysis. The ratio between cells in G1 and cells in G2/M phase was larger following treatment with the extract (65% of the cells in G1 / 7% cells in G2/M) than in control cells (45% in G1 / 20% in G2/M) indicating on cell cycle arrest. In contrast when this extract was subjected to rat fibroblasts, no effect was observed. Studies for identifying the active compound/s in extract #21 and revealing the mechanism of action are being performed. A CYTOSKELETAL DEMOLITION WORKER: MYOSIN II ACTS AS AN ACTIN DEPOLYMERIZATION AGENT

L. Haviv, F. Backouche, D. Gillo, A. Bernheim-Groswasser

Department of Chemical Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Myosin II motors play several important roles in a variety of cellular processes, some of them involving active assembly/disassembly of cytoskeletal sub-structures. Myosin II motors have been shown to function in actin-bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. A close examination has shown an intimate relationship between myosin II motors ATPase activity and actin turnover rate. Yet, the direct implication of myosin II in actin turnover is still not understood. Here, we show, using high resolution cryo-transmission electron microscopy (cryo-TEM), that myosin II motors control the turnover of actin-bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin-bundles occurs through two main stages; the first comprises unbundling into individual filaments, and the second, their subsequent depolymerization. These evidences suggest that, in addition to their "classical" contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo, for example, in the disassembly of the contractile ring, by fine tuning of their local concentration/activity. REGULATORY DISULFIDES OF THE SULFHYDRYL OXIDASE Ero1

N. Heldman, D. Fass

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

The sulfhydryl oxidase Ero1 is the primary catalyst of disulfide bond formation in the endoplasmic reticulum (ER). The yeast ortholog contains a pair of essential disulfide bonds that are directly involved in the electron transfer pathway from thiol substrates to oxygen. Other disulfides appear to be regulatory and to affect enzyme kinetics. In particular, a C150A/C295A mutant of Ero1 has an enhanced rate of thiol oxidation in vitro and in vivo, a decreased lag phase in the in vitro reaction with model substrates, and a negative impact on redox homeostatis in yeast cells. We set out to understand the structural basis of how this disulfide regulates Ero1 activity. We have determined the X-ray crystal structure of this de-regulated, hyper-oxidizing Ero1 mutant. The structural data together with enzymatic assays suggest that this mutant is hyper- sensitive to activation rather than constitutively active, and that further changes after reduction of the C150-C295 disulfide are necessary to complete the Ero1 activation cascade. IN VIVO ROLE OF DNA POLYMERASE ETA IN TRANSLESION DNA SYNTHESIS ACROSS UV-INDUCED DNA LESIONS IN HUMAN CELLS

A. Hendel1, N.E. Geacintov2, Z. Livneh1

1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Chemistry, New York University, NY, USA

DNA lesions that have escaped repair inhibit replication, leading to arrested replication forks and formation of single-strand gaps. These replication impediments are relieved by translesion DNA synthesis (TLS), a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases are employed to bypass the blocking lesions. We have investigated the role of DNA polymerase eta (pol eta) in TLS across two major UV-induced DNA lesions in cultured human cells, a cis-syn cyclobutane TT dimer (TT CPD), and a (6-4) TT photoadduct. To that end we have employed a quantitative TLS assay previously developed in our lab, based on transfection of cultured cells with gapped plasmids carrying site-specific defined DNA lesions. The cells analyzed included primary fibroblasts from a healthy individual, and from a xeroderma pigmentosum variant (XPV) patient lacking pol eta. In addition, transformed cells from a healthy individual and from an XPV patient were assayed. The results show distinct mutational signatures in cells proficient or deficient in pol eta. Interestingly, in the absence of pol eta, when TLS across the TT CPD is impaired, product analysis revealed a significant proportion of deletions and insertions. These are consistent with a mechanism in which inability to fill in the gap by TLS leads to the formation of a break at the ssDNA region flanking the lesion. This double-strand break is then repaired by non-homologous end joining or homologous recombination. These events are similar to those observed in the genome of cells in which TLS is defective. SP1 AS A NOVEL SCAFFOLD BUILDING BLOCK FOR SELF-ASSEMBLY NANOFABRICATION

A. Heyman1, I. Medalsy2, O. Dgany1, O. Bet-Or1, A. Altman1, D. Porath2, O. Shoseyov1

1The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, and the Otto Warburg Minerva Center for Agricultural Biotechnology, Hebrew University of Jerusalem, Rehovot, Israel, 2Department of Physical Chemistry, Hebrew University of Jerusalem, Israel

Self-assembly is a prerequisite for the fabrication of nanoscale structures. Biomolecules in general and proteins in particular are capable of self-assembling into a wide variety of structures that can be readily manipulated and functionalized. An ideal protein scaffold provides a rigid folding unit, which spatially brings together several potential engineerable domains. We present stable protein 1 (SP1), a novel highly stable homo-oligomeric protein, as a molecular scaffold. This ring-shaped homododecamer protein is utilized to present both nano-particles and protein domains in a pre-defined manner. Using genetic modifications, the protein was engineered to specifically bind various surfaces and nano particles such as gold, silicon and titanium. Assemble two and three dimensional arrays and structures, and present various protein domains. By genetically fusing Glucose oxidase (GOx) to SP1 we observed the formation of active multienzyme nanotube particles containing hundreds of GOx molecules per tube. The presented work demonstrates the value of SP1 as a self-assembly molecular scaffold. A POTENTIAL ANTIMICROBIAL TREATMENT OF KLEBSIELLA PNEUMONIAE BY THE TELLURIUM COMPOUND AS101

M. Hoffmann1, R. Dror1, I. Petchtnikov1, O. Avidan1, M. Albeck2, B. Sredni1, Y. Nitzan1

1The Mina and Everard Goodman Faculty of Life Sciences, The Safdie Institute for AIDS and Immunology Research, Bar-Ilan University, Ramat-Gan, Israel, 2Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel

K. pneumoniae an opportunistic nosocomial pathogen is known to be one of the most common Gram-negative pathogens that can cause pneumonia, urinary tract infections, and sepsis in intensive care unit patients and among immunosuppressed patients. Recently these bacteria became very resistant to most antibiotics. In this study we examined the direct effect of the immunomodulator, tellurium compound, AS101 on Klebsiella pneumoniae. AS101 inhibits the bacterial growth in a dose dependent manner which is also dependant on the nutritional content. Growth inhibition by 3 orders of magnitude was exhibited only in protein rich media. Addition of thiols to the tellurium compound resulted in growth inhibition even in poor nutritional media. AS101 shows bacteriocidic activity towards K. pneumoniae at log phase. X-ray microanalysis indicates damage to Na/K pumps which are located in the bacterial cytoplasmic membrane, after a treatment with AS101. Scanning microscopy exhibited cell wall damage in bacteria treated with or without cysteamine. Using a liposome technique it was shown that AS101 can penetrate liposomes containing porins of K. pneumoniae. Penetration of AS101 can be also suggested by Transmission electron microscopy that demonstrate chromosomal breakage and cell lysis following AS101 treatment. In conclusion, the results above revealed that AS101 acts as a potential antimicrobial bacteriocidal agent. INVOLVEMENT OF MATRIX METALLOPROTEINASES IN THE CUTANEOUS AND OCULAR RESPONSE TO SULFUR MUSTARD

V. Givant-Horwitz, A. Amir, L. Cohen, Y. Shalem, H. Gutman, M. Cohen, E. Fishbine, J. Turetz, T. Kadar, S. Dachir

Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, Israel

The gelatinases, MMP-2 and MMP-9, are involved in extracellular matrix remodeling during physiological and pathological processes. Sulfur mustard (SM) causes acute cutaneous and ocular lesions, followed by delayed pathology. This study aimed to evaluate the involvement of gelatinases in the acute and long term pathology following SM exposure. Hairless guinea pig (HGP) skin and rabbit eyes were exposed to SM vapor. Cutaneous and ocular clinical evaluation was performed daily for the first week and once a week thereafter for up to 1 month. Gelatinase activity was measured by zymography at various time points post exposure. Constitutive MMP-2 ve rabbitןactivity, with no MMP-9 activity, was found in naive HGP skin and in na corneas. MMP-9 and MMP-2 activities increased in both skin and cornea followed exposure to SM vapor. While MMP-9 activity decreased parallel to spontaneous healing, MMP-2 activity remained elevated. At the late phase, MMP-9 was found only in corneas displaying neovascularization, the main characteristic of corneal delayed pathology. These impaired corneas had elevated MMP-2 activity as well. In tear fluid, MMP-9 activity was found throughout the follow-up period post exposure, ve rabbits. The pattern ofןin contrast to gelatinase negligible activity in tears of na gelatinase activity following SM exposure contributes to the understanding of mechanisms involved in SM injury and may have therapeutic implication, such as the use of MMPs inhibitors. MOLECULAR CHARACTERIZATION OF ARGININE AND LYSINE TRANSPORTERS FROM LEISHMANIA DONOVANI

E. Inbar1, I. Darlyuk1, M. Suter-Grotemeyer2, N. Holand1, D. Rentsch2, D. Zilberstein1

1Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel, 2Institute of Plant Sciences, University of Bern, Switzerland

We have cloned and functionally characterized from Leishmania donovani two genes, LdAAP3 and LdAAP7 encoding for permeases that transport arginine and lysine, respectively. Expressing these genes in Saccharomyces cerevisiae mutants revealed that both permeases obtain high affinity (Km around 2 µM) to their corresponding substrates, with optimum activity at acidic pH. Both permeases localize to the surface membranes and are constitutively expressed in both promastigotes and amastigotes. Interestingly, the two permeases are highly specific to their corresponding substrates. This was unexpected since macrophages that host Leishmania, transport arginine and lysine via a common permease; the cation amino acid permease 1 (CAT1). Moreover, whereas LdAAP3 and LdAAP7 belong to the Auxin/amino acid permease family, CAT1 belongs to the amino acid polyamine family. Interestingly, Starvation of L. donovani promastigotes to amino acids induced an increase of arginine transport which paralleled up regulation of LdAAP3 mRNA and protein abundance. Starvation had no effect on lysine transport and level of expression. The results suggest that L. donovani obtain a mechanism that specifically controls cellular arginine homeostasis. HEPATITIS C VIRUS CORE PROTEIN ASSOCIATES WITH DETERGENT RESISTANT MEMBRANES DISTINCT FROM CLASSICAL PLASMA MEMBRANE RAFTS

M. Matto1,2, J.S. Glenn2, B. Aroeti1

1Department of Cell and Animal Biology, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Division of Gastroenterology and Hepatology, Stanford University School of Medicine, CA, USA

Cholesterol sensitive domains (rafts) are dynamic membrane assemblies enriched with cholesterol and sphingolipids. Rafts have been implicated as membrane platforms for the assembly of several enveloped viruses, including HIV, Ebola and Influenza. The major aim of our study is to analyze the association of HCV core with lipid rafts during the course of HCV life cycle. We show here that a significant fraction of HCV core in Huh7 cells expressing the full-length replicon of HCV is associated with Triton X 100-insoluble membranes, or rafts. Cholesterol depletion with methyl-betaƒ{cyclodextrin abolishes the association of core with the buoyant membrane fraction, indicating that cholesterol is essential for core interactions with the detergent-resistant membranes (DRMs). Analysis by immunofluorescence microscopy reveals two types of morphologically distinct core populations: one appears as cytoplasmic patches while the other surrounds the periphery of lipid bodies. However, none of them colocalize with respect to the classical plasma membrane raft markers caveolin-1 and subunit B of Cholera toxin, raising the possibility that core is bound to DRMs in intracellular organelles. Furthermore, expression of core by itself behaved similarly to core expressed within the context of the full-length HCV replicon. Finally, an HCV core mutant that lacks amino acids 78- 118 in Domain 1 displays weak interactions with DRMs. Based upon these data we conclude that HCV core is associated with some, perhaps specialized, class of cytoplasmic DRMs. This association may be critical for budding of HCV and for recruitment of host cellular proteins to assist the process. These results have exciting implications for the HCV life cycle and may reveal a valuable probe of host cell biology. DISSECTION AND RECONSTITUTION OF A COMPLEX REQUIRED FOR SORTING OF GENERAL AMINO ACID PERMEASE 1 (Gap1)

K. Kogan

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

The Saccharomices cerevisiae general amino-acid permease, Gap1p, is a model for membrane proteins that are regulated by intracellular sorting. At low concentrations of amino acids, the sorting machinery directs the Gap1p to the plasma membrane, whereas at high concentrations Gap1p is driven to the vacuole for degradation. Recently a complex called GSE (GTPase-contaning complex for Gap1p Sorting in the Endosome) was identified. As its name suggests, GSE interacts with Gap1p and is required for proper sorting of the permease to the plasma membrane. The GSE complex comprises five proteins: two small GTPases (Gtr1p and Gtr2p) and three other small proteins (Gse1, Gse2, Ltv1). However, the sum of the molecular masses of these five proteins (~170 kDa) is much less then the mass estimated from gel filtration (~600 KDa) of the native complex from yeast lysate. Though extensive biochemical work has been done in an attempt to map the interactions between members of the GSE complex, the stoichiometry is unknown and the manner in which it assembles and interacts with Gap1p is poorly understood. We are working to establish a recombinant expression system to produce sufficient GSE complex for in vitro biochemical and structural characterization, as well as analyzing various elements individually. We have found over-expression of GSE complex components in bacteria to be challenging: Gtr1 and Gtr2 are very unstable even when co- expressed. Gse1 and Gse2 can be expressed at marginally higher levels, and we are attempting their co-expression with Gtr1 and Gtr2 in various combinations. Initial trials have revealed the sensitivity of protein expression level, solubility, and stability to the nature of the purification tags used and to their location within the fusion proteins. CE-EMERIN AND CE-MAN1 OVERLAPPING FUNCTIONS ARE REQUIRED FOR MUSCLE INTEGRITY

R. Barkan1, A. Margalit1, E. Neufeld1, N. Feinstein1, J. Liu2, Y. Gruenbaum1

1Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

Emerin and MAN1 are LEM domain proteins and integral proteins of the metazoan inner nuclear membrane. Emerin is known to interact with nuclear lamins, barrier-to- .autointegration factor (BAF), nesprin-1ƒׁ, actin and regulates ƒׂ-catenin activity MAN1 also interacts with lamins, BAF, Smad2, Smad3 and antagonizes signaling by TGFbeta. Mutations in emerin cause X-linked recessive Emery-Dreifuss muscular dystrophy. Mutations in MAN1 cause Osteopoikilosis- bone density disorders. Emerin and MAN1 are both evolutionarily conserved from human to Caenorhabditis elegans. Loss of either Ce-emerin (emr-1) or Ce-MAN1/LEM-2 (lem-2) has no detectable phenotypes. In contrast, downregulation of both Ce-emerin and Ce-MAN1, which removes both maternal and zygotic mRNAs, causes lethality before the 100- cell stage with abnormally condensed nuclei and anaphase chromatin bridges. To study phenotypes caused by the loss of LEM-domain proteins at later stages of development, we have generated a lem-2 null strain that is also heterozygous for emr- 1 gene. This strain produces emr-1 and lem-2 null progeny, which survive embryogenesis due to emr-1 maternal contribution. These worms arrest at L2 larvae stage with severe phenotypes including lobulated and open nuclei and dystrophic muscles. Gene profiling of emr-1 deletion strain and lem-2 deletion strain showed alter transcription of genes and many of these genes overlapped in the two strains, including many muscle genes. This research can have an impact on the understanding of EDMD disease especially on the potential role of MAN1 in its evolvement. . These results probably have direct implications on the mechanism of human muscular dystrophies caused by mutations in emerin. BIOCHEMICAL AND FUNCTIONAL ANALYSIS OF A FAMILY OF PLANT PROTEIN KINASES WITH AN ATYPICAL ATP BINDING SITE

Y. Koifman1, J. Allen2, E. Shani3, N. Ori3, K. Shokat2, G. Sessa1

1Department of Plant Sciences, Tel-Aviv University, Ramat Aviv, Israel, 2Department of Cellular and Molecular Pharmacology, University of California - San Francisco, CA, USA, 3The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel

Protein kinases are highly conserved and constitute one of the largest gene families. Their catalytic activity consists in binding and orientation of ATP in a complex with divalent cations, and transfer of the ATP gamma-phosphate to the acceptor hydroxyl residue (Ser, Thr, or Tyr) of the protein substrate. Our study focuses on a family of Arabidopsis protein kinases with atypical structural characteristics that we refer to as “alanine protein kinases” (APKs). The hallmark of the APKs is an alanine residue in the ATP binding site at a position, which is occupied in all protein kinases by a bulky amino acid, commonly defined as the gatekeeper. The gatekeeper residue normally restricts access to a pre-existing cavity within the ATP binding pocket and its mutation to a small residue allows the kinase to use N6-substituted ATP analog as phospho-donors. Interestingly, N6-substituted molecules share structural similarity with cytokinin nucleotides, which naturally occur in plants. Based on the unusual structure of the APK ATP binding site and the similarity between ATP analogs and cytokinin nucleotides, we hypothesize that APKs have a role in cytokinin signaling or synthesis in plant cells. Biochemical analysis of GST-fusions of three representative APKs revealed that they are active protein kinase in vitro with Tyr, Thr and Ser specificity, defining them as dual-specificity protein kinases. In addition, APKs showed highest activity in basic pH conditions and preferentially used as cofactor Mn rather than Mg divalent cations. Interestingly, APKs were able to bind cytokinin nucleotides in vitro and to use them as phosphodonors. Moreover, APKs showed a higher affinity to cytokinin nucleotides than to ATP. To analyze the link between APKs and cytokinin signaling in plants, we are currently analyzing phenotypes of T- DNA insertion mutants of APK family members. REDOX SWITCH: FERREDOXIN AS A NOVEL RNA-BINDING PROTEIN

M. Kolton1, F. Shaya1, A. Zehavi2, A. Danon2, O. Ostersetzer-Biran1

1Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Bet-Dagan, Israel, 2Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel

The expression of organellar genomes is essential for plant growth and development and is regulated in response to various developmental and environmental signals. Much of this control is thought to be at the post-transcriptional level, via nuclear- encoded RNA-binding proteins that are targeted to organelles. Yet, only few such proteins have been characterized and even less is known about their specific roles in these activities. In particular, post-transcriptional regulation of gene expression by redox active proteins is still in its infancy with many basic questions remaining unanswered. Ferredoxin (Fd) are small iron-sulfur proteins, carrying a key redox function in photosynthesis throughout their role in electron transport. Plants genomes contain several homologues of Fds, whose members display diverse roles in different tissues. Intriguingly, indirect observations suggest that Fds may associate with RNA, thus, implicating a second role of Fd’s in post-transcriptional regulation of organellar genome expression. As mobile electron-carriers, able to partition between the stroma and the thylakoid membranes, Fds are excellent candidates for regulating organellar gene expression, and thus, to participate in the complex intracellular cross-talk between the plastids and nucleus. Here, we propose a novel scenario in which the role of Fd, as a redox active protein in photosynthetic electron transport, is reflected in a second role as an RNA-binding protein involved in light-dependent post- transcriptional regulation of chloroplast gene expression. Accordingly, the aim of this research are to (ii) identify whether Fd’s carry RNA-binding activity in vitro; (ii) identify RNA ligands associated with each Fd in vivo. Our preliminary data suggest that Fd’s are indeed associated with RNA in vivo. Moreover, detailed biochemical analysis demonstrated that At_Fd5 binds to psbA RNA with high affinity, mainly throughout its association with regions found in the mRNA but also to the 5’UTR and 3’UTR regions. COMBINING TWO DNA FINGERPRINTING TECHNIQUES FOR THE STUDY OF THE INTESTINAL MICROBIOTA IN HEALTH AND DISEASE

A. Kovacs, U. Gophna

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel

The intestinal microbiota is known to play an essential role in the healthy function of the gut. On the other hand, evidence shows that in genetically susceptible individuals, the gut microbiota is involved in the pathogenesis of various intestinal disorders, such as inflammatory bowel diseases. Sequencing of 16S rRNA clone libraries and fingerprinting analyses, as well as qualitative PCR approaches, have been applied in recent years for the study of the intestinal microbiota. ARISA (Automated Ribosomal Intergenic Spacer Analysis) and LH-PCR (Length Heterogeneity PCR) are both DNA fingerprinting techniques originally developed for the study of the microbiota in aquatic environments. Before applying these techniques to the intestinal ecosystem, we created a small database, containing the theoretical amplicon lengths of representative intestinal bacteria. LH-PCR, which is based on variability in length of either one or more variable regions in the 16S rRNA gene, gives low resolution groupings of bacterial taxa. ARISA is based on variability in length of the internal transcribed spacer (ITS) and provides specie-level and in some cases even strain-level resolution. Each technique has its disadvantage - while ARISA doesn't allow observations of shifts in large divisions such as the order or phylum, LH-PCR may erroneously group together genetically remote organisms. Combining the two techniques, using appropriate databases and algorithms, makes it possible to get a better picture of the microbiota in the studied environment. Here we demonstrate the application of this LH-ARISA combination for the study of two different model populations - mice fecal samples and human tissue samples. CANNABINOID REGULATION OF MICROGLIAL ACTIVITY

E. Kozela, M. Pietr, R. Levy, Z. Vogel, A. Juknat

Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

Cannabinoids have been shown to exert anti-inflammatory activity in various in vivo and in vitro experimental models, including several neurodegenerative diseases. However, the mechanisms of their anti-inflammatory effects are currently unknown. Using BV-2 mouse microglial cell line we investigated the signaling pathways involved in the anti-inflammatory effects of cannabinoids as well as their influence on expression of genes known to be involved in inflammation. Using lipopolysaccharide (LPS, a bacterial endotoxin) to activate microglial cells, we found that LPS induced the production and secretion of proinflammatory cytokines including interleukin- 1beta (IL-1beta) and IL-6. The production and secretion of IL-1beta and IL-6 were reduced by the two major cannabinoids present in marihuana, delta9- tetrahydrocannabinol (THC) and cannabidiol (CBD). However, the mechanisms of their anti-inflammatory action did not involve the currently known cannabinoid receptors (CB1 and CB2). Moreover, we found that THC and CBD act through different mechanisms. NFkB is an important modulator of the inflammatory response. LPS leads to the degradation of IL-1 receptor kinase (IRAK-1) and IkB (NFkB cytoplasmic inhibitor) proteins and via this pathway activates NFkB. We found that CBD, but less so THC, reversed the LPS-induced IRAK-1 and IkB degradation. On the other hand, the expression (mRNA) of IRAK-M (inducible negative regulator of inflammatory response) was enhanced after THC treatment, but not by CBD. Gene array profiling showed that CBD reversed many of the LPS induced changes in BV-2 microglial gene expression while THC had a relatively minor effect. In conclusion, although both THC and CBD exert inhibitory effects on the production of inflammatory cytokines in microglial cells, their activity seem to involve different intracellular pathways. Supported by the Dr Miriam and Sheldon G. Adelson Center for the Biology of Addictive Diseases (TAU). INVESTIGATION OF FIBRILLAR DEFORMATION IN ANTLER USING SYNCHROTRON RADIATION

S. Krauss1, H.S. Gupta1, J. Seto1, J.D. Currey2, T. Landete-Castillejos3, S.S. Funari4, S.V. Roth4, P. Fratzl1

1Department of Biomaterials, Max-Planck Institute of Colloids and Interfaces, Golm, Germany, 2Department of Biology, University of York, UK, 3IREC (Sec. Albacete) y ETSI Agronomos, IDR, University Castilla-La Mancha, Albacete, Spain, 4HASYLAB- DESY, Hamburg, Germany

Introduction: Deer antler is a unique example of an annually regenerated biomineralized tissue, and exhibits extraordinary toughness. Because the apatite weight content (59%) is close to that of bone (65%), we hypothesized that the high work of fracture in antler arises due to a difference in the fibrillar and mineral deformation mechanisms in the mineralized matrix. Approaches: Cortical antler specimens from Iberian red deer (Cervus elaphus hispanicus) were mechanically stretched to failure (under wet conditions) while in – situ small angle X – ray scattering (SAXS) with synchrotron X – ray radiation (lambda = 1.5 nm) was used to measure fibril strain concurrently. Apatitic mineral particle size and orientation were determined using scanning SAXS. Results: At each external load the strain of fibrils was half of that of the bulk tissue. This ratio is remarkably similar to bone in the elastic regime. In the inelastic regime, the fibrillar strain in antler showed a continuous linear increase with strain. In addition an inhomogeneous fibril straining is observed to commence in the inelastic regime. At the tissue level specimens exhibited a mean Young’s modulus of 6.9 ± 0.5 GPa and a mean work of fracture of 1 ± 0.1 MJ m-3. The mineral particles had a mean thickness of 3 nm, which is comparable to that of bone from young (10 years old) human, and a similarly high degree of orientation. Conclusions: Because the fibrils sustain only half of the external load, a hierarchy in strain in antler compact tissue is developed. Inhomogeneous fibril elongation is associated with damage in post-elastic deformation. The mean thickness of the mineral particles in antler is in the range of young human bone although antler is grown in a much shorter time interval. MOLECULAR GENETIC ANALYSIS OF DROSOPHILA COP9 SIGNALOSOME SUBUNIT 8

O. Krieger2, P. Oren-Giladi1, D.A. Chamovitz2, D. Segal1

1Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel, 2Department of Plant Sciences, Tel Aviv University, Ramat Aviv, Israel

The COP9 signalosome (CSN) is a multi-protein complex active in the ubiquitin- proteasome pathway. The CSN was first identified in Arabidopsis where it is required for the repression of photomorphogenesis. CSN or CSN-related complexes have been implicated in varied processes in diverse eukaryotes. The CSN directly interacts with CRL-type E3 ubiquitin ligases, and removes the post-translational modification Nedd8 from the cullin component of the SCF. In addition, the CSN also affects proteolysis by its association with protein kinases, deubiquitylating enzymes, and proteasome substrates. We use Drosophila for studying the roles of the CSN in animals in the context of the whole organism. We have previously shown that fly mutants in subunits CSN4 and CSN5 have defects in various developmental processes ranging from oogenesis, and embryogenesis to axon guidance and immune response. Here we report our initial characterization of the Drosophila CSN8 subunit. Two csn8 deletion mutants were generated by imprecise excision of a nearby P element. Gel filtration analysis of wild type and mutant Drosophila indicated that the CSN8 subunit is found exclusively as part of the entire CSN complex, and not as a monomer, and that proper assembly of the CSN complex is CSN8-dependent. The abnormal assembly of the complex in the csn8null mutants affects CSN function since de- neddylation of cullin1 in them was found to be defective. The csn8null mutants are larval lethal. The mutant larvae have defects in molting from the 2nd to the 3rd instar, evident by double mouth hooks, similar to csn4null mutants. They also harbor melanotic capsules in their hemolymph, as do csn5null mutants. These phenotypes suggest that the CSN complex is required for molting and hematopoiesis. Mosaic analysis indicated that CSN8 is required for development of various adult structures including the wing and the notum, and is also necessary for normal oogenesis. PRODUCTION OF NOVEL COMPOSITE BIOMATERIALS MADE FROM CELLULOSE AND RESILIN

S. Lapidot1,2, M. Dekel1,2, S. Meirovitch1,2, J. Fierer1,2, T. Danieli3, N. Lapidot4, O. Shoseyov1,2

1The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricutural Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, 2The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricutural Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, 3The Protein Expression Facility, Wolfson Center for Applied Structural Biology, Faculty of Science, Hebrew University of Jerusalem, Israel, 4CollPlant Ltd., Rehovot, Israel

Resilin is a rubber like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Its unique mechanical properties allow the outstanding jumping ability of fleas to jump up to 30 cm high equivalent to a human high jump of 400 meters. The polymerization of the protein results in assembly of a natural protein-carbohydrate composite material with the cuticle chitin. Recently the Drosophila melanogaster gene for Resilin that contains an elastic repetitive domain and a putative chitin binding domain type R&R was identified, which enables successful production and polymerization of recombinant resilin-like proteins. Cellulose is the most common biopolymer on earth used in numerous applications for thousands of years. Utilizing cellulose binding domain (CBD) for cellulose fiber modification and production of cellulose CBD fusion proteins composites is a well established technology. The current research project aims to produce CBD-resilin fusion proteins that will enable binding and polymerization of resilin on cellulose to obtain a new composite biomaterial with superior mechanical properties for the use of many applications. For that purpose we have engineered recombinant fusion proteins of CBD and resilin and overexpressed them in E. coli. Recently we showed that these proteins are highly expressed and their binding to carbohydrates such as cellulose and chitin was proven. In addition we managed to polymerize these proteins into solid resilin by photochemical crosslinking. Production of the cellulose-resilin composites is currently under progress DYNAMIC PATCHES OF MEMBRANE PROTEINS

Y. Lavi1, M.A. Edidin2, L.A. Gheber1

1Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Biology, Johns Hopkins University, Baltimore, MD, USA

The Major Histocompatability Complex class I (MHC-I) presents peptides from within the cell to the inspection of T-cells. It was shown to be organized in patches, 300nm-700nm in diameter (Hwang et al, 1998), on the plasma membrane of cells. A model, explaining the mechanism leading to this organization, suggested these patches are dynamic in nature, and are maintained due to a combination of barriers to lateral free diffusion and vesicles traffic to and from the membrane (Gheber and Edidin, 1999). Using TIRFM in real time, we explored the dynamic nature of GFP- tagged MHC-I patches on plasma membrane of mouse fibroblasts. The dynamic characterization revealed the life course of these patches is comprised of delivery of molecules at a short instant, followed by a slow, exponential decay, corresponding to diffusion of the molecules over dynamic barriers to free lateral diffusion. The characteristic lifetime of the patches, extracted from the measurements, is approximately 30 s (Lavi et al, 2007). Using phalloidin to reinforce barriers to lateral diffusion, we further tested the model. The distribution of patches’ lifetimes in cells treated with phalloidin shifted towards higher values, when compared with untreated cells, with the most probable value of approximately 80s. These results are in excellent agreement with our suggested model. Further experiments involve actin destabilizing factors, such as cytochalasin D, which is expected to shorten the measured lifetimes of the dynamic MHC-I patches. The persistence of MHC-I patches and their lifetime will be further correlated with T-cell response.

References: 1. J. Hwang, L. A. Gheber, L. Margolis, and M. Edidin, Biophys. J. 74, 2184 (1998). 2. L. A. Gheber and M. Edidin, Biophys. J. 77, 3163 (1999). 3. Y. Lavi, M. Edidin and L. A. Gheber, Biophys. J. 93, L35 (2007). TWO MITOCHONDRIA-ASSOCIATED FACTORS – GC CONTENT AND METABOLIC RATE EXPLAIN OVER 3/4 OF VARIATION IN MAMMALIAN MAXIMUM LONGEVITY

G. Lehmann1, E. Segal1, K. Muradian2, V. Fraifeld1

1The Shraga Segal Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Physiology, Institute of Gerontology, Kiev, Ukraine

In animal cells, mitochondria are semiautonomous organelles of virtually ‘hostile’ (bacterial) origin, with their own code and genome (mtDNA). The semiautonomy and restricted resources could result in occasional ‘conflict of interests’ with other cellular components, in which mitochondria have greater chances to be ‘the weakest link’, thus limiting longevity. Two principal questions are addressed: 1) to what extent the mammalian maximum life span (MLS) is associated with mtDNA base composition? 2) Does mtDNA base composition correlate with another important mitochondria- associated variable – resting metabolic rate (RMR), and whether they complement each other in determination of MLS? Analysis of 140 mammalian species revealed significant correlations between MLS and the content of the four mtDNA nucleotides, especially noted for GC pairs. The most remarkable finding of this study is that the GC content and RMR, taken together explained 77% of variation in MLS (p < E-25). To our knowledge, it is the highest coefficient of MLS determination that has ever been reported for a comparable sample size. Taking into account substantial errors in estimation of MLS and RMR, it could mean that the GC and RMR explain most of the MLS biological variation. Other putative players in MLS determination either should have relatively small contribution or their effects should be realized via the same channels. Although further research is clearly warranted, the extraordinary high correlation of mtDNA GC and RMR with MLS suggests a “direct hitting" of the core longevity targets, inferring mitochondria as a primary object for longevity-promoting interventions. ANTIBIOFILM ACTIVITY OF NANOSIZED METAL FLUORIDES

J. Lellouche2, E. Banin1, A. Gedanken2

1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel, 2Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel

A biofilm is a complex aggregation of microorganisms marked by the excretion of a protective and adhesive matrix. Biofilms are also often characterized by surface attachment, structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix. Resistance to antimicrobial agents is the most important feature of biofilm infections. As a result, infections caused by bacterial biofilms are persistent and very difficult to eradicate than their planktonic counterparts. More notably, biofilms are also a major cause of infections associated with medical implants. The number of implant-associated infections approaches 1 million/yr in the US alone and their direct medical costs exceed $3 billion annually. Thus, there is an urgent need to find novel approaches to eradicate biofilms. Here we propose a simple and fast method to synthesize Magnesium Fluoride nanoparticles (MgF2 Nps). The synthesis is carried out in an ionic liquid solvent using a domestic microwave oven. The product was characterized with X-ray diffraction and transmission and scanning electron microscopes which identified the product as nanocrystalline MgF2. When evaluating the antibiofilm activity of the above mentioned product against common biofilm pathogens excellent results were obtained. Our results prove strong antibiofilm activity against Escherichia coli and Staphylococcus aureus with 70% and 100% inhibition, respectively. We further confirmed this result on different MgF2 coated surfaces such as polymers and glass. The inhibition activity was in the micromolar concentration. We suggest that the spherical nanosized form of the magnesium fluoride increase bacteria-nanoparticle interactions and killing by membrane disruption. THE MECHANISM OF PROTEIN FOLDING: COOPERATIVE OR DOWNHILL?

E. Lerner, M. Wolf, D. Amir, E. Haas

Institute of Life Sciences, Bar Ilan University, Ramat Gan, Israel

Cooperative protein folding invokes discrete folded and unfolded ensembles separated by a free-energy barrier. In contrast, downhill folding involves just one ensemble of protein molecules within a single free-energy well. Common method of monitoring the folding transition which yield mean values cannot resolve the two mechanisms. Time-resolved dynamic resonance nonradiative excitation energy transfer (trFRET), which can yield distributions of conformers in ensembles of partially folded protein molecules was applied. Two model proteins were studied. E. Coli adenylate kinase (AK) (a large globular protein) and the B domain of protein A (a small fast folding single domain) were studied. For both protein the analysis of trFRET monitored chemically induced unfolding/refolding transition yielded a clear evidence for the presence of two distinct sub-population at the transition conditions. One sub- population was native like and the other was unfolded. The proportion of the size of the two sub-populations was varied as function of the concentration of the denaturant. These experiments yielded solid evidence in support the model of cooperative, barrier crossing, mechanism of folding of both the large and the small model proteins. At least for these two cases, the model of downhill mechanism of folding is not applicable. AMMONIA SECRETION - A DUAL PARASITE AND HOST SIGNAL

N. Alkan, R. Fluhr, D. Prusky

Department of Postharvest Science, Agricultural Research Organization, Bet-Dagan and Department of Plant Science, Weizmannn Institute of Science, Rehovot, Israel

Secretion of ammonia is known to be associated with virulence of the pathogenic fungi Colletotrichum coccodes. Consistent with this, we show that nit- mutants that are compromised in ammonia secretion are less virulent. While ammonia causes alkalinization and a subsequent increase in fungal pathogenesis factors, we examined if ammonia can also directly contribute to host-pathogen interactions. Pre-treatment of the infection court with low pH was found to elevate total ammonia secretion and increase aggressiveness in comparison to pre-treatment with high pH. This indicates that the effect of ammonia secretion goes beyond direct pH control. Indeed, we demonstrate that ammonia activates the tomato host membranal NADPH oxidase (Rboh) and stimulates ROS accumulation. Direct application of ammonia to plant tissue without infection leads to membrane leakage and local host cell death. The results suggest that activation of the membranal Rboh by ammonia is important for fungal virulence. Indeed, tomato Rboh mutants produce less ROS and demonstrate less ammonia-induced ion leakage and less cell death. As necrotrophic fungus prospers by host cell death, the subversion of Rboh activity by the fungus through ammonia secretion is a novel virulence strategy. This work demonstrates that Colletotrichum coccodes uses ammonia secretion as a novel dual-edged strategy; optimizing the infection court pH which is conducive to fungal virulence gene activation and at the same time stimulating host ROS production and local cell death. INVOLVEMENT OF TYPE 4 PILI IN PATHOGENICITY AND BIOFILM FORMATION OF ACIDOVORAX AVENAE SUBSP. CITRULLI

O. Bahar, T. Goffer, S. Burdman

Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel

The Gram-negative bacterium Acidovorax avenae subsp. citrulli (Aac) is the causal agent of bacterial fruit blotch (BFB), a destructive disease of cucurbits. BFB has gained importance since the late 1980’s, after several devastating outbreaks in the US. Since then, the disease spread to many parts of the world including the Americas, Asia, Europe, Middle and Far East and Australia. To date, little is known about basic aspects of the biology of the pathogen and of the interactions with its host plants. To improve our knowledge on these aspects, we generated a transposon mutant library on the background of M6, an Aac wild type strain isolated from melon. Mutants are being screened for reduced or enhanced virulence on melon, and several genes, likely contributing to bacterial virulence, have been identified so far. Here we report one of these genes, pilM, which encodes a type 4 pili (TFP) assembly protein. Bacterial TFP are involved in twitching motility, surface adherence, biofilm formation and genetic material uptake. TFP also play a role in virulence of several pathogenic bacteria. Indeed, the Aac pilM mutant was shown to possess a significantly reduced virulence as compared to the wild type strain in seed transmission assays as well as in seedling inoculation. Microscopy observations showed that, in contrast to the wild type, the pilM mutant does not exhibit twitching motility. Biofilm formation assays also revealed an impaired ability of the mutant to form biofilms on different surfaces. We are currently studying the role of TFP in Aac pathogenicity. To date, a role of TFP in virulence of plant pathogenic bacteria has been demonstrated only for vascular pathogens (eg., those possessing the ability to colonize and spread via the plant xylem vessels). Evidence suggests Aac does not belong to this class of bacterial phytopathogens. REGULATION OF THE EFFECTOR PROTEIN AvrA OF SALMONELLA ENTERICA

Z. Ben-Barak Zelas1, W. Streckel2, H. Tschaepe2, S. Yaron1

1Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel, 2National Reference Centre for Salmonellae and Other Enterics, Robert Koch Institute, Wernigerode, Germany

The effector protein AvrA of Salmonella enterica contributes to the complexity of the virulence network of Salmonella. AvrA is a cysteine protease, which turns the inflammatory response of the host by inhibition of the production of IL8 and helps to induce apoptosis. The avrA gene is prevalent in most S. enterica serovars, but absent in several serovars such as Typhi, which usually cause severe systemic disease. Comparsion between the avrA sequence and expression in different Salmonella serovars revealed that the avrA sequence is highly conserved. The transcription of avrA was constitutive and its mRNA levels were very similar in all the Salmonella serovars. In contrast, the expression of AvrA took place either constitutively or after acid induction only or not at all in different serovars. Cloning of avrA with its promoter on low copy plasmids gave rise to a strong AvrA production in all tested S. enterica serovars. This was found to be in contrast to Escherichia coli which transcribed the plasmidial avrA gene but failed to produce AvrA. These results demonstrated that the control of AvrA expression is due to post-transcriptional regulation, probably controlled by a factor, which might be absent in E. coli. Using 5'- rapid amplification of cDNA ends (5'-RACE) strategy and by creation of nested deletions, we mapped the promoter region of avrA. We defined the minimal regulatory region essential for expression using gfp as a reporter gene and determined the transcriptional start site which located 55 bases upstream of the translation start point that was identified by Edman-sequencing. The untranslated region in the transcript contains 3 suspected binding motifs of the RNA binding protein CsrA. CsrA is part of a global regulatory system Csr which control numerous genes post- transcriptionally. csrA deletion mutants did not allow the translation of chromosomal as well as of plasmid encoded AvrA. This may imply that CsrA also influences the AvrA production. TLR2 SIGNALING IN PERIODONTAL DISEASE

E. Burns, G. Nussbaum

Institute of Dental Sciences, Faculty of Dental Medicine, Hadassah Medical center, Hebrew University of Jerusalem, Israel

The tissue destruction and alveolar bone loss characteristic of periodontal disease is the result of both the presence of oral pathogens and an exaggerated host immune response. Surface components (e.g. LPS) of Porphyromonas gingivalis (Pg), a major perio-pathogen, interact with host expressed toll-like receptors (TLRs), key control elements in the innate immune response. TLR activation leads to expression of inflammation related genes by signaling through adaptor proteins (usually MyD88) and the involvement of specific transcription factors (mainly NFkappaB). Isolated components of Pg have been demonstrated to activate TLR2 and TLR4 on a variety of cell types. In our study we determine the importance of TLR2, TLR4 and MyD88 in recognition of live Pg and development and progression of periodontitis. Pg infection in subcutaneous chambers yielded high amounts of pro-inflammatory cytokines in WT and TLR4del mice in contrast to moderate-to-absent cytokines levels in TLR2-/- mice. Surprisingly, the moderate response found in TLR2-/- mice correlated with efficient bacterial clearance while the robust response found in WT and TLR4del mice correlated with bacterial survival. Injection of FITC-labeled bacteria revealed that TLR2-/- mice efficiently phagocytose Pg in comparison to significantly reduced phagocytosis observed in WT and TLR4del mice. Next, we conducted experiments using MyD88-/- mice to help determine the signaling pathways involved after Pg recognition by TLR2. Results of MyD88-/- mice resemble those of WT mice i.e. a robust pro-inflammatory cytokine response and high bacterial survival. Results therefore suggest existence of a TLR2-dependent MyD88-independent pathway triggered by Pg. Oral infection of WT, TLR4del and MyD88-/- mice with live Pg resulted in significant alveolar bone loss compared to control mice treated with vehicle alone. TLR2-/- mice were resistant to bone loss, further supporting the existence of a TLR2-dependent MyD88-independent signaling pathway activated by Pg infection. PLANT-INSECT COMMUNICATIONS VIA VOLATILE COMPOUNDS AND THEIR GLUCOSIDES

A. Chernyahovsky, O. Shoseyov

Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel

Developing safe, sustainable crop protection is a top priority for modern agriculture all over the world. The best way to reduce chemical contamination (and the harm it causes) in our environment is to use environmental-safe pest control methods. Wild plants have developed means to survive the attack of herbivores or plant pathogens in the course of evolution in part due to their ability to communicate (attract or repeal) with their environment via volatile signals. In this research we hypothesize that cultured plants have lost some of these traits in the course of the breeding. However these inheritable traits may still be possessed in the wild ancestors of cultured plants. In the laboratory of Shoseyov it was shown in the past by that the over expression of the A.niger beta-glucosidase gene (BGL1) in the different plant cell compounds can enhance the emission of many volatile compounds with probable insecticidal activity (Shu W.et. al., 2004). Whiteflies (Heteroptera) and thrips (Thysanoptera) are the key pest of many field and horticultural crops all over the world. They are known as one of the most damaging insects in world agriculture. Moreover, whiteflies and thrips are a primary vector for many viral plant diseases, which cause noticeable damage to crops. In our research we are testing the following hypotheses. Plants in the wild and some commercial varieties store glycosides and beta-glycosidase in different subcellular compartments which upon cell disruption release volatiles that may attract or repeal relevant insects. Composition analysis of glycosides of volatile compounds of certain wild type and transgenic plants may reveal important volatile compounds that may serve as efficient attractant or repellents of major insects. PNEUMOCOCCAL CAPSULAR POLYSACCHARIDE IS IMMUNOGENIC WHEN PRESENT ON THE SURFACE OF MACROPHAGES/DENDRITIC CELLS: TLR-4 SIGNALING INDUCED BY A CONJUGATE VACCINE OR BY LPS IS REQUIRED

N. Cohen1, M. Stolarsky-Bennun1, H. Amir-Kroll1, R. Margalit1, G. Nussbaum2, M. Cohen-Sfady1, M. Pevsner-Fischer1, M. Fridkin3, H. Bercovier4, L. Eisenbach1, S. Jung1, I.R. Cohen1

1Department of Immunology, Weizmann Institute of Science, Rehovot, Israel, 2Hadassah School of Dental Medicine, Hebrew University of Jerusalem, Israel, 3Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel, 4Hadassah Medical School, Hebrew University of Jerusalem, Ein Karem, Jerusalem, Israel

Previously, we reported that a peptide, p458, from the sequence of the mammalian 60 KD heat shock protein (HSP60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR-4- dependent manner; other PS4 conjugates, or PS4 or peptide alone did not do so. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Pn challenge. The TLR-4 ligand LPS could also induce the late appearance on the surface of un-conjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4, or by macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone-marrow derived dendritic cells activated by LPS and pulsed with un-conjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by a cellular vaccine was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM antibodies. Thus, activation of TLR-4 on APC can lead to the appearance on the cell surface of polysaccharide antigens immunogenic for IgG. THE CELL CYCLE OF BDELLOVIBRIO BACTERIOVORUS, A PROKARYOTIC PARASITE – PREDATOR OF BACTERIA: A PROTEOMIC ANALYSIS

M. Dori-Bachash1, B. Dassa2 S. Pietrokovski2, E. Jurkevitch1

1Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, 2Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Bdellovibrio and like organisms exhibit a simplified model of parasitic / predatory interaction between prokaryotes. They are phylogenetically diverse and ubiquitously found in the environment. B. bacteriovorus exhibits a dimorphic life-cycle comprising an attack phase cell that penetrates and settles within the periplasm of a gram-negative prey. There, it develops into a filament, within which DNA replication occurs. Subsequently, the cell splits into progeny attack cells that burst the host envelope. Therefore, these bacteria can constitute a simplified model of differentiation, as well as of predatory interactions. Spontaneous host-independent (HI) mutants are facultative predators. They can be grown in the presence of prey but also axenically, in a prey-free system, and still exhibit a complete dimorphic life-cycle. We use proteomic analysis to track protein expression during the bacterium's growth cycle. Proteins were extracted from wild-type (wt) and HI mutant attack phase cells, and from the filamentous and replication growth phases of a synchronized HI culture at three time points. They were analyzed by differential 2DE and mass spectrometry. In addition, an LC-MS/MS analysis of the excreted complement was performed revealing many hydrolytic enzymes and excreted hitherto unidentified proteins ("ORFans"). About 745 proteins (21% of the total estimated proteome) were detected. Approximately 140 spots were differentially expressed during the HI strain’s life- cycle and about 40 between wt and HI attack phase cells. The former included proteins involved in cell cycle control, transcription, translation, signal transduction, cell motility, chaperones and other functions, as well as many "ORFans“. Additional functions were found by identifying operons, and co-transcription was further studied by RT-PCR. In some cases post-translational modifications were extensive, like in a protein containing a bacterial intein-like domain. Its auto-catalytic properties were further confirmed in-vitro. This study provides a first large-scale understanding of the cellular processes occurring in a dimorphic obligate predatory bacterium. INVOLVEMENT OF THE MOUSE NKp46 RECEPTOR (mNCR1) IN STREPTOCOCCUS PNEUMONIA INFECTION

S. Elhaik, D. Kafka, Y. Mizrahi, A. Porgador

Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer Sheva, Israel

Natural killer cells are involved in the innate immune response against pathogens. NK cells use two groups of receptors: inhibitory and activating. The major group of activating receptors is the natural killer cytotoxicity receptors (NCRs) including NKp30, NKp44, and NKp46. In mice, only the NKp46 receptor (mNCR1) has been identified. There are increasing evidences of detrimental roles for NK cells and IFNƒn gamma in streptococcus pneumonia infections but their exact role is not yet defined. Streptococcus pneumonia is a member of the gram-positive bacteria family. Gram- positive bacteria accounts for 30% to 50% of sepsis cases, a syndrome that causes 1400 deaths world wide each day. Following Streptococcus pneumonia infection functional reciprocal relationship between Dendritic cells (DCs) and NK cells occur. We explored the involvement of mNKp46 in NK cells activation following streptococcus pneumonia challenge using C57BL/6 mice that are knock-out for the NKp46 receptor (mNCR1). We found that co-incubation of streptococcus pneumonia infected WT BMDCs (bone marrow DC) with NK cells from WT and mNKp46 KO mice resulted in higher secretion of IFN gamma and expression of CD107 in the WT mice. Accordingly, infection of WT and mNKp46 KO mice with streptococcus pneumonia for 3 and 6 hours resulted in lower bacterial load in the WT mice. In addition, histological analysis of lung tissue from streptococcus pneumonia infected mice after 48 hours, demonstrated an acute pneumonia inflammation in mNKp46 KO mice as opposed to interstitial chronic inflammation in WT mice. These results suggest that mNKp46 plays a role in the interaction between NK cells and DCs during early infection of streptococcus pneumonia. This interaction results in secretion of IFN gamma which might mediate the type of inflammation developed in infected mice. REGULATION OF RELEASE OF ANTIBACTERIALS FROM STRESSED SCLERACTINIAN CORALS

Y. Geffen, E.Z. Ron, E. Rosenberg

Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Recently, we showed that mechanical stress on scleractinian corals caused a rapid release of antibacterial material (referred to as coral antibacterial activity, or CAA) which killed various bacterial species, including the coral pathogen Vibrio coralliilyticus (Geffen and Rosenberg 2005). We now report on studies on the regulation of CAA release from stressed scleractinian corals. Corals can repeatedly release highly active CAA as a result of sequential stress inductions. Fragments of Pocillopora damicornis and Stylophora pistillata were transferred 19 times from one beaker into another with a stress induction each time after 10 min. Both corals showed a decrease in the level of antibacterial activity released during the first 4-5 transfers. After the fifth transfer, the corals kept releasing CAA for the rest of the experiment with no significant decrease. When CAA was released into a large volume of sea water, it was accumulated until a maximal level was reached. Apparently, the release of CAA is down regulated by feedback inhibition, depending upon the concentration of CAA in the surrounding water. CAA was bound irreversibly to bacterial cells in a stoichiometric manner. Approximately 4x102 bacterial cells were sufficient to bound 1 unit of CAA. It was also found that the ability of corals to repeatedly release CAA was affected by changing the location of the point of induction on the coral surface. CAA release was obtained from corals after removal of the mucus layer, and the mucus itself contained antibacterial activity. It is suggested that scleractinian corals regulate CAA release in response to sequential injuries, such as the bite of a predator, and the release is localized to the site of the wound. VIRULENCE FOR MICE OF THREE PLAQUE-ISOLATES OF THE LISTER STRAIN OF VACCINIA VIRUS AND THE PROTECTION AGAINST AN ORTHOPOX VIRUS INDUCED BY THEM

I. Gurt, E. Katz

Department of Virology, Hebrew University - Hadassah Medical School, Jerusalem, Israel

Three plaques of the Lister strain of vaccinia virus were isolated, based on their size in BS-C-1 cells: large plaque (L), medium plaque (M) and small plaque (S). They differ in their virulence to mice when inoculated either intranasally or intracerebrally. Intranasal infection with the S variant revealed that it was more virulent than the other two variants, while the parent virus, the Lister strain of vaccinia virus, was the most virulent of all four viruses, as judged by the loss of weight of the mice following infection. Intracerebral injection revealed that the S variant was more virulent than the parent virus and the L and M variants; the latter was the most attenuated of all. The titer of the virus in the mouse brain, when the mice reached their critical weight (loss of 25% of initial weight) was high for S and L, and significantly lower for the parent virus. The three variants, when infecting mice intranasally, induced high levels of protection against a lethal orthopox virus. THE INFLUENCE OF SALICYLIC ACID ON THE ACTIVITY OF CIPROFLOXACIN AGAINST EXTRACELLULAR AND INTRACELLULAR SALMONELLA TYPHIMURIUM

E. Hartog, D. Shachar, L. Ben Shalom, S. Yaron

Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel

Salmonella enterica serovar Typhimurium is food-borne pathogenic bacteria which is able to invade, survive, and replicate within the macrophages in the host. Salicylic acid (SAL), an active component of aspirin, is frequently used in medical applications. The primary goal of our research was to examine the influence of SAL on the activity of ciprofloxacin, the antibiotic of choice to treat salmonellosis, against extracellular and intracellular Salmonella. Viable counts showed that intracellular bacteria (in J774 macrophages) were less susceptible to ciprofloxacin than extracellular bacteria. However the tolerance of extracellular Salmonella to ciprofloxacin was enhanced significantly after exposure to SAL, in contrast to the tolerance of intracellular bacteria that slightly reduced. In order to investigate the causes of this observation, we studied the influence of SAL on the expression of the marA, acrAB and micF genes. In Escherichia coli and Salmonella the MarA confers a low level of resistance to different antimicrobials, including ciprofloxacin. The MarA dependent resistance is mediated mainly through the activation of AcrAB efflux pumps and the reduction of influx by repression of micF. To examine the transcriptional activation of these genes after exposure to ciprofloxacin and SAL, we used two methods: reporter plasmids, in which GFP is under the control of one of the promoters of these genes, and qRT-PCR. The distribution of the expression was analyzed by flow cytometry. Results showed that SAL but not ciprofloxacin induced the marA, micF and acrAB genes in extracellular and intracellular Salmonella. Induction of AcrAB by SAL may explain the enhanced tolerance to ciprofloxacin in extracellular bacteria. This hypothesis was reinforced by using the efflux pump inhibitor Phe-Arg-beta-naphthylamide, which diminished the tolerance enhancement in SAL-induced extracellular salmonella. Interestingly, the enhanced tolerance to ciprofloxacin obtained only with extracellular Salmonella, although the induction of marA, micF and acrAB by SAL was observed in intracellular Salmonella as well. This difference probably relies on the high expression levels of acrAB in intracellular Salmonella even without induction. INDUCED RESISTANCE RESPONSE TOWARDS THE SOFT ROT BACTERIUM PECTOBACTERIUM CAROTOVORUM IN THE ORNAMENTAL MONOCOT CALLA LILY INVOLVE PRIMING OF ANTIMICROBIAL PHENOLICS

T. Luzzatto-Knaan1,2, A. Golan1,2, M. Yishay1,2, I. Bilkis2, A. Ion1, A. Lipsky1, I. Yedidia1

1Department of Ornamental Horticulture, ARO, The Volcani Center, Bet-Dagan, Israel, 2Department of Biochemistry and Food Sciences, Hebrew University of Jerusalem, Rehovot, Israel

Calla lilies are herbaceous monocotyledonous plants highly sensitive to Pectobacterium carotovorum, the causal agent of soft-rot disease. Two plant defense activators, Benzothiadiazole (BTH-Bion®), acting through the salicylic acid pathway, and methyl jasmonate, involving the jasmonate-dependent signaling pathway, differed in both their capacity to induce accumulation of polyphenols, and resistance against the pathogen. While Bion afforded protection only during the first 24 h, methyl jasmonate, completely inhibited P. carotovorum development in calla lily leaves presenting long lasting effect. Methyl jasmonate elicitation brought about higher accumulation of free phenolics and polyphenol oxidase (PPO) activity, relative to Bion, suggesting priming of bioactive polyphenols as a principal factor in calla lily defense against P. carotovorum. Results demonstrate enhanced biological activity against the necrotrophic P. carotovorum only in the methyl jasmonate induced plants. The polyphenolic nature of the induced compounds was supported by auto- fluorescence, absorbance spectra, and reaction with Folin-Ciocalteu reagent. To further characterize the nature of induced compounds, two major compounds were collected and identified as c-glycosylflavonoids, swertisin and isovitexin by mass and NMR spectroscopies.

The Israeli Chief Scientist grant No. 256-0717 ANTIBODIES AGAINST V ANTIGEN OF Y. PESTIS AND PROTECTIVE IMMUNITY AGAINST PLAGUE: NEUTRALIZATION OF MACROPHAGE CYTOTOXICITY AS EFFECTIVE ASSAY FOR PREDICTING IN-VIVO SURVIVAL

A. Zauberman1, S. Cohen1, Y. Levy1, G. Halperin2, S. Lazar1, B. Velan1, A. Shafferman1, Y. Flashner1, E. Mamroud1

1Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel, 2Department of Biotechnology, Israel Institute for Biological Research, Ness-Ziona, Israel

Infection by Y. pestis, the etiological agent of plague, occurs by intra-dermal fleabites or inhalation, leading to fatal bubonic or pneumonic plague. Various vaccines against plague have been developed during the last decade and the most promising of them include the immunogenic virulence factor V antigen (LcrV). LcrV is a multifunctional protein, part of the Type III secretion system (TTSS), which plays a crucial role in the process of effectors translocation into host cells. We developed a highly sensitive in- vitro assay that allows correlating the in-vitro serum anti-LcrV neutralizing activity with protective immunity against Y. pestis in a mouse model. This assay is based on inhibition of macrophage cytotoxicity mediated by Y. pestis, using a recombinant Y. pestis strain, expressing Y. enterocolitica yopP, thereby enhancing the limited ability of Y. pestis to cause macrophage cell death. Mice passively immunized with rabbit anti-LcrV IgG preparations or actively immunized against recombinant LcrV were protected against lethal doses of virulent Y. pestis strain in a mouse model of bubonic plague. In all passive and active immunization regimes, protection and survival of mice demonstrated clear-cut correlation to the in-vitro antibodies neutralizing activity, but not to their ELISA titers. Focusing on mouse survival, a cutoff value for serum neutralizing activity could be calculated for each vaccination regime, and activities above this level unexceptionally predicted survival. The reliable prediction of survival offered by this assay system provides a useful method for appraisal of vaccination efficacy. THE INVOLVEMENT OF A TOMATO MAPKKK IN PLANT IMMUNITY

S. Melech-Bonfil, G. Sessa

Department of Plant Sciences, Tel-Aviv University, Ramat Aviv, Israel

Resistant plants have evolved the capability to protect themselves from disease- causing organisms by activating a wide array of defense responses. In our lab, we investigate resistance of tomato (Solanum lycopersicum) to two different Gram- negative bacteria: Xanthomonas campestris pv. vesicatoria (Xcv) and Pseudomonas syringe pv. tomato (Pst), which are the causal agents of spot and speck diseases, respectively. These pathogens colonize the aerial parts of the plant and cause economically important loss to tomato yield worldwide. Control of speck and spot diseases by cultural practices or chemicals is not effective and genetic sources of resistance are only limited to certain Pst and Xcv strains. To isolate genes involved in these resistance responses, we used a functional screen based on virus-induced gene silencing (VIGS). Silencing of a MAP kinase kinase kinase, SlMAPKKK, compromised resistance to both bacterial pathogens and its overexpression elicited a pathogen-independent cell death. In addition, by silencing SlMAPKKK in N. benthamiana plants, we found that this gene is a key regulator of cell death mediated by the specific Pto and AvrPto interaction. Moreover, we found that this gene is specifically involved in the development of HR activated by paires of resistance genes and avirulence genes form tomato and the leaf mold fungus Cladosporium fulvum, respectively. We also tested the hypothesis that SlMAPKKK might play a role in disease susceptibility. Using VIGS, we showed that SlMAPKKK kinase did not affect the bacterial growth in susceptible plants and it is not essential for disease susceptibility. These results reveal a role for SlMAPKKK gene in plant disease resistance and in the elicitation of cell death. REAL TIME ANALYSIS OF EFFECTOR TRANSLOCATION BY THE TYPE III SECRETION SYSTEM OF ENTEROPATHOGENIC E. COLI

E. Mills, K. Baruch, X. Charpantier, S. Kobi, I. Rosenshine

Department of Molecular Genetics and Biotechnology, Faculty of Medicine, Hebrew University of Jerusalem, Israel

Pathogenic bacteria use type III secretion systems (TTSS) to translocate effector proteins into host cells. We designed a real time, high-throughput translocation assay and used it to analyze simultaneously and quantitatively the translocation kinetics of the six core-effectors of enteropathogenic E. coli, namely EspF, EspG, EspH, EspZ, Map and Tir. We observed a distinct order in the efficiencies of effector translocation and show that translocation efficiency is determined by multiple factors. These include the intrabacterial effector concentration, effector-chaperone interactions, the efficiency of bacterial attachment to the host cells, and possibly also by translocation autoinhibition. STUDYING THE INTERACTION OF T-20 WITH GP120 BY NMR

A. Moseri, J. Anglister

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

The HIV envelope spike is formed as a complex between gp120 and gp41. The gp120 mediates attachment of the virus to the target cell, whereas gp41 is required for the fusion of virus and target cell membranes. The gp41 experiences a structural re- organization that enables the interaction between the heptad repeat region HR1 and HR2 (corresponding to the N and C terminus respectively) forming a thermo-stable, six helix bundle leading to viral fusion. T-20 is an HR2 based 36 amino acids peptide and is already FDA approved fusion inhibitor. The mechanism by which T-20 acts is still under investigation. While it is widely accepted that the N-terminus of T-20 interacts with segment in HR1 there is no direct evidence for similar interactions for its C-terminus, as this region is not included in the gp41 crystal structures. Accumulating experimental data suggest additional targets for T-20 on gp120. Several studies had demonstrated that T-20 directly interacts with gp120; this interaction is enhanced by CD4 binding and mapped to the C4 and V3 segments of gp120 which also form the co-receptor binding site. This data indicates the T-20 may act in a different manner then other HR2 peptides and targets multiple sites on gp120 and gp41 to prevent viral fusion. We have establish an expression system in E-coli for a 64 amino acid peptides which include the C4 and V3 segments of gp120 MN strain (C4V3-MN) and for T-20 enabling isotope labeling for NMR studies. We are studying the interaction between T-20 and C4-V3 segment by NMR. Our data my provide information on T-20 mode of action, resistance mutation and the functionality of T-20 C-terminus in the viral spike. DIFFERENTIAL GENE EXPRESSION OF RECOVERY IN THE ENTOMOPATHOGENIC NEMATODE HETERORHABDITIS BACTERIOPHORA

A. Moshayov1, H. Koltai2, I. Glazer1

1Department of Entomology, Nematology Division, ARO - Agricultural Research Organization, Volcani Center, Bet Dagan, Israel, 2Department of Ornamental Horticulture, ARO - Agricultural Research Organization, Volcani Center, Bet Dagan, Israel

Nematodes of the genus Heterorhabditis are insect parasites that are widely used as biological control agents. In Heterorhabditis bacteriophora the free-living, third juvenile stage that is well adapted to long-term survival in the soil and it is the infective stage. When a suitable host is infected, the infective juvenile (IJ) recovers from developmental arrest and resumes growth and development. Recovery is a critical step in the parasite-host interaction and is a part of the host recognition by the parasite. Recovery of entomopathogenic nematodes is a very important process from a commercial point of view. To characterize the process of recovery in H. bacteriophora we aimed to isolate genes involved in this process. For this purpose, we constructed subtraction library of recovering IJs subtracted by arrest IJs. Two hundreds twenty expressed sequence tags (ESTs) were sequenced and annotated resulting with 109 useful ESTs that were categorized into functional categories according to Kyoto Encyclopedia of Genes and Genomes. Most of ESTs (38%) belongs to the metabolism category, which shows that different metabolic pathways are highly active in the recovering IJs. Genes of several functional groups were identified. These included protease, dauer pathway genes (daf-4 & daf-16), heat shock genes (hsp-20, hsp-40 and hsp-16.1), and signaling proteins like GTP binding proteins (tag-210 & tag-308). This system can help us understand parasitism, mutualism and vector born disease in other systems like mammalians parasitic nematodes. ISOLATION AND CHARACTERIZATION OF THE Omp-PA PORIN FROM PORPHYROMONAS ASACCHAROLYTICA AND ITS ROLE IN PATHOGENICITY

Y. Nitzan1, L. Magalashvili1, I. Pechatnikov1, H. Wexler2

1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel, 2Veterans Administration Wadsworth Medical Center, Greater Los Angeles Health Services, Los Angeles, CA, USA

Porphyromonas asaccharolytica is a Gram-negative anaerobic bacterium which is resistant to a large number of the beta lactam antibiotics. A single monomeric porin, Omp-PA (37kDa), was isolated from the outer membrane of P. asaccharolytica. Further characterization revealed that Omp-PA consists of two fractions: a heat- modifiable fraction which in its denatured form migrated on SDS-PAGE as a protein with molecular weight of 41kDa and a heat-resistant fraction which did not change its migration on SDS-PAGE after boiling. Liposome swelling assay revealed that only the heat-resistant fraction was able to transport sugars after its incorporation into the liposomes, although it did not discriminate between differently sized sugars. We hypothesize that the heat-modifiable fraction corresponds to the "closed" conformer of Omp-PA, whereas the heat-resistant fraction corresponds to the "open" conformer of the protein. Cloning of the omp-PA gene revealed open reading frame of 1098 bases encoding a protein of 366 amino acids. The C-terminal domain of Omp-PA is homologous to the characteristic OmpA signature domain (71% similarity). Alignment with other anaerobes from the Bacteroides family demonstrated homology across the entire ORF. Secondary structure prediction indicated that the “open” conformation contains 12 transmembranal beta-strands, whereas the “closed” conformation spans the outer membrane 8 times with amphiphilic beta-strands. In addition, we have investigated the ability of the Omp-PA to regulate the induction of several pro-inflammatory and immunoregulatory cytokines by murine splenocytes in vitro. Our findings suggest that Omp-PA affects the release of IL-1alpha, TNF-alpha, IFN-gamma, IL-6 and IL-10 in a dose-dependent fashion. THE C-TERMINAL SEGMENT OF T-20 CONTRIBUTES TO THE STABILIZATION OF THE SIX-HELIX BUNDLE FORMED WITH MATCHING gp41 N-PEPTIDE

E. Noah, J. Anglister

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

Hiv-1, like other enveloped viruses utilizes a protein embedded in its membrane, termed envelope protein (ENV), to facilitate the fusion process. The ENV glycoprotein is organized as trimers on the membrane of the virus and is composed of two non-covalently associated subunits. The surface subunit (SU), gp120, mediates host tropism, whereas the transmembrane subunit (TM), gp41, is responsible for the actual fusion event. The extracellular part of gp41, is composed of several functional regions, including the fusion peptide (FP), the N-terminal heptad repeat (NHR), the C-terminal heptad repeat (CHR), and the membrane proximal external region (MPER) domain. Since the revelation of gp41’s post fusogenic conformation, the structure and function of the protease-resistant six helix bundle core has gained much focus. In this work, we describe additional stability determinants between the membrane proximal region and the fusion peptide proximal region of gp41. This information sheds new light on the activity mechanism of Fuzeon, the only FDA approved HIV-1 fusion inhibitor, and warrant more detailed characterization of MPER interactions. Our data may enable a better understanding of the structure-function relationship of gp41 and its role in HIV-1 fusion. INTERACTION WITHIN HIV-1 V3 REGION - IMPLICATION FOR PHENOTYPE CONVERSION

O. Rosen, A. Samson, J. Anglister

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

HIV-1 co-receptor usage plays a critical role in virus tropism and pathogenesis. A switch from CCR5- to CXCR4-using viruses occurs during the course of HIV-1 infection and correlates with subsequent disease progression. A single mutation at position 322 within the V3 loop of the HIV-1 envelope glycoprotein gp120, from a negatively to a positively charged residue, was found to be sufficient to switch an R5 virus to an X4 virus. In this study, the NMR structure of the V3 region of an R5 strain, HIV-1JR-FL, in complex with an HIV-1 neutralizing antibody was determined. Two oppositely charged residues at positions 304 and 322, were found to be juxtaposed in the beta-hairpin structure, enabling a favorable electrostatic interaction that stabilizes the postulated R5 conformation. Comparison of the R5 conformation with the postulated X4 conformation of the V3 region (positively charged residue at position 322), reveals that electrostatic repulsion between residues 304 and 322 in X4 strains triggers the observed one register shift in the N-terminal strand of the V3 region. We posit that electrostatic interactions at the base of the V3 beta-hairpin can modulate the conformation and thereby influence the phenotype switch. In addition, we suggest that inter-strand cation-pi interactions between positively charged and aromatic residues induce the switch to the X4 conformation as a result of the S306R mutation. The existence of three pairs of identical (or very similar) amino acids in the V3 C-terminal strand facilitates the switch between the R5 and X4 conformations. BYPASSING Pto KINASE ACTIVITY: INSIGHTS TO THE RECOGNITION OF PSEUDOMONAS EFFECTORS

D. Salomon1, A. Bonshtien1, C. Zhang2, M. Mayrose1, K. Shokat2, G. Sessa1

1Department of Plant Sciences, Tel-Aviv University, Ramat Aviv, Israel, 2Department of Cellular and Molecular Pharmacology, University of California - San Francisco, CA, USA

Pseudomonas syringae pv. tomato (Pst) is the causative agent of bacterial speck disease in tomato. The molecular basis of tomato resistance to speck disease resides in the specific recognition by the plant surveillance system of either of the two Pst effectors AvrPto and AvrPtoB. This recognition is mediated by the physical interaction of AvrPto or AvrPtoB with the resistance protein Pto, a serine/threonine protein kinase. To examine the role of Pto kinase activity in effector recognition, we used a recently developed chemical-genetic approach and sensitized Pto to specific kinase inhibitors. The sensitized Pto was functional in the elicitation of the hypersensitive response and able to bind AvrPto and AvrPtoB in the yeast two-hybrid system, albeit with lower efficiency as compared to wild-type Pto. Surprisingly, addition of specific kinase inhibitors increased the binding ability of sensitized Pto back to that of wild-type Pto. This increase was independent of Pto kinase activity since it was also observed for kinase-deficient forms of the sensitized Pto. These results indicate that neither Pto autophosphorylation, nor its phospho-transfer activity, are required for Pto interaction with AvrPto and AvrPtoB. Furthermore, they suggest that occupancy of the Pto ATP-binding site by a ligand is the prerequisite for these interactions. Additional experiments revealed that binding of ATP to kinase deficient forms of Pto is not sufficient to confer to the protein the ability to interact with effectors. We thus hypothesize that Pto ATP-hydrolase activity and the occupancy of the ATP-binding site by ADP are required for Pto binding of AvrPto and AvrPtoB. CHARACTERIZATION OF THE pHD1 PLASMID OF SALMONELLA ENTERICA SEROVAR VIRCHOW

D.B. Schlisselberg, H. Solnik-Isaac, S. Yaron

Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel

Salmonella enterica serovar Virchow is highly prevalent in humans and farm animals in Israel. Recently we have detected a remarkably high incidence of resistance of S. Virchow isolates from human and poultry origin to multiple antibiotics. In frame of an extensive study aimed to elucidate mechanisms of resistance of S. Virchow to different antibiotics, we identified a plasmid (pHD1) that was correlated with resistance to ampicilin. The plasmid was isolated and sequenced. Sequence analysis of the ~8500bp of the plasmid revealed that it contains three open reading frames (ORFs) with homology to known proteins. ORF1 showed very high homology (99% identity) to blaTEM from Escherichia coli, a gene that encodes to beta-lactamase and confers ampicilin resistance. ORF2 was homolog to the LasA from Vibrio cholerae (72% identity). Proteins of the LasA family are extracellular proteases. They possess both elastolytic and staphylolytic (hydrolysis of pentaglycine cross-links in the cell wall peptidoglycan) activities. LasA had never been identified in Salmonella enterica plasmids or genomes. Between both genes a Tn3 transopsone was identified. A third ORF was similar to a Rom-like protein from Escherichia coli (96% identity), a protein which is involved in determination of plasmid copy number. Screening for the presence of pHD1 in 90 S. Virchow isolates from humans and poultry revealed 14 positive isolates, most of them were resistant to ampicilin. Transformation of this plasmid to E. coli DH5alpha was obtained in order to approve its ampicilin resistance. Furthermore, we found a high conjugation ratio to non-ampicilin resistant GFP expressing S.Virchow. The pHD1 plasmid is probably a conjugative small plasmid that harbors virulence and resistance genes. Further study is undertaken in order to study the contribution of the pHD1 plasmid to the virulence properties of Salmonella. ADHESION AND COLONIZATION OF SALMONELLA TYPHIMURIUM ON PLANTS – THE ROLE OF BACTERIAL CELLULOSE

N. Shirron, A. Lapidot, S. Yaron

Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel

Fresh fruits and vegetables are increasingly recognized as a significant source of food- borne pathogens associated with diseases outbreaks. The current prevention and disinfection methods are ineffective, and novel effective methods are not available. Salmonella enterica serovars are among the most commonly isolated human pathogens associated with plants. The objective of this research was to investigate the role of the bacterial cellulose in the plant-bacteria interactions. By using a GFP labelled Salmonella Typhimurium we demonstrated the ability of S. Typhimurium wild type (WT) to penetrate parsley after drip-irrigation with contaminated water as compare to the cellulose deficient mutant. Salmonella foci were found in the soil and upper parts of treated plants even 21 days after irrigation. In contrast, the cellulose deficient mutant accumulated mainly in the soil and colonized the plant at significantly lower concentrations than the WT. To reinforce the hypothesis that cellulose produced by Salmonella is important for the adhesion to the plant, we compared the cellulose deficient mutant with the WT and with the non-pathogenic Escherichia coli K12 for their ability to adhere to Arabidopsis thaliana plant cells (AT-202) and tobacco BY-2 cell lines. After incubation for 24 h and washings, confocal laser scanning microscopy (CLSM) images illustrated specific attachment of S. enterica to the plant cell wall. The attachment was dependent on synthesis of bacterial cellulose, but not on biofilm formation, since it was not affected with the curli encoding mutants (curli deficient mutant does not form biofilm). Furthermore the amount of WT S. Typhimurium attached to plant cells was approximately 3 orders of magnitude than the cellulose deficient mutant. These results place the bacterial cellulose as an important factor in adhesion of S. Typhimurium to the cells. This is in agreement with the proposed mechanism for attachment of phytopathogenic bacteria to roots, which dependents on tight and irreversible cellulose-to-cellulose binding to the roots. THE EspM1 AND EspM2 ARE REDUNDANT EFFECTORS OF ENTEROHAEMORRHAGIC E. COLI ACTIVATING THE RHOA SIGNALING PATHWAY

M. Simovitch1, H. Sasson2, B. Aroeti2, I. Rosenshine1

1Department of Molecular Genetics and Biotechnology, Hadassah Faculty of Medicine, Hebrew University of Jerusalem, Israel, 2Department of Cell and Animal Biology, Institute of Life Sciences, Hebrew University of Jerusalem, Israel

Enterohaemoragic E.coli (EHEC) is a major cause of food-borne gastroenteritis. Essential for its virulence is a type III secretion system (TTSS) employed to deliver around 40 different effector proteins into infected host cells. We describe here a functional analysis of two of these effectors: the closely related EspM1 and EspM2. We show that EspM2 is expressed and translocated by EHEC under native conditions and that this translocation is dependent upon the TTSS and the CesT chaperon. In the host cell, EspM2 induces formation of stress fibers, lamellipodia formation and rounding up of the cells. These morphologies are similar to those induced by activated RhoA. The localization profile of EspM2 to membrane ruffles, tips of lamellipodia, cell-cell contacts and the cleavage furrow of dividing cells also matches that of activated RhoA. Moreover, wild type EHEC induced pronounced stress fibers in infected cells, but was no longer capable of doing so upon deletion of both espM1 and espM2, showing their redundancy. Finally, EspM2 was toxic to wild type yeast, but not to mutants in the Rho1p signaling pathway. Taken together, these results indicate that EspM1 and EspM2 are redundant effectors, which activate the RhoA signaling pathway, likely via RhoA mimicry. MOLECULAR AND FUNCTIONAL ANALYSIS OF STRESS-INDUCED GRAS TRANSCRIPTIONAL ACTIVATORS IN TOMATO PLANTS

M. Mayrose1, S. Ekengren2, P. Kumar1, S. Melech-Bonfil1, Y. Sterlin1, G. Martin2, G. Sessa1

1Department of Plant Sciences, Tel Aviv University, Ramat Aviv, Israel, 2Boyce Thompson Institute for Plant Research, Ithaca, NY, USA

Members of the GRAS family of transcriptional regulators have been implicated in the control of various aspects of plant growth and development, and in the interaction of plants with symbiotic bacteria. We examined the complexity of the GRAS gene family in tomato (Solanum lycopersicum) and investigated its role in disease resistance and mechanical stress. As a first step, a large number of tomato ESTs corresponding to GRAS transcripts was retrieved from public databases and assembled in 17 contigs of putative genes. Expression analysis of these genes by real- time RT-PCR revealed that six SlGRAS transcripts are induced during the onset of disease resistance to Pseudomonas syringae pv. tomato. Further expression analysis of two selected family members showed that they accumulate in tomato plants in response to different avirulent bacteria or to the fungal elicitor EIX, and their expression kinetics correlate with the appearance of the hypersensitive response. In line with their proposed function as transcription factors, all six pathogen-induced GRAS genes encode proteins that localize to the nucleus of tomato leaf tissues and have the capability to activate transcription in a heterologous yeast system. In addition, eight SlGRAS genes, including all the Pseudomonas-inducible family members, were induced by mechanical stress much earlier than upon pathogen attack. Activation of SlGRAS genes by mechanical stress was in part dependent on the signaling molecule jasmonic acid. Remarkably, suppression of SlGRAS6 gene expression by virus-induced gene silencing impaired tomato resistance to Pseudomonas syringae pv. tomato. These results reveal a novel function for GRAS transcriptional regulators in the plant response to biotic and abiotic stress. A RECOMBINASE-IVET APPROACH REVEALED THAT citH IS REQUIRED FOR WILD TYPE LEVELS OF VIRULENE OF XANTHOMONAS CAMPESTRIS PV. VESICATORIA ON TOMATO

D. Tamir-Ariel, N. Navon, S. Burdman

Department of Plant Pathology and Microbiology, Hebrew University of Jerusalem, Rehovot, Israel

The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease of tomato and pepper. The disease process is interactive and intricate, involving a plethora of both pathogen and host genes. In the pathogen, many genes are activated in response to the host environment to enable it to survive, adapt, evade defense responses, propagate, and cause disease. To understand the disease process it is imperative to broaden our understanding of the gene machinery that participates in it, and the most reliable way is to identify these genes in vivo. For this purpose we have adapted a Recombinase-based In Vivo Expression Technology (RIVET) approach to identify genes activated in Xcv during its interaction with tomato. RIVET revealed 61 unique Xcv in vivo overexpressed (ivx) genes/operons. To further explore the role of some of these genes, we generated knockout mutants for 13 genes and characterized their ability to grow in planta and to cause disease symptoms. This analysis revealed that a citH homolog encoding a citrate:H+ symporter is required for wild type levels of virulence. The citH homolog has 61 % identity to the characterized citrate transporter CitN of Bacillus subtilis. The citH mutant was unable to grow in minimal medium with citrate as the sole carbon source, suggesting the citH product facilitates citrate transport in Xcv. Analysis of the promoter activity of this gene showed that relative to its induction in rich media, it is induced to more than two orders of magnitude in planta. In planta, it was shown to be induced to up to 20 times more than the promoter of hrpA, a gene which is essential for pathogenicity, and is selectively induced in vivo. Our goal is to better understand how citH contributes to the virulence of Xcv. ENTAMOEBA HISTOLYTICA DNA METHYLTRANSFERASE 2 INTERACTS WITH THE MULTI FUNCTIONAL PROTEIN ENOLASE

A. Tovy

Department of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

One of the ways to better understand the role of DNA methyltransferase DNMT2 is by examining its interaction with other proteins. Up to date, no interacting candidate was ever reported for this family of DNA methyltransferase. Therefore we searched for such interaction with Ehmeth, a DNMT2 homolog present in the parasite Entamoeba histolytica. A two-hybrid screening between Ehmeth and E.histolytica cDNA library was performed and after several verification steps a single clone was retrieved. The insert within this clone matched the E.histolytica glycolytic protein enolase. Enolase, is an essential enzyme which catalyzes the conversion of 2- phosphoglycerate (2PG) to phosphoenolpyruvate. In addition to this conventional activity, enolase was shown to have multiple functions like the binding to human plasminogen in the pathogens Streptococcus pyogenes or the binding to nuclear DNA in the human adrenal cortex cells. The binding of Ehmeth to enolase was confirmed by in vitro and in vivo pull down assays validating enolase as a binding partner to Ehmeth. Western Blot analysis with a specific Enolase antibody shows that this protein is secreted following contact of E.histolytica trophozoites with mammalian cells. Enolase is also present in the cytoplasm and in the nucleus of the parasite. Preliminary results indicate that this protein may inhibit the binding Ehmeth to one of its DNA target, the scaffold/matrix attachment regions (S/MAR) DNA. This suggests that enolase may act as negative regulatory protein. INVOLVEMENT OF TOLL-LIKE RECEPTOR 2 IN THE HUMAN NEUTROPHIL RESPONSE TO PORPHYROMONAS GINGIVALIS

M. Tzemach1, Y. Houri-Hadad2, G. Nussbaum1

1Institute of Dental Sciences, Faculty of Dental Medicine, Hadassah Medical Center, Hebrew University of Jerusalem, Israel, 2Department of Prosthodontics, Faculty of Dental Medicine, Hadassah Medical Center, Hebrew University of Jerusalem, Israel

Introduction: Periodontitis is a chronic inflammatory disease characterized by a constant presence of bacteria, alongside a persistent host response. Porphyromonas gingivalis (Pg) is a black-pigmented gram-negative anaerobic coccobacillus that is considered to be one of the major periodontal pathogens. Toll-like receptors (TLRs) are key molecules of the innate immune system, which detect various structures of invading pathogens and trigger inflammation. We discovered that TLR2 plays a major role in the innate response to Pg. Surprisingly, we discovered that TLR2-mediated activation leads to enhanced bacterial survival in a murine model of Pg infection. Objectives: 1. To determine whether TLR2 activation by live Pg affects the phagocytic response of human neutrophils. 2. To understand the role of monocytes in regulating the phagocytic response of human neutrophils to live Pg. Methods: Neutrophils and monocytes were isolated from whole blood of healthy donors by density gradient separation. Pg 381 was grown for 48h and then labeled with fluorescein isothiocyanate (FITC). The cells were exposed to the FITC-labeled Pg and phagocytosis was allowed to proceed. To asses the role of TLR2 in Pg neutrophil activation, we used blocking antibodies to TLR2 vs. TLR4, and evaluated the following parameters: 1. phagocytosis level 2. apoptosis 3. cytokine secretion Results: In the presence of monocytes, there was a decrease in the phagocytosis of Pg by neutrophils. After blocking TLR2, in a mixed population, there was a significant enhancement in the phagocytosis. Exposure of neutrophils to Pg delayed cell apoptosis, but TLR2 blockage increased the apoptosis level, and decreased the cytokine levels. Conclusion: 1. Neutrophil phagocytosis of Pg is actively down- regulated by monocytes. 2. TLR2 plays a role in neutrophil cytokine secretion and delayed apoptosis in response to live Pg. These results are consistent with the hypothesis that TLR2-mediated activation benefits Pg by promoting its survival in periodontal lesions. PHASE DEPENDENT IMMUNE EVASION OF HERPESVIRUSES

T. Vider-Shalit, V. Fishbain, S. Raffaeli, Y. Louzoun

Department of Mathematics, Bar-Ilan University, Ramat Gan, Israel

Viruses employ various modes to evade immune detection. Two possible evasion modes are a reduction of the number of presented epitopes and mimicry of self epitopes. The immune evasion efforts are not uniform among viral proteins. The number of epitopes and their similarity to self in a given viral protein can be used as a measure of the viral attempts to hide these proteins. We perform a genomic analysis of the escape attempts from immune detection in four human herpesviruses (HSV1– HHV1, EBV-HHV4, HCMV-HHV5, KSHV-HHV8) and one murine herpesvirus (MHV68) using bioinformatic tools. We compute the full presented CTL epitope repertoires from each virus protein, and show that herpesvirus proteins present much less epitopes than expected. Furthermore, the epitopes that are presented are more similar to self epitopes than random viral epitopes, minimizing the immune response. We define a SIR score (Size of Immune Repertoire) representing the number of epitopes in a protein. The number of epitopes in proteins expressed in the latent and early infection phases is significantly smaller than in the lytic phase in all tested viruses. The latent and immediate early epitopes are also more similar to self than lytic epitopes. A clear trend emerges from the analysis. In general herpesviruses demonstrate an effort to evade immune detection. However, within a given herpesvirus, proteins expressed in critical phases of infection fate (e.g. early lytic and latent) evade immune detection more than all others. The application of the SIR score to specific proteins allows us to quantify the importance of immune evasion, and to detect optimal targets for immuno-therapy and vaccine development. AN ENDOCYTIC MECHANISM OF HEME-IRON UTILIZATION IN CANDIDA ALBICANS

Z. Weissman, D. Kornitzer

Department of Molecular Microbiology, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Like many bacterial pathogens, the fungal pathogen Candida albicans is able to utilize hemin and hemoglobin as iron sources. Heme-iron utilization is facilitated by Rbt5, an extracellular, GPI-anchored, hemin- and hemoglobin-binding protein. However, the pathway through which the heme iron is extracted and incorporated into the cell is unknown. Here, we show that Rbt5 and its close homolog Rbt51 are short-lived plasma membrane proteins, degradation of which depends on vacuolar activity. Rbt5 facilitates the rapid endocytosis of hemoglobin into the C. albicans vacuole. We relied on recapitulation of the Rbt51-dependent heme-iron utilization in S. cerevisiae to identify mutants defective in hemoglobin utilization. Homologs of representative mutants in S. cerevisiae were deleted in C. albicans and tested for hemoglobin iron utilization and hemoglobin uptake. These mutants define a novel endocytosis- mediated hemoglobin utilization pathway that depends on acidification of the lumen of the late secretory pathway, on the activity of the ESCRT pathway, and on a Type I myosin. A specific vacuolar iron transporter is required for hemoglobin-iron utilization, suggesting that heme extraction from hemoglobin and iron extraction from the heme occur in the vacuole. INTERNALIZATION AND INDUCTION OF APOPTOSIS BY THE IBARAKI STRAIN OF THE EPIZOOTIC HEMORRHAGIC DISEASE VIRUS

R. Yaniv, M. Ehrlich

Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, Israel

The structurally conserved Reoviruses are pathological agents of humans (Rotaviruses) and farm animals (Orbiviruses).In addition they recently have been proposed as promising oncotherapeutic agents (Orthoreoviruses). Although less well studied, a recent outbreak in cattle farms in Israel underscores the need of characterizing the Epizootic Hemorrhagic Disease Virus (EHDV), an Orbivirus. In this context, the interactions of Orbiviruses, in general, and of the EHDV in particular, with the membrane trafficking and signal transduction machineries remain poorly characterized. Furthermore, the Bluetongue virus (BTV), a related Orbivirus has been shown to induce the apoptotic cell death of infected cells. However, the ability of EHDV to induce this process, the precise mechanism by which apoptosis is triggered, the viral and cellular proteins involved, the stage of the infection in which it occurs and the function of this process in viral replication/spread or host defense are all currently under investigation. In the present study we have identified the clathrin mediated endocytic pathway as the infectious internalization route of the Ibaraki strain of the Epizootic Hemorrhagic Disease Virus (EHDV). In addition we show that the Ibaraki virus induces the apoptotic cell death of certain types of tissue culture cells. Moreover, an apoptotic-resistant cell type, Bend-Mouse Brain Endothelial cells, became sensitive upon transformation with oncogenic H-Ras G12V. These cells will serve as a platform for the elucidation of the involvement of Ras-based signaling in virally induced apoptosis. THE PATHOGENECITY OF VARIOUS PECTOBACTERIUM CAROTOVORUM ISOLATES FROM DICOTS AND MONOCOTS CORRELATES WITH EARLY GENOMIC DIVERGENCE

M. Yishay1,2, S. Bordman2, T. Luzzatto1,2, R. Ophir1, I. Yedidia1

1Department of Horticulture, ARO, Volcani Center, Bet-Dagan, Israel, 2Department of Microbiology and Phytopathology, Hebrew University of Jerusalem, Rehovot, Israel

Pectobacterium carotovorum is a member of the former genus Erwinia. While some of these former Erwinias show high specificity to a certain host, others such as P. carotovorum have world wide distribution and a broad host range. P. carotovorum strains capable of infecting hosts in the monocotyledonous plants clade were occasionally reported in the scientific literature, yet no consideration was given to study the association of such isolates and their natural hosts. We have used two P. carotovorum susceptible monocots hosts, the ornamentals Zantedeschia and Ornithogalum as well as two common dicots hosts, Solanum tuberosum and Brassica oleracea, as model plants for the study of aggressiveness and specificity of isolates obtained from both plants clades. Forty seven P. carotovorum isolates from several geographical locations in four unrelated orders of monocots and five orders of dicots plants were phenotypically and genetically characterized using virulence assays, 16S rRNA sequence analysis, ITS-PCR and AFLP. The aggressiveness level of the isolates originating from monocots hosts towards the monocots model plants, was higher and independent of the isolates geographical origin or the specific plant from which they were isolated as long as it was a monocot. Moreover, this aggressive phenotype was associated with genotypic characteristics such as typical ITS-PCR banding pattern, AFLP marker band, and 16S rRNA clustering. It is thus suggested that the analysis of P. carotovorum isolates in relation to their host classification, offers a valuable tool to unravel the machinery underlying the shared evolution of plant/P. carotovorum associations. IDENTIFICATION AND CHARACTERIZATION OF TRAFFICKING MOTIFS OF THE IBARAKI VIRUS NS3 VIROPORIN

N. Ziv, R. Yaniv, M. Ehrlich

Department of Cell Research and Immunlogy, Tel Aviv University, Ramat Aviv, Israel

The Ibaraki virus, an Orbivirus, is an etiological agent of Epizootic Hemorrhagic Disease. Orbiviruses are dsRNA, non enveloped arboviruses of the Reovirus family. The genome of Orbiviruses is composed by 10 small dsRNA fragments. The s10 genome segment encodes for the only orbiviral transmembrane proteins, NS3 and NS3A.These proteins which exert an important function in viral pathogenesis are suggested to induce membrane permeabilization, and thus are candidate viroporins. Due to their prominent role in virus-host interactions, viroporins have been the object of intense study. However, their precise mechanism of action is still unknown. Furthermore, the intracellular location where the membrane perturbation occurs as well as the virally encoded factors which regulate NS3 localization are yet to be determined. In the present study we have generated GFP and RFP fusion proteins of NS3 and monitored their localization in transfected and infected cells. In both transfected and infected cells NS3-GFP localizes to the endoplasmic reticulum and to later secretory/endocytic compartments. We have initiated a dissection of the candidate trafficking motifs present on NS3, identified by sequence analysis, and are addressing their function in the determination of the viroporin potential of NS3. COUPLING CONSTITUTIVE DENDRITIC CELL ACTIVATION TO MHC-I PEPTIDE PRESENTATION THROUGH mRNA ENCODING PEPTIDE- BETA2 MICROGLOBULIN-TLR4

E. Amram1,2, G. Cafri1,2, S. Fishman1, A. Margalit1,3, G. Gross1,3

1Laboratory of Immunology, MIGAL, Kiryat Shmona, Israel, 2Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 3Department of Biotechnology, Tel-Hai Academic College, Upper Galilee, Israel

Dendritic cells (DCs) play a pivotal role in the induction of cytotoxic T lymphocyte (CTL) response. Residential DCs at the infection site pick up pathogen constituents, stress-induced products or apoptotic cell particles, undergo maturation, migrate to the secondary lymphoid organs, present the antigenic peptide in the context of MHC-I and provide costimulatory signals necessary for CTL priming. Recently, we have developed a genetic platform for peptide presentation based on membranal beta-2 microglobulin (b2m). We have shown that incorporation of the intracellular signaling domain of toll-like receptor 4 (TLR4) as the b2m anchor in couples antigen presentation to cell activation. To test the utility of mRNA as a vector for DC transfection in a mouse tumor model, we have employed 2 DC lines, DC2.4 (H-2b) and XS52 (H-2d). We transfected these cells by electroporation with a mRNA encoding a peptide-b2m engrafted with TLR4 or H-2Kb cytosolic domains, the latter as a reference, inert anchor. Transfection efficiency of both cells approached 90%. Level of expression and antigen presentation was exceptionally high. The antigen was detected on MHC-I for over 36 hours. The decrease in peptide presentation was estimated to be 10% per day, taking cell proliferation into account. Although the two anchors provided efficient presentation platforms, only TLR4 induced cell activation, as measured by elevation in the basal level of COX-2 and IL-1b mRNA. These results provide evidence that mRNA encoding the peptide-b2m-TLR4 can be utilized as an effective vehicle to drive high level and persistent presentation of the linked antigenic peptide following DC transfection. DIFFERENT MUTATIONS IN THE Bcl-6 P53RE, CARRIED BY B-NHL PATIENTS, RESULTED IN PARTIAL INDUCTION OF Bcl-6 TRANSCRIPTION BY p53 PROTEIN

H. Amram1, O. Margalit1, A.J. Simon1, D. Givol2, N. Amariglio1, G. Rechavi1

1Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Ramat Aviv, Israel, 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

The Bcl-6 transcriptional repressor mediates survival, proliferation, and differentiation blockade of B cells during the germinal center reaction and is frequently misregulated in B-cell non-Hodgkin lymphoma (B-NHL). The p53 tumor suppressor gene is considered to be the 'guardian of the genome'. In response to genotoxic stress, p53 is activated, and leads cells to either cell cycle arrest or to apoptosis. p53 is central in tumorigenesis, and undergoes mutations in about 50% of human cancers. We have shown that an active p53RE resides within the Bcl-6 5' UTR. During B-cell development, the immunoglobulin variable (V) region genes are subjected to somatic hypermutation (SHM) in the germinal center. In B-NHL patients the SHM process targets, in addition to immunoglubulins sequences, oncogenes such as Bcl-6. The Bcl-6 p53RE contains a motif which is preferentially targeted by the SHM mechanism. In B-NHL patients the mutations within Bcl-6 genes are distributed in the 5' UTR or coding sequences, and affect also the p53RE. We performed reporter assays which were based on the wild type Bcl-6 p53RE and on mutated sequences. Each mutated sequence was identical to a Bcl-6 p53RE found in B-NHL patient. Using that method we were able to show that that mutations in the Bcl-6 p53RE sequence found in B-NHL patients lowers the induction of Bcl-6 by p53. There was not a complete loss of the Bcl-6 induction, but a partial reduction in the ability of p53 to induce Bcl-6 transcription. This phenomenon, along with the minority of B-NHL patients which carry p53 mutations may imply that the p53-Bcl-6 balance has a role in the lymphoma development and progression, as well as in the physiological state at the germinal center niche. IFN-gamma ENHANCES NEUROGENESIS IN WILD-TYPE MICE AND IN A MOUSE MODEL OF ALZHEIMER'S DISEASE

R. Baron1, A. Nemirovsky1, I. Harpaz2, H. Cohen2, A. Monsonego1

1The Shraga Segal Department of Microbiology and Immunology, The National Institute of Biotechnology in the Negev, Faculty of Health Sciences, Ben-Gurion University in the Negev, Beer-Sheva, Israel, 2Ministry of Health, Mental Health Center, Anxiety and Stress Research Unit, Faculty of Health Sciences, Ben-Gurion University in the Negev, Beer-Sheva, Israel

The generation of new neurons and glia from a precursor stem cell takes place in the adult brain. However, the number of new neurons generated in the dentate gyrus declines sharply with age and to an even greater extent in neurodegenerative diseases. Here we raise the question whether peripheral immune mechanisms can generate immunity to such deficits in neuronal repair. Transgenic mice expressing IFN-gamma in the brain were injected with bromodeoxyuridine (BrdU) at 1, 3, and 9 months of age and examined for neuronal precursor cells in the hippocampus. Cognitive performance and gene expression in the brain were examined at 3 months of age. We demonstrate that in contrast to primarily innate immunity cytokines, such as IL-6 and TNF-alpha, the adaptive immunity cytokine IFN-gamma enhances neurogenesis in the dentate gyrus of adult mice associated with upregulation of neurotrophic factors and improved spatial learning and memory performance. In older mice, the effect of IFN- gamma is more pronounced in both wild-type mice and mice with Alzheimer's-like disease and is associated with neuroprotection. In addition, IFN-gamma reverses the increase in oligodendrogenesis observed in a mouse model of Alzheimer’s disease. It is thus suggested that limited amounts of IFN-gamma in the brain shape the neuropoeitic milieu to favor neuronal repair through aging and the progression of Alzheimer's-like disease, possibly representing the normal function of the immune system in controlling brain inflammation and repair. RECOMBINANT HUMAN ANTIBODIES WITH TCR-LIKE SPECIFICITY DIRECTED TOWARD CEA (CARCINOEMBRYONIC ANTIGEN) HLA-A2 RESTRICTED EPITOPE

Y. Carmi, Y. Reiter

Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel

MHC class I molecules play a central role in the immune response against a variety of cells that have undergone malignant transformation by shaping the T cell repertoire and by presenting peptide antigens from endogeneous antigens to CD8+ cytotoxic T cells. Because of their unique specificity such MHC-peptide complexes are a desirable target for novel approaches in immunotherapy and diagnosis. Antibodies that have the ability to recognize these complexes with high affinity and specificity can be used for structural and functional studies of TCR-peptide-MHC interactions and are an important tool for obtaining accurate information about the presence and the expression of tumor derived antigens. These antibodies, termed TCR-like antibodies, can also be used for therapeutic applications of these diseases. In this work, we focused on the CEA, a 180 kDa oncofetal glycoprotein, member of the Ig supergene family. This molecule is highly expressed in the vast majority of colorectal, gastric, and pancreatic carcinomas. In addition, CEA is found in 50% of breast cancers and 70% of non-small cell lung carcinomas. CEA is also expressed in the fetal colon, and, at a lower level, by normal colonic epithelium. We have screened a Fab antibody phage display library with the produced MHC complexes containing the CEA-Cap1 peptide (YLSGANLNL). Several clones were obtained from the screened library and were further analyzed using ELISA assays and FACS analysis using APC loaded with the CAP1 peptide. One clone, H10 showed highly specific binding to JY cells loaded with the CAP1 peptide and not to control peptides. The H10 clone was then tested for the ability to bind CEA positive, HLA-A2 positive cancer cells, but showed weak binding or no binding to any of the cell line tested. Further work should be done in order to determine the H10 clone activity, specificity and future uses. THE N-TERMINAL PRO-PIECE OF INTERLEUKIN 1 ALPHA IS A DNA BINDING PROTEIN

I. Cohen, S. Dotan, R.N. Apte

Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Interleukin-1 alpha (IL-1 alpha) is a potent pleiotropic cytokine that primarily affects inflammatory and immune responses, angiogenesis and hematopoiesis, but also other homeostatic functions of the body such as growth, differentiation and apoptosis. Although most cytokines are known for their biological effects after interaction with their specific cell surface receptors, IL-1 alpha, which is rarely secreted actively from most cells, has an additional intracellular function that is mediated by a range of intracellular mechanisms. IL-1 alpha contains a large N-terminal pro-piece formed as a result of proteolytic processing of the 31- to 33-kDa precursor protein. This 17-kDa N-terminal pro-piece was shown to be a nuclear localized transforming oncoprotein. Here we show that the N-terminal pro-piece of interleukin 1 alpha is a DNA binding protein. By using cyclic amplification and selection of targets (CAST’ing), a method mainly used for identifying transcription factor binding sites, we isolated 49 clones which interact with a recombinant GST-IL-1 alpha pro-piece fusion protein. In order to determine the IL-1 alpha pro-piece consensus binding site we analyzed the oligonucleotide sequences of all the 49 clones with motif sampler (V 2.0), a bioinformatic softwear, and revealed two major groups of potential DNA binding consensus motives. A preliminary electro mobility shift assay (EMSA) with biotin labeled oligonucleotides obtained from the CAST’ing experiment reconfirmed that IL-1 alpha pro-piece is indeed a DNA binding protein with binding affinity in the micro molar range. This result can highly promote the search of genes regulated by IL-1 alpha pro-piece and contribute to the understanding of the intracellular function and the physiological role of this important cytokine. DESIGN OF DISULFIDE STABILIZATION FOR A RECOMBINANT MHC CLASS II

R. Dahan1, N. Adir2, Y. Reiter1

1Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel, 2Department of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel

MHC class II molecules are membrane anchored heterodimers on the surface of antigen presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Therefore, Recombinant MHC molecules plays a central role in the field of immunological research and immunotherapy. We propose a novel approach for stabilizing a recombinant molecule of MHC class II in E.coli expression system. Our approach is designed uniformly for all MHC alleles, based on alignments of MHC class II from different organisms. Successful results will potentially overcome a global problem of very low yields in the production of recombinant MHC molecules. MHC class II molecules present an inherently difficult for in vitro folding. Our designed molecule consist of the extracellular domains of MHC II and therefore lack the two anchor transmembrane domains. All previous reported attempts to fold in vitro the extracellular chains of MHC II have been limited to a very few alleles but with low yield efficiency and completely failed for some other alleles. Using a rational design we engineered a recombinant MHC molecule by introducing a stabilizing mutated disulfide bond between the two extracellular chains. We performed a 3D structural model which outlined a conserved hydrophilic pocket between the chains. This pocket is our target region for the mutated disulfide bond and a pair of two structurally conserved residues within it were mutated to cystoids. We were able to fold in-vitro the HLA-DR4 allele covalently linked to GAD65 555- 567 peptide. The dimmeric molecule contains a disulfide bond between its subunits. The structural and functional feature of this newly constructed molecule are under investigation. DOWNREGULATION OF THE WISKOTT-ALDRICH SYNDROME PROTEINS CRUCIAL FOR ACTIN POLYMERIZATION

A. David, L. Frishman, S. Jakubowicz, I. Wortzel, O. Perl, M. Barda-Saad

Department of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

The Wiskott–Aldrich Syndrome (WAS) is a hematopoietic disorder characterized by thrombocytopenia, increased susceptibility to infections, autoimmunity and lymphoid malignancies. The disease is caused by mutations of the gene encoding the Wiskott– Aldrich Syndrome protein (WASp), which is exclusively expressed in hematopoietic cells, where it plays a key regulatory role in cytoskeletal dynamics. Our studies demonstrated that following T cell antigen receptor (TCR) activation, actin polymerization was driven by the Wiskott-Aldrich Syndrome protein (WASp) and was dependent on its dynamic localization. Late in the activation process, vesicles containing WASp left the periphery and tracked along the microtubules towards the interior of the cell to a central structure for internalization and most probably degradation. In the current study, we identify the molecular mechanisms governing the degradation of active signaling molecules that lead to downregulation of TCR signaling complexes. Our data indicate that following TCR activation, WASp is ubiquitinated through the WASp- homology 1 domain (WH1) which is the region with the vast majority of WAS mutations. We demonstrate that WASp protein levels are tightly regulated via proteasome-dependent degradation. Using high resolution molecular-imaging technique in combination with standard biochemical analysis, we explore the degradation mechanisms of WASp in activated T-cells in vivo. Special focus is put on exploring both the molecular mediators and the functional consequences of the ubiquitination process. This knowledge may explain the susceptibility of WASp to degradation in the hematopoietic cells of WAS patients and will give novel insights into the pathogenesis of the WAS immunodeficiency. ANTIGEN SPECIFIC PATHWAY FOR T-CELL ENTRY INTO THE ALZHEIMER'S DISEASE BRAIN

Y. Fisher, A. Nemirovsky, A. Monsonego

The Shraga Segal Department of Microbiology and Immunology, The National Institute of Biotechnology in the Negev, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Alzheimer's disease (AD) is a neurodegenerative age related disease and the most common cause for dementia. Accumulation of amyloid beta (Abeta) in the hippocampous and cortex areas of the brain is a hallmark of AD. Transgenic mice carrying a mutant form of the human amyloid precursor protein (APP) develop aging- dependent Abeta plaques with features similar to the disease in humans and serve as a mouse model for AD. One of the most promising therapeutic approaches for treating AD deals with the ability of the immune system to alter the deposition of Abeta and enhance neuroprotection. As Abeta vaccination of APP Tg mice has shown remarkable antibody-mediated clearance of Abeta from the brain, the role of specific lymphocytes in brains affected with AD has not been characterized. We crossed the APP Tg mice with mice expressing interferon-gamma (IFN-gamma) in the brain to generate a unique mouse model of the disease, which in contrast to previous models, enables lymphocyte migration to sites of Abeta plaques in the brain. These APP/IFN- gamma double Tg mice elicit a strong anti-Abeta immune response upon Abeta vaccination resulting in significant infiltration of bone marrow-derived cells to the brain. While B cells and dendritic cells (DCs) remained in meningeal spaces, both CD4 and CD8 T cells migrated to sites of Abeta plaques and induced their clearance. T cells appeared to enter from blood vessels coated or colocalized with Abeta plaques and harbor peripheral DCs. These data demonstrate an antigen-specific lymphocyte entry into the brain, affecting the progression of AD-like disease, via antigen presentation by peripheral DCs targeted to Abeta-coated blood vessels. EXPRESSION OF DNA OR mRNA ENCODING PEPTIDE-BETA2 MICROGLOBULIN-TLR4 IN MOUSE ANTIGEN-PRESENTING CELLS RESULTS IN EFFICIENT PEPTIDE PRESENTATION AND CONSTITUTIVE CELL ACTIVATION

S. Fishman1, E. Amram1,2, G. Cafri1,2, A. Margalit1,3, G. Gross1,3

1Laboratory of Immunology, MIGAL, Kiryat Shmona, Israel, 2Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 3Department of Biotechnology, Tel-Hai Academic College, Upper Galilee, Israel

Activation of peripheral dendritic cells (DCs) by pathogen constituents or stress- induced products is mediated primarily by members of the toll-like-receptors (TLR) family and occurs in parallel to acquisition of antigen. Priming of cytotoxic T lymphocytes (CTLs) entails their productive encounter with these activated DCs in secondary lymphoid organs. Effective CTL-inducing vaccines should therefore drive sufficient and persistent MHC-I presentation of antigen by DCs, which provide the optimal costimulatory environment for the CTL precursors. We have recently created a potentially universal platform for CTL-inducing genetic vaccines, which is based on membrane-anchored beta2 microglobulin (b2m), the monomorphic MHC-I light chain. This design combines MHC-I stabilization with exceptional presentation of pre-selected peptides covalently linked to b2m, and we previously demonstrated its potency in a mouse melanoma model. This new platform offers an ideal backbone for supplementing b2m with TLR intracellular signaling domains operating as genetic adjuvants. In the present study we explored the functional potency of the intracellular portion of mouse TLR4 (mTLR4). To this end we assembled a series of genetic constructs encoding peptide-human b2m (hb2m)-H-2Kb (harboring the intracellular domain of H-2Kb as an inert anchor) and a corresponding series of constructs in which the H-2Kb-derived anchor was replaced with that of mTLR4. We first generated stable transfectants of the macrophage cell line RAW264.7. We show that surface expression of hb2m was consistently higher for the constructs harboring the H-Kb anchor than those with TLR4. Yet, using semi-quantitative reverse-transcriptase PCR analysis we showed that constitutive cell activation was only evident with TLR4, as judged by markedly elevated mRNA level of the pro-inflammatory TNF-alpha and COX2 products. In another set of experiments we used two mouse DC lines, XS52 and DC2.4, and showed that electroporation of mRNA encoding similar constructs led to exceptionally high level of expression and antigen-presentation, and constitutive cell activation. CONSERVED EPITOPES OF AVIAN INFLUENZA ELICITED ANTIBODIES THAT RECOGNIZE THE WHOLE VIRUS

T. Gefen, J. Pitcovski1

Department of Immunology and Virology, MIGAL - Galilee Technology Center, Kiryat-Shmona, Israel

Influenza virus is highly contagious and causes an acute respiratory disease in animal and human. Hemagglutinin (HA) and neuraminidas (NA) are two viral membrane glycoproteins. Nowadays, all known subtypes of influenza A virus (15 subtypes of hemagglutinins (H1 -H15) and 9 subtypes of neuraminidases (N1- N9)), are found in wild waterfowl. Variation in these two proteins contributes to the inability to develop effective vaccines. HA is involved in the attachment of viral particles to host cell receptor and the entry into the target cells. Due to its critical role, most of the neutralizing antibodies are directed to this protein, and a subunit vaccine or peptide- based vaccine based on HA has the potential to eliminate viral infection. It has been demonstrated that HA protein has conserved regions. Some epitopes of HA were shown to elicit neutralizing antibodies and immunization with them conferred protection against challenge by several influenza strains. The purpose of this study was to test multi- epitope vaccine based on peptides derived from the conserved regions of the HA protein. For this purpose, we designed and expressed a recombinant polypeptide, comprising repeats of six neutralizing-epitopes of HA1 protein of influenza virus (H9). Following vaccination, the recombinant multi-epitope protein induced antibodies that showed specificity to the whole virus. These antibodies were able to inhibit hemagglutination. These results suggest that the recombinant multi- epitope vaccine can induce antibodies that will detect various sub-types of the most abundant avian influenza, H9. The results of this study may provide a new approach to develop effective vaccines against influenza virus. HSP DERIVED PEPTIDE SERVES AS AN IMMUNOGENIC CARRIER, FOR WEST NILE VIRUS (WNV) EPITOP, IN CONJUGATED VACCINES

O. Gershoni-Yahalom, B. Rager, A. Porgador

Laboratory of Immuno-Virology, Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel

WNV is a single stranded plus-sense RNA virus that is classified within the Flaviviridae family. WNV is naturally maintained in a mosquito–bird-mosquito cycle, with humans and other mammals serving as incidental hosts. The renewed interest in WNV stems from the fact that it has emerged and appeared across the USA and Canada. Recently, it has been shown that WNV can also be transmitted through blood transfusion, organ transplantation and breast feeding. Clearly, an effective WNV vaccine is needed to protect populations at-risk. HSP60 belongs to a family of chaperone molecules highly conserved throughout evolution. Mammalian HSP60 is highly homologous to bacterial cognates, showing about 50% amino acid identity. Thus, HSP60 is shared by the host and its parasites, and is immunogenic, cross- bacterial or ,ד reactive, and universally expressed in inflammation. Heat shock, IFN viral infection, and inflammation, all result in the presentation of endogenous HSP60 epitopes on MHC class II molecules leading to the activation of HSP60-specific T cells, even in healthy individuals. Thus, natural T cell reactivity to a HSP60 derived peptide, such as p458 or Ec27 (derived from Gro EL - bacterial counterpart of HSP60), in conjugate vaccines can be mobilized to help induce resistance to a lethal infection. To develop a subunit vaccine by combining an immunogenic WNV epitope with a T cell carrier HSP60 epitope to recruit T-cell help for the induction of effective antibody or CTL response . Our results demonstrate the efficacy of this vaccination approach in a WNV mouse model. Immunization with the subunit vaccine resulted ד with an effective and specific immune response, including T cell proliferation & IFN secretion (after in-vitro stimulation), specific antibodies. Furthermore, the subunit vaccine gave full in-vivo protection against WNV challenge. SEQUENCE AND PHYLOGENETIC ANALYSIS OF SIGMA C PROTEIN OF AVIAN REOVIRUS ISRAELI ISOLATES

D. Goldenberg1, J. Pitcovski1, M. Pasmanik-Chor2

1Department of Virology and Immunology, MIGAL - Galilee Technology Center, Kiryat-Shmona, Israel, 2Department of Bioinformatics, Tel-Aviv University, Ramat Aviv, Israel

Introduction: Reovirus in poultry cause viral arthritis, tendosynovitis, liver infection, heart, intestine, and immunosuppressant. Birds are sensitive mainly at young ages. Protection is conferred by antibodies transferred from maternal flocks that have been vaccinated. The vaccines in use are attenuated (s1133 strain) or inactivated virulent isolates. Despite vaccination, many flocks are still infected by reovirus. In this study we amplified, cloned and sequenced sigma c protein from 23 Israeli isolates. Viral protein sigma c was used for molecular analysis and comparisons, since it is known as the most variable protein of the virus and is most important in induction of neutralizing antibodies. High degree of variability has been found in sigma c protein, with amino acid identities among the isolates ranging from approximately 35% to 98%. Sequence analysis reveals that all variants may be assembled into four separate groups with at least 75% amino acid identity within group. The vaccine was classified to one of the groups, and differs gradually from field isolates. This may explain the mobility of the vaccine to protect birds against the disease. In spite of the high variability, multiple sequence alignment of reovirus sigma c proteins (including the new Israeli strains and previously characterized from the world), revealed conserved regions, mainly localized at the carboxy-terminal of the protein. Sequence conservation data may suggest functional importance. Overall, this information may shed new light on the inefficiency of the traditional present vaccine, and enable the design of new relevant vaccine. PCERA-1 INHIBITS PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE STIMULATED MACROPHAGES

M. Goldsmith, T. Zor

Department of Biochemistry, Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel

PCERA-1, a ceramide-1-phosphate analog, down regulates inflammatory processes both in-vivo and in-vitro. We have shown that PCERA-1 inhibits the production of the pro-inflammatory cytokines TNFalpha and IL-12p40, and simultaneously up regulates production of the anti-inflammatory cytokine IL-10. To further elucidate the anti-inflammatory activity of PCERA-1, we investigated the effect of PCERA-1 on the production of the inflammatory modulator Prostaglandin E2 (PGE2), a lipid mediator crucial for the onset and the maintenance of the inflammatory process, including the visible inflammation symptoms of swelling, redness, pain, and temperature. Here we show that PCERA-1 effectively inhibits the production of PGE2 in lipopolysaccharide (LPS)-stimulated macrophages. We demonstrate that the inhibitory activity is not due to suppression of LPS-induced expression of COX2, the enzyme which initiates prostaglandins production and is the target of most common non-steroidal anti-inflammatory drugs. Further evaluation reveled that PCERA-1 blocked the LPS-induced release of arachidonic acid, indicating that PCERA-1 inhibits PLA2 activity. Interestingly, PCERA-1 was unable to affect Zymosan- induced release of arachidonic acid. Since LPS is thought to induce expression of PLA2 and to activate it by phosphorylation in a calcium-independent mechanism, while Zymosan activates PLA2 by a calcium-dependent mechanism, our data suggest that PCERA-1 blocks the expression and/or phosphorylation of PLA2, and thus inhibits the production of PGE2, contributing to its robust anti-inflammatory character. NOVEL INVOLVEMENT OF THE IMMUNOMODULATOR AS101 IN IL-10 SIGNALING, VIA THE TYROSINE KINASE Fer

R. Hayun1, S. Shpungin1, M. Albeck2, E. Okun1, U. Nir1, B. Sredni1

1Safdie Institute for AIDS and Immunology Research, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel, 2Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel

Interleukin-10 (IL-10) plays a major proliferative role in many tumors, and activates the transcription factor Stat3 by tyrosine phosphorylation. The immunomodulator ammonium trichloro (dioxoethylene-o,o') tellurate (AS101) has a direct anti-tumor activity, and is able to sensitize several tumors to chemotherapy, by inhibiting the tumor IL-10 autocrine loop. The tyrosine kinase Fer is essential for the proliferation of numerous malignant cell-lines and in some cases was related to Stat3 activation. This study examined the role of AS101 in IL-10 signaling, and the correlation between Fer and Stat3, in human peripheral blood mononuclear cells (PBMC). We show that Fer was associated with Stat3 in PBMC and RAW 264.7, a macrophage cell line. Recombinant IL-10 (rIL-10) increased the tyrosine phosphorylation of Stat3, up regulated the levels of Fer, and increased the association of Fer with phosphorylated Stat3 (pYStat3). All of IL-10’s activities mentioned above were reversed by AS101. The effects conferred by AS101 were totally abolished by exogenous addition of rIL- 10. These results indicate that AS101 down regulates the Stat3 IL-10 loop, and inhibits Fer association with pYStat3. We conclude that anti-IL-10 treatment using AS101, may be beneficial in certain malignancies and other pathologies in which IL- 10 secretion is elevated and Stat3 is continuously phosphorylated. CHARACTERIZATION OF SIGNALING MOLECULAR COMPLEXES CRUCIAL FOR ACTIN REORGANIZATION IN T-CELLS

M. Hazani, O. Perl, M. Barda-Saad

Department of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

Following T-cell antigen receptor (TCR) engagement, multiprotein signaling complexes are formed at the T-cell and antigen-presenting-cell (APC) interface. These complexes are required for biochemical signal transduction events that regulate numerous basic T-cell functions including cell proliferation, adhesion, and motility, all crucial for the T-cell-mediated immune response. One fundamental cellular process regulating T-cell functions is the reorganization of actin-cytoskeleton and accumulation of actin filaments at the site of the T-cell/APC interface. A key component in hematopoietic cells required for this process is the Wiskott-Aldrich syndrome protein (WASp), an actin nuclear-promoting factor. Mutations or deficiency of WASp cause human primary immunodeficiency (WAS) associated with defective T-cell-actin reorganization and impaired T-cell activation in vivo. A triple molecular complex consisting of the adapter molecules, SLP76, Nck, and VAV, regulates WASp recruitment to the T-cell/APC contact site and its activation. This is enabled by SLP76, which serves as a scaffold protein and is simultaneously associated with Nck and VAV. Using multidisciplinary approaches of high-resolution imaging techniques on live activated T-cells, together with gene silencing and biochemical analysis, we study the dynamics of this signaling complex formation. Our data reveal that SLP76, Nck, and VAV molecules do not exclusively act as components of a single ternary signaling complex, but form binary molecular complexes that have the potential to stimulate actin machinery. This research provides novel insights into the regulation of signaling complex formation critical for both normal immune function and the induction of pathophysiological conditions. RESTRICTED SPECTRUM OF SOMATIC HYPERMUTATION OF THE REARRANGED VH GENE IN ANTIBODY FORMING EBV- TRANSFORMED LYMPHOBLASTOID CELL LINE

M. Steinitz1, I. Chezar2, L. Lobl-Lavi2, Y. Gil2, R. Laskov2

1Department of Pathology, Hebrew University - Hadassah Medical School, Jerusalem, Israel, 2Department of Experimental Medicine and Cancer Research, Hebrew University - Hadassah Medical School, Jerusalem, Israel

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs), which produce specific antibodies. LCLs express the activation- induced cytidine deaminase (AID) gene, a key enzyme in the generation of somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes. We studied an established LCL that secretes a rheumatoid factor (RF: an IgM anti-IgG antibody) and previously identified the accumulation of SHM in the rearranged IgV heavy chain gene (VH) with a mutation frequency of 1.5x10-3 mut/bp. The aim of the present work was to find out if SHM was initiated as an early event, following EBV transformation. Our results show that already the earliest RF-culture, mutates its VH, at a frequency of 1.7x10-3. Overall, we detected 16 point mutations in the 26 cellular clones derived from the RF-cultures. The mutations were biased to C/G transitions, indicating that they were AID-mediated. A genealogical tree demonstrates that 1-2 mutations/cell division were accumulated in a stepwise manner. However, no mutations were found in the rearranged V-lambda gene from the same RF-cultures and their subclones (i.e., <1.2x10-4 mut/bp). To our knowledge this is the first reported cell line that generates SHM in the VH, but not in the Vƒ{lambda. Out of the 16 mutations detected in the VH, there was one stop codon, 4 mutations which obliterated antibody binding to its IgG antigen, and 1-2 mutations which enhanced antigen-binding affinity. These results show that the evolutionary developed germline encoded antibody combining site is highly sensitive to amino acid replacements. Accumulation in a stepwise manner of 1-2 point mutations/sequence/cell division in combination with the generation of a high percentage of functionally deleterious mutations, support the 'multiphase-recycling model' of SHM, which states that B cells in the germinal center are subjected to multiple rounds of somatic mutations interchanged with periods of antigenic selection. DEVELOPMENT OF CTL-INDUCING GENETIC VACCINES THAT COUPLE MHC-I PEPTIDE PRESENTATION TO CD40 ACTIVATION

N. Levin1,2, R. Ben-Shushan1,3, A. Margalit1,4, G. Gross1,4

1Laboratory of Immunology, MIGAL, Kiryat Shmona, Israel, 2Sharett Institute of Oncology, Hadassah University Hospital, Jerusalem, Israel, 3Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 4Department of Biotechnology, Tel-Hai Academic College, Upper Galilee, Israel

The interaction between CD40 ligand (CD40L) on the surface of activated CD4 Th1 cells and CD40 on dendritic cells (DCs) is essential for full DC maturation and acquisition of cytotoxic T lymphocyte (CTL)-priming capacity. Cross-linking of CD40 by agonistic antibodies or soluble CD40L can replace CD4 T cells. We have recently developed a new genetic modality for the induction of antigen-specific CTLs. It is based on converting beta2 microglobulin (b2m), the invariant MHC-I light chain, into an integral membranal protein and the genetic linking of an antigenic peptide to its N terminus. This modality prompts exceptionally efficient peptide presentation on MHC-I molecules and is effective in eliciting tumor protection and tumor suppression in-vivo. In particular, it allows the engraftment of the intracellular signaling domains of DC activation receptors onto the C-terminus of b2m, turning them into potential 'genetic adjuvants'. In order to obviate the need in CD4 T cells we sought to harness the intracellular portion of CD40 as the signaling moiety in this design. Here we show that cross-linking of MHC-I heavy chain on the surface of stably transfected mouse cells expressing b2m-CD40, but not on parental cells, leads to transient reduction in IkappaBalpha, which is similar to that induced by CD40 cross-linking. This activation is also manifested by substantial elevation in membranal CD80. Human cells transfected with mRNA encoding peptide-b2m-CD40 present high level of the peptide on MHC-I, as revealed by a complex-specific antibody and a CTL hybridoma. Importantly, these transfectants are specifically activated by cross-linking of the resulting complexes. Presence of these complexes at the surface of these dividing cells is detectable at least 7 days post-transfection, attesting to remarkable stability. These findings demonstrate that the CD40 signaling domain is functional in the context of b2m and underscore its potential application in CTL-inducing genetic vaccines. ACTIVATION-DEPENDENT EXPRESSION OF FOXP3 IN HUMAN CD4+CD25- T CELLS IS LIMITED AND STRICTLY REPRESSED BY DNA METHYLATION OF THE FOXP3 GENE

M. Nagar1,5, H. Vernitsky1, Y. Cohen1, D. Dominissini1, Y. Berkun4, G. Rechavi1,4, N. Amariglio1,3, I. Goldstein1,2

1Sheba Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer and Tel Aviv University, Ramat Aviv, Israel, 2Rheumatology Unit, Chaim Sheba Medical Center, Tel Hashomer, Israel, 3Institute of Hematology, Chaim Sheba Medical Center, Tel Hashomer, Israel, 4Division of Pediatrics, Edmond and Lily Safra Children's Hospital, Chaim Sheba Medical Center, Tel Hashomer, Israel, 5The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

The transcription factor FOXP3 is the master regulator of regulatory T cells (Treg). In mice CD4+CD25- T cells do not express Foxp3 following T cell receptor activation. Whether FOXP3 is a common activation induced molecule in all human T cells – hence not Treg restricted – is currently a controversial issue. Given the importance of FOXP3 in regulating the T cell’s effector functions, resolving this question in human T cells, mainly in the context of the epigenetic regulation of the FOXP3 gene, is of major significance. Here we show that in conventional CD4+CD25- T cells, the de novo expression of FOXP3 after TCR-activation is a limited phenomenon, and moreover it is particularly rare in activated tissue infiltrating CD45RO+ effector memory T cells. We next demonstrate that the usual silencing of FOXP3 transcription in resting and activated conventional T cells is associated with complete methylation of an evolutionary conserved intronic CpG-island that is, in contrast, unmethylated in natural Treg. Moreover, the inhibition of the maintenance DNA methyltransferase, DNMT-1, by the drug 5-aza-2’-deoxycitidine induced significant FOXP3 expression in cycling conventional T cells; a phenomenon which was further amplified by co- treatment with TGF-beta. These results indicate that the epigenetic inheritance of DNA methylation has a central role in the repression of significant FOXP3 transcription within the majority of recently activated conventional human CD25- T cells. Finally, our novel findings may facilitate the development of clinical therapies designed to amplify, in vivo, the number and function of Treg in patients with resistant autoimmune disorders. CD33: IS IT JUST A MYELOID MARKER?

H. Novak, K. Oved, Y. Reiter

Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel

CD33 (Cluster of differentiation 33) is a member of the Siglec (sialic acid–binding immunoglobulin-like lectin) family. CD33 is not found on normal hematopoietic stem cells but is considered to be specifically expressed on the myeloid lineage. CD33 is characterized by an amino-terminal V-set immunoglobulin (Ig) domain, one C2-set Ig domains, and a transmembrane domain, followed by a short cytoplasmic tail. The cytoplasmic domain contains two immunoreceptor tyrosine-based inhibitory motif (ITIM), which can recruit SHP-1 and SHP-2. CD33 function is unknown. We found that a subpopulation of about 6-13% of the T cells express CD33 following activation. We isolated T cells from peripheral blood of healthy donors. T cells were activated for 10 days and CD33 expression was detected starting day 3 and the following activation days. Our findings imply that CD33 might functions not only as a myeloid marker but participate during T cells activation. REDIRECTING T CELLS WITH CHIMERIC RECEPTORS COMPOSED OF T CELL RECEPTOR LIKE ANTIBODIES TOWARD THE TUMOR DERIVED ANTIGEN WT1 PRESENTED ON MHC COMPLEX

R. Oren1, Z. Eshhar2, Y. Reiter1

1Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel, 2Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Therefore, antibodies that have the ability to recognize these complexes with high affinity and specificity can be used for structural and functional studies of TCR- peptide-MHC interactions and are an important tool for obtaining accurate information about the presence and the expression of tumor derived antigens. These antibodies, termed TCR-like antibodies, can also be used for therapeutic applications of these diseases. The T-body approach combines antibody recognition and T cells effector function. It is based on T cells expressing chimeric receptors composed of antibody- derived scFV as their extra-cellular recognition elements joined to lymphocyte triggering molecules. Since T cells are well suited to penetrate and destroy solid tumors, the generation of T cells expressing chimeric receptors composed of TCR-like-scFV as their extracellular elements, will enable us to redirect them toward MHC-complexes presented on tumor cells. The use of redirected effector cells has potential in the immunotherapy of virtually any type of cancer. In this work we focused on the WT1 protein, a tumor antigen which fulfill some oncogenic functions in human acute myeloid and in progressing lymphoid leukemias. Overexpression of WT1 has been reported also in several other human malignancies including breast, lung, brain, ovary and skin. We have screened a Fab antibody phage display library with the produced MHC complexes containing the WT1 Db126 peptide (RMFPNAPYL). Several clones were obtained from the screened library and were further analyzed using ELISA assays and FACS analysis using APC loaded with the WT1 peptide. One clone, F2 showed highly specific binding to WT1/HLA-A2 positive tumor cells. Further work should be done in order to clone the F2 Fab into the T-body construct. COMBINING A FAST PROTOCOL FOR EX-VIVO DIFFERENTIATION OF HUMAN DENDRITIC CELLS WITH mRNA TRANSFECTION AS A NOVEL STRATEGY FOR THE GENERATION OF CANCER VACCINES

A. Pato1,2, G. Cafri1,3, M. Lotem2, A. Margalit1,4, G. Gross1,4

1Laboratory of Immunology, MIGAL, Kiryat Shmona, Israel, 2Sharett Institute of Oncology, Hadassah University Hospital, Jerusalem, Israel, 3Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 4Department of Biotechnology, Tel-Hai Academic College, Upper Galilee, Israel

Most traditional protocols for ex-vivo generation of human dendritic cells (hDCs) from blood monocytes require at least 5-7 days for differentiation into immature DCs and another 2-3 days of maturation and activation. This long period in culture is time- and labor-consuming. Most importantly, it narrows the time frame remaining for further genetic manipulation of these cells and their subsequent administration to the patients as vaccines, due to their limited life span. To shorten the culturing period substantially we employed recently published protocols and adjusted them to our culturing facilities. In this protocol we culture adherent monocytes for 24 hours in medium containing GM-CSF and IL-4 to drive DC differentiation. We then add a ‘maturation cocktail’ comprising TNF-alpha, IL-1-beta, IL-6 and prostaglandin E2 (PGE2) and grow the cells for another 24 hours. Here we show by flow cytometry analysis that hDCs grown according to the slow protocol (analysis performed at day 7 post cell thawing) or the fast protocol (at day 3) express comparable levels of the costimulatory markers CD83 and CD86. In our ongoing studies we are exploring genetic vaccines in the form of peptide-beta2 microglobulin (b2m)-anchor, where the anchor is either inert (derived from an MHC-I molecule) or is from a DC activation receptor, such as toll-like receptor (TLR) or CD40. We then went on and showed that transfection by electroporation of day-3 fast differentiated hDCs with mRNA encoding gp100(209-217)-b2m-TLR4, but not gp100(209-217)-HLA-A2 (with the inert anchor), results in DC activation in the absence of any added adjuvants of cytokines. This activation is manifested by upregulation of the surface co-stimulatory molecules CD80, CD83 and CD86. The combination of the fast protocol for hDC ex- vivo propagation and mRNA transfection offers a powerful tool for the generation of DC-based cancer vaccines with significantly extended life-span and elevated in-vivo potency. NATIVE HUMAN AUTOANTIBODIES TARGETING GIPC1 IDENTIFY DIFFERENTIAL EXPRESSION IN MALIGNANT TUMORS OF THE BREAST CANCER

S. Rohkin1, V. Yavelsky1, A. Tzikinovsky1, R. Shaco-Levy2, T. Amir1, H. Kohn1, B. Delgado2, A. Rabinovich3, B. Piura3, I. Trakht4, L. Lobel1

1Department of Virology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Pathology, Soroka University Medical Center and Faculty of Health Sciences, Ben Gurion University of the Negev, Beer- Sheva, Israel, 3Unit of Gynecologic Oncology, Soroka University Medical Center and Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel, 4Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY, USA

We have studied the native humoral immune response to cancer and isolated a library of fully human autoantibodies to a variety of malignancies. We have also isolated and characterized two fully human monoclonal antibodies, 27.F7 and 27.B1, from breast cancer patients that target the protein known as GIPC1/TIP-2, an accessory PDZ- domain binding protein involved in regulation of G-protein signaling. The current study employs cELISA, flow cytometry, immunocytochemistry, and immunohistochemistry. Immunohistochemical data were analyzed statistically with the Wilcoxon Signed Ranks Test and Spearman’s correlation. We have screened breast (MCF7), ovarian (SKOV3) and pancreas (PaCa) cancer cell lines with 27.F7 and 27.B1 antibodies using fibroblasts as a control. The cELISA results have revealed that both 27.B1 and 27.F7 antibodies demonstrate positive immunoreactivity against all cancer cell lines in contrast to fibroblasts. We have extended our study by further analysis of 27.B1 and 27.F7 antibodies interaction with breast cancer cell line and different types of malignant and benign breast tumors. The results of immunocytochemical experiments demonstrated specific staining of MCF7 cell line, by 27.F7 and 27.B1 and their internalization into these cells in contrast to fibroblasts. The immunohistochemical examination of different types of breast cancer with 27.F7 and 27.B1 demonstrates specific binding to malignant breast cancer tissue with no reactivity with normal breast tissue. Moreover, the results reveal that the level of GIPC1 expression depends on tumor invasiveness and displays a higher expression than in benign tumors. The present study demonstrates that the GIPC1 protein is overexpressed in breast cancer, which may provide an important diagnostic and prognostic marker and will constitute the basis for further study of the role of this protein in malignant diseases. In addition, this study suggests that human monoclonal antibodies 27.F7 and 27.B1 should be further evaluated as potential diagnostic tools. ALLOIMMUNE MEMORY IN THE SCLERACTINIAN REEF CORALS STYLOPHORA PISTILLATA AND ACROPORA HEMPRICHI

B. Rosental1, K. Amar2, B. Rinkevich2

1Department of Immunology and Microbiology, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Oceanographic and Limnological Research, The National Institute of Oceanography, Haifa, Israel

Immunological memory though a known phenomenon in vertebrate adaptive immunity, is still a debatable issue in the invertebrate immune system. The occurrence of a memory component in allorecognition responses was suggested to exist in several invertebrate groups including sponges, cnidarians, annelids, echinoderms, and tunicates. However the expression of alloimmune memory in invertebrates is not ubiquitous, even within the same taxonomic group. We studied alloimmune memory possible expression in the allogeneic reactions of Stylophora pistillata and Acropora hemprichi. The study objectives: 1. To elucidate a possible memory component in the allorecognition response and to evaluate its range of specificity. 2. To examine the impact of repeated allogeneic challenges on the establishment of the hierarchies between tested coral genotypes. In this study, tissue contacts between three allogeneic colonies in situ were established in order to elucidate a possible memory component in this response. First set assays (phase I) were established from all possible pairings between 3 colonies (different set for each species). Thereafter, interacting pairs were detached and challenged in second set assays (phase II). In phase I of S. pistillata allogeneic hierarchies were established. Results of phase II suggest enhancement in the alloimmune responses by reduction in time of first response. Alloimmune response specificity was not conclusive and therefore memory component has not yet been proven. During phase I of allogeneic encounters (7 weeks long) of A. hemprichi no visible responses were documented. A shift was observed in the hierarchies directionality between tested coral genotypes of S. pistillata. Although not conclusive, this might suggest an impact of repeated allogeneic challenges on coral physiology. STUDY OF ANTIGEN PRESENTATION USING T-CELL RECEPTORS

K. Sinik, Y. Reiter

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

For the immune system to function properly it must differentiate foreign/damaged cells from its own. In order to achieve this nuclear cells display peptides bound to MHC-I molecules on their surface. Our laboratory isolated unique antibodies that bind specific pMHC complexes similarly to T-cell receptor with high affinity as antibodies, thus named 'TCR-like antibodies'. During an immune response, T-cells secrete the cytokine INF-gamma, inducing effects on the target cells such as increased antigen presentation and transformation of constitutive proteosomes to immunoproteosomes. Immunoproteosomes, differ from constitutive proteosomes by three catalytic beta subunits. These subunits are associated with 'three major peptidase activities' of the proteosomes and the replacement may alter the peptidase activity of the proteosomes. In our research we examine sets of melanoma cells that have been subjected/not subjected to INF-gamma. Using different TCR-like antibodies specific to melanoma on the presentation of ד-characteristic pMHC complexes, we study the effect of INF melanoma specific antigen presentation. Western analysis enables us to examine the expression level of the melanoma proteins these epitopes are derived from and of immunoproteasome subunits and detect the differences between cells stimulated/not stimulated by INF-gamma. Preliminary results show that while INF-gamma had no effect on the presentation of gp100 epitope, epitopes derived from MART and This effect .ד-Tyrosinase show higher levels of presentation after stimulation by INF was not in correlation with MART and Tyrosinase expression levels within the cells. The effect of INF-gamma was dependant on the time of stimulation. Each of the cell lines studied showed increased presentation to a different extent. These effects of INF-gamma were accompanied by replacement of constitutive proteosome to immunoproteosome. Our TCR-like antibodies enabled us for the first time to study in a qualitative and quantitative manner to better understand the ד-the effects of INF molecular mechanisms involved in antigen presentation. SIGNAL SUMMATION OF CD8+ T-CELLS FOLLOWING POST PRIMING ANTIGEN DENSITY-DEPENDANT ANERGY

R. Wolchinsky, K. Oved, Y. Reiter

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Following an encounter between professional APC and naive T-Cell clone, taken place in a secondary lymphoid organ, the naive antigen-specific CD8+ T cells become activated, proliferate, acquire effector functions, and differentiate into effector CD8+ T cells. Most effector CD8+ T cells die by apoptosis, but 5%–10% survive and differentiate into memory CD8+ T cells. These memory CD8+ T cells, following second activation, exert their cytotoxic effects on targeted pathogenic cells. The minimal antigen densities required for proper CTL mediated killing were the focus of extensive investigation in the past decade. During the past year a novel mode of action was described, which transform human activated memory cytotoxic CD8+ T cells anergic. The molecular machinery for this Ag-induced postpriming control mechanisms entail changes in the expression of surface molecules that control synaptic activity, changes in cytokine profiles, and extensive alteration in gene expression profiles related to energy metabolism, membrane potential, impaired TCR signaling and function, and cell cycle arrest. We have shown that the conglomeration of these results manifests a unique gene expression signature that confers anergy on memory T cells that encounter high Ag density on their target cells. We have begun to elaborate on the processes taking place in specific niches on which the gene-profiling shed light on, such as iNOS up-regulation and changes in membrane potential following varying antigen densities encounters. Mainly, we intend to demonstrate the process under which the cell, after encountering an APC and sampling the specific complexes which it presents, process them and respond to these different signals arising from sub-optimal, optimal and supra-optimal antigen densities. CHARACTERIZATION, CONTROLLED ASSEMBLY AND PATTERNING OF AROMATIC DIPEPTIDES NANOSTRUCTURES

L. Adler-Abramovich, E. Gazit

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Organic and inorganic self-assembled tubular nanostructures were suggested to have key potential in nanotechnological devices and applications. The diphenylalanine peptide efficiently self-assembles into discrete, well-ordered peptide nanotubes. Using different microscopy and spectroscopy tools we describe a remarkable thermal stability of aromatic dipeptide nanotubes (ADNT) both in aqueous solution and under dry conditions. In addition, the ADNT exhibit substantial chemical stability in various organic solvents. Furthermore, we studied the ADNT mechanical properties, which were directly measured by indentation type experiments using an atomic force microscopy. We found that the ADNT maintain high averaged point stiffness of 160 N/m, and correspondingly high Young’s modulus of approximately 19 GPa, which places these nanotubes among the stiffest bio-inspired materials presently known. Moreover, we explored the potential of a self-immolative dendritic system to serve as a transporter platform for control assembly of the ADNT. Self- immolative dendrimers are a novel class of molecules that can amplify a single cleavage event, which is received at a focal point, into multiple releases of tail groups at the periphery. Additionally, we used the inkjet technology for the application of ADNT on non-biological surfaces. The ADNT were used as an "ink" and patterned on transparency foil and ITO plastic surfaces using a commercial inkjet printer.

References: 1. Kol, N., Adler-Abramovich, L., Barlam, D., Shneck, R. Z., Gazit E., & Rousso, I. Nano Lett. 5, 1343 -1346 (2005). 2. Adler-Abramovich, L., Reches, M., Sedman, V. L., Allen, S., Tendler, S. J. B., & Gazit, E. Langmuir 22, 1313-1320 (2006). 3. Adler-Abramovich L., Perry R., Sagi A., Gazit E., Shabat D. ChemBioChem. 8, 859-862 (2007). 4. Adler-Abramovich, L., and Gazit, E. J. Pep. Sci. (2007). SPECTROSCOPIC AND MICROSCOPIC ANALYSIS OF THE SELF- ASSEMBLY OF AROMATIC DIPEPTIDES NANOSPHERES

I. Cohen-Or, L. Adler-Abramovich, E. Gazit

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Self-assembled nanostructures provide key direction for the controlled fabrication of novel materials and devices at the nano-scale. Peptide nanotubes and nanospheres are especially intriguing assemblies as they have the scope for numerous chemical modifications and allow the use of biological systems specificity. Furthermore, peptide-based nanomaterials are extremely attractive as potential building blocks for various nanotechnological application, for instance, electronic devices, sensors, catalysis, magnetic devices, drug delivery, tissue engineering, ion channels, and medical imaging because their physical and chemical properties are tuneable via control of their shape and size. The diphenylalanine peptide, the core recognition motif of Alzheimer's beta-amyloid efficiently self-assembles into discrete, well- ordered peptide nanotubes. Ordered structures are also form by the self assembly of the amine-modified analogue of the diphenylalanine peptide, t-butyl-carbamate-Phe- Phe-OH (Boc-Phe-Phe-OH). Recently we discover that this amine modified peptide self assembled into two types of nanostructures under different conditions: nanotubes or nanospheres. When the Boc-Phe-Phe-OH is dissolved in fluorinated alcohol then diluted in water, tubular structures are formed. However, diluting the peptide solution in 50% ethanol, results in the formation of nanometric spherical structures. Using different spectroscopy and microscopy tools, we examined the effect of different parameters such as the solvent contents, relative humidity and temperature on the formation of either spherical or tubular structures. These finding will lead to a better understanding of the self assembly mechanism of aromatic dipeptide nanostructures and might allow the use of these self assembled nanostructure in future applications such as nano electromechanical system (NEMS) or medical application. SYNTHETIC GENE BRUSHES: RESPONSE OF EXPRESSION TO CROWDING AND CONFINEMENT

A. Buxboim1, S.S. Daube2, R. Bar-Ziv1

1Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel, 2Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

Synthetic gene expression systems that function autonomously on materials platforms are a vision of nanotechnology. A chip with immobilized genes is an innate environment to separate, cascade, and control biosynthetic reactions. We assembled gene brushes made of double-stranded DNA molecules coding for entire genes that are attached to a surface at one end. This was made possible by a novel photolithographic biochip approach with which we could tune the mean distance between neighboring molecules, from 300 nm, where 2000 base-pair DNAs assume a relaxed conformation, down to 30 nm, where DNAs extend in a dense configuration. We carried out a biochemical “structure-function” study of gene brushes and found that RNA transcription and protein biosynthesis are exquisitely sensitive to DNA density, conformation, and orientation. We observed simple linear gene/protein relationship for stretched genes but non-linear, non-monotonous behavior for dilute relaxed conformations, with attenuation at dense gene packing due to molecular crowding. An additional hallmark feature of gene brushes is the local buildup of product concentration at the proximity of the brush, and localization of RNA polymerase at distinct depths within the brush, depending on promoter positioning. This study shows that on-chip gene expression can be controlled at the nano-scale by locally adjusting the structure of the gene brush. In turn, gene brushes as a synthetic model could deepen our understanding of biochemical reactions taking place under confinement and molecular crowding in living cells. MICRO-BIOREACTORS FOR CULTURE OF HUMAN EMBRYONIC STEM CELLS

N. Korin, A. Bransky, U. Dinnar, S. Levenberg

Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel

Micro-technologies can be used to pattern and control cells at cellular length scale. Thus, micro-bioreactors have great potential for Embryonic Stem cell (ES) studies and applications. The present work studies the conditions required for culturing human ES in a novel microchannel system. The role of design parameters is evaluated and discussed. We have previously shown the ability to long-term culture of mammalian cells under constant perfusion in plan a microchannel configuration. However, human ES cells are highly responsive to flow and culture conditions. Recently, we have successfully used micro-wells to shear protects and co-culture human ES cells with HFF within a microchannel bioreactor. Additionally, we examine the use of a novel automated periodic perfusion system to co-culture human ES cells with HFF in a microchannel bioreactor. PDMS microchannels (depth: 100 micron) are fabricated using conventional soft-lithography methods. The channels are sterilized, coated with a human fibronectin solution and seeded with cells. Following static incubation, the microchannels are connected to a perfusion system. The perfusion system consists of a syringe pump, a custom-made oxygenator and a waste container. The system is kept inside an incubator (apart from the syringe pump). Cell growth is monitored throughout the culture process. Mass Transport and fluid mechanics models are used to estimate the culture conditions within the micro- bioreactor (shear stress, oxygen level, nutritious etc.). 2D and 3D finite elements simulations (Comsol 3.3) are preformed giving insight into design parameters. Altogether, the experimental results and the theoretical models indicate that the use of novel perfusion bioreactors may be suitable and promising to culture human ES in micro-reactors. Culturing undifferentiated human ES cell colonies in a micro- bioreactor is an initial step toward utilizing microfluidic techniques to investigate embryonic stem cell biology. SELF-ASSEMBLED PEPTIDE-BASED HYDROGEL AS BIOCOMPATIBLE AGENT FOR DRUG DELIVERY, TISSUE ENGINEERING AND REGENERATION

R. Orbach, A. Mahler, E. Gazit

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Hydrogels are of great interest as class of materials for drug delivery as well as tissue engineering and regeneration as they offer 3D scaffolds that can support the growth of cultured cells. Peptide-based hydrogels incorporate the advantages of both synthetic and naturally derived hydrogel forming materials. They are easy to manufacture in large quantities and can also be easily decorated chemically and biologically. Such decoration gives the ability to design an ultra-structure that presents ligands, as well as other functional groups. The diphenylalanine peptide, the core recognition motif of the beta-amyloid polypeptide, efficiently self-assembles into discrete, well-ordered nanotubes. During the process of its characterization, we discovered that dissolving the Fmoc-diphenylalanine peptide at higher concentration in aqueous solution resulted in the formation of a rigid material with macroscopic characteristics of a gel. Although the hydrogel contained less than 1% peptide material, it kept its 3D spacious volume exceptionally well. This newly discovered hydrogel is formed under mild conditions in an aqueous solution. In spite of the short building-block size, it has physical properties that exceed those of hydrogels formed by longer polypeptides. We suggest that this rigidity is facilitated by the aromatic nature of the peptide building block and can be very advantageous in applicative technologies. In addition, the hydrogel is very stable under extreme conditions, can be injected, and can be shaped in accordance to the vessel it is assembled in. In order to get better understanding about the properties of Fmoc-peptide fragments which comprise aromatic residue, we study the self-assembly properties of various Fmoc-Peptides to form hydrogels and other nano-ordered assemblies as well as the possibility to change the hydrogel macroscopic properties. A NOVEL APPROACH FOR SENSING PEPTIDE-NANOPARTICLE BINDING

D. Reichmann1, T. Karakouz2, A. Vaskevich2, I. Rubinstein2, G. Schreiber1

1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel

The study of biomolecules for nanobiotechnology applications has grown enormously in recent years with hopes of exploiting self-assembly and molecular recognition for integrating advanced materials into medicine and industry. However, despite the remarkable speed of development of nanosceince, relatively little is known about general principles governing specific interaction between peptides/proteins with inorganic surfaces, and this is now a serious bottleneck in nanomedicine and nanotoxicology enterprise. A deep understanding of the biological effects on nanopraticles requires knowledge of both chemical composition of the proteins or peptides and the kinetics properties of the interaction, such as binding affinities and stochiometries for different combination of proteins and nanoparticles. Our understanding of protein - nanoparticle interactions and their biological consequences may be advanced by development methods to identify and measure wide kinetic range of binding between proteins and nanoparticles. Here, we introduce an approach to measure binding between different proteins and gold nanoparticles in real time using Surface Plasmon Resonance (SPR). We developed a method of specific immobilization of the particles to the sensor surface through avidin protein, which does not restrict the measurement only to gold. To evaluate binding of specific peptides we identified a protein, which is inert to gold and used as a linker for peptide of interest. This approach was applied to study a mechanism of binding between gold and oligopeptides containing Histidine residues with varying length. The results clearly showed an increase in gold binding as the length of the peptide grows, up to a maximum of 6xHis. Clear pH dependence was seen for His binding, which was enhanced upon lower pH values, suggesting that the protonated form of His binds gold, while the non-protonated form does not. Currently, this approach is implemented to investigate binding between amino acid composition and different inorganic nanoparticles. ULTRA-POROUS AND HIGHLY PERMEABLE SCAFFOLD FOR TISSUE ENGINEERING THROUGH HYDROSPINNING

R. Tzezana1, E. Zussman1,2, S. Levenberg1,3

1Russel Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa, Israel, 2Faculty of Mechanical Engineering, Technion-Israel Insitute of Technology, Haifa, Israel, 3Faculty of Biomedical Engineering, Technion-Israel Insitute of Technology, Haifa, Israel

One of the most important aspects of tissue engineering is the scaffold on which the tissue is grown in vitro. That scaffold must be porous enough so that the cells can infiltrate the scaffold and have the space to grow inside, but must also have a structural integrity leading to mechanical stability. Electrospinning is a method to create nano-fibrous scaffolds, that are highly porous (up to 90%), but their pores size – and therefore their permeability to the cells - is limited. We have devised a novel method, called hydrospinning. Using a wet coagulation bath and a layer-by-layer build-up, we have managed to create a new scaffold with porosity of 97%, which proved more permeable to cells than its electrospun counterpart. The resulting scaffold also exhibits high interconnectivity and biocompatibility, as usually is the case with ordinary electrospun scaffolds. Experimental evidence shows that myoblasts have managed to infiltrate up to 700 micro-meters into a hydrospun scaffold, with an average 800 nm diameter fibers, while in the traditional electrospun scaffold, woven of the same size of nanofibers, the cells managed to infiltrate only 200 micrometers deep. Further experiments have demonstrated that there are at least 30% more cells per unit area inside the novel hydrospun scaffold, than in the traditional electrospun scaffold. Based on this evidence, we propose to establish hydrospinning as a novel and efficient way to create highly porous and permeable scaffolds for tissue engineering applications. THE USE OF SELF ASSEMBLED PEPTIDE NANOSTRUCTURES TO ENCAPSULATE MAGNETIC NANO-PARTICLES

I. Yanai, E. Gazit

Department of Molecular Biology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

Lose of cerebral neural cells is observed in several age-associated disorders such as Alzheimer's disease, Parkinson's disease and other neurodegenerative disorders. Currently, the medical procedures for the diagnosis of those diseases are not very conclusive. The need for new methods is relevant now more than ever with longer life expectancy and the increase of the elderly population. The work at our laboratory of the mechanism of self-assembly had led to the discovery of aromatic dipeptides that self assemble into various structures such as nanotubes, nanospheres and others. These structures are well-ordered and discrete. The current research is directed towards the development of novel contrast agents that are based on peptide nanostructures loaded with ferromagnetic nano-particles. The experimental model includes peptides which self assemble into closed caged nanospheres under certain conditions. The spheres are formed in the presence of ferromagnetic nanoparticles. The peptides then encapsulate the nano-particles and form closed spheres. In order to develop efficient diagnostic tool the nanospheres will be decorated with recognition motifs that could specifically bind antigens that are associated with various degenerative diseases. FUNCTIONAL ORGANIZATION OF A PLANT PHOTOSYSTEM I

A. Amunts, O. Drory, N. Nelson

Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel

Photosystem I (PSI) catalyzes the light-driven electron transfer from the soluble electron carrier plastocyanin, located at the lumenal side of thylakoid membrane, to ferredoxin, which is located at the stromal side. Being one of the most intricate membrane complexes, plant PSI performs the photochemical activity with an unprecedented quantum yield of close to 1.0. Basing on intense analysis of membrane protein crystallization procedures, we developed a high-yield purification method of PSI out of thylakoid membrane. Large amounts of purified, intact and homogenic material allowed optimization of crystallization conditions, which enabled the recent determination of the X-ray crystal structure at 3.4ֵ resolution [1]. The model reveals locations of 17 out of 19 currently known protein subunits. Positions of 168 chlorophylls, 2 phyloquinones, 3 Fe4S4 clusters and 5 carotenoids are assigned. The remarkable feature of PSI structure is the unprecedented extremely high content of non-protein components – approximately one third of the total mass of about 650 KDa consists of different co-factors. The structure reveals unique interactions between the LHCI and the RC complexes and provides a structural basis for the state transitions phenomenon. In addition, putative docking sites of the soluble electron carriers are described for the first time at almost atomic resolution.

[1] Amunts, A., Drory, O. & Nelson, N. (2007) Nature, 447, 58-63. NOVEL THERMOPLASTIC CYANOBACTERIA

J. Dinamarca1, O. Kerner1, D. Kaftan2, A. Scherz1

1Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel, 2Institute of Physical Biology USB, Institute of System Biology and Ecology ASCR, Nove Hrady, Czech Republic

The maximal rate of solar energy conversion and biomass production by mesophilic photosynthetic organisms is restricted to a relatively narrow range of temperatures between 20-30 C. This temperature range is mainly a reflection of constraints upon the photosystems and the Rubisco complex. PSII RC is a key player in the acclimation of energy conversion rates to ambient temperatures. We have discovered a sequence motif in the D1 protein of the PSII RC and identified adaptive mutations capable of shifting the maximal photosynthetic rates to higher temperature (Kerner- Shlik et al, Nature, 2006). Here we present thermoplastic D1 mutants of Synechocystis sp. PCC 6803 which acclimatize their photosynthetic rates reaching high biomass and chlorophyll contents under stress temperature 43 C. The phenotypes of both the mutants and wild type did not show a significant difference at standard growth temperature (30 C), however, complete bleaching of the wild type was observed after 7 days of incubation at 43 C and at 43-10-43 C temperature cycle. Mutants were found to contain an increased D1, psaC and Rubisco protein levels compared with the wild type under stress conditions. These findings offer new insights and provide guidelines for the generation of novel organisms capable of enhanced photosynthesis and the provision of biomass over a wider temperature range.

Shlyk-Kerner, O., Samish, I., Kaftan, D., Holland, N., Sai, P.S. M., Kless, H. & Scherz, A. Protein Flexibility Acclimatizes Photosynthetic Energy Conversion to the Ambient Temperature. Nature 442, 827-830 (2006). CHANGES IN THE PHOTOSYNTHETIC REACTION CENTER II RESULTS IN NON-PHOTOCHEMICAL FLUORESCENCE QUENCHING IN THE DIATOM PHAEODACTYLUM TRICORNUTUM

D. Eisenstadt1, I. Ohad2, N. Keren1, A. Kaplan1

1Department of Plant and Environmental Sciences, Hebrew University of Jerusalem, Israel, 2Department of Biological Chemistry, Hebrew University of Jerusalem, Israel

Diatoms are an important group of primary producers in the aquatic environment. They are able to adapt to fast changes in the light intensity by various mechanisms including a fast rise in non-photochemical quenching (NPQ). The NPQ has been attributed to the xanthophyll cycle (XC) following activation of diadinoxanthin de- epoxidase by the acidification of the thylakoid lumen. To examine whether fluorescence quenching depends on the delta pH generated by the photosynthetic electron transport, we arrested the latter by DCMU. This treatment hardly affected the NPQ or XC, even when methylviologen was present. Dissipation of the delta pH by 4- Dinitrophenol inhibited the XC but did not alter NPQ. Similar results, i.e. inhibition of the XC but normal fluorescence quenching were observed when the experiments were performed at 3 C. Measurements of thermoluminescence showed that high light treatment caused a marked decline in the signals obtained due to recombination of QA- and QB- with the primary donor. The light treatment also diminished the QA oxidation signals. The analysis of the data suggested that changes in PSII core center due to exposure to high light conditions plays an important part in the acclimation to the high light treatment. ACCLIMATION FROM PHOTOAUTOTROPHIC TO PHOTOMIXOTROPHIC GROWTH - HOW DOES IT WORK?

M. Haimovich1, S. Kahlon1, Y. Hihara2, J. Lieman-Hurwitz1, A. Kaplan1

1Department of Plant and Environmental Sciences, Hebrew University of Jerusalem, Israel, 2Bioscience Center, Nagoya University, Nagoya, Japan

Presenting author number 1

The cyanobacterium Synechocystis sp. PCC 6803 grows photoautotrophically under light conditions and survives in the dark by catabolizing storage carbohydrates such as glycogen. A glucose-tolerant (GT) mutant of Synechocystis that was isolated can grow photomixotrophically under light conditions when provided with glucose as well as heterotrophically in the dark if provided with a daily pulses of white light. Little is known about the regulation of the switching from photoautotrophically growth to photomixotrophically growth. An histidine kinase, Hik31, was found to be involve at glucose sensing. Inactivation of hik31, resulted in inactivation of glucokinase at the post transcriptional level. Glucokinase mediate the conversion of glucose to G6P in the oxidative pentose pathway and its inactivation causes the GT strain to be unable to grow on glucose. PmgA an homolog to an anti sigma factor, is essential for the acclimation of Synechocystis to changing light intensity, but the growth of its mutant, pmgA, was found to be impaired under photomixotrophic conditions. This phenotype could be rescued by inactivation of ndhF3 (involved in co2 uptake) in the background of the pmgA mutant. The mechanism involved is being examined. GENETIC MODIFICATIONS OF PHOTOSYSTEM II OF SYNECHOCYSTIS PCC 6803 TO PROMOTE ELECTRON TRANSFER FROM THE NATIVE ELECTRON-TRANSFER CHAIN TO AN ELECTRON ACCEPTOR MOLECULE.

F. Salama1, S. Larom2, G. Schuster2, N. Adir1

1Department of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel, 2Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Oxygenic photosynthetic organisms such as plants, algae and certain bacteria efficiently use light energy and water to produce both storable chemical energy and reducing power via the photosynthetic process. The energy of light is converted into the motion of energized electrons in the reaction center at the heart of photosystem II, a membrane protein complex located in the thylakoid membranes. The proteins D1 and D2 are two polypeptides located at PSII reaction center, between them a pair of chlorophyll a molecules, P680. When a photon is absorbed, P680 is excited and the electron is rapidly transferred to a pheophytin, and then to plastoquinone. QA accepts the electron from the pheophytin. After axcepting two electrons from QA, the QB plastoquinone is converted into a plastoquinol, released from the photosystem, and delivers its electrons to the next link in the electron-transfer chain. In this work we are genetically modifyingy the PSII proteins of Synechocystis PCC 6803, D1 and D2, in a way that a high binding affinity site will be introduced in close vicinity to the plastoquinone binding site. Binding of a putative electron acceptor molecule (a hemoprotein) within 10-15A of the quinone site may be sufficient to promote electron transfer to the photoreduced QA and then to the carrier protein A NEW FAMILY OF ARABIDOPSIS SHORT MEMBRANE PROTEINS LOCALIZING TO ER, PLASTIDS, PEROXISOMES AND MITOCHONDRIA

M. Abu-Abied1, E. Belausov1, Z. Kam2, E. Sadot1

1Institute of Plant Sciences, Volcani Center, Bet-Dagan, Israel, 2Department of Molecular Biology of the Cell, Weizmann Institute of Science, Rehovot, Israel

In a functional genomic screen done by combining Arabidopsis YFP fused cDNA library, rat fibroblasts as host and automatic microscopy we found an unidentified short protein with a predictable trans membrane domain. In rat fibroblasts it localized to the ER. From the data bases we learned that it has one more family member in Arabidopsis and homologs in Rice and in the fungus Ustilago maydis. We named these two proteins from Arabidopsis SMP1 and 2 (Short Membrane Protein). No SMP homologs were found in mammalians. Surprisingly, when the full length cDNAs of SMP1 and 2 were fused to YFP under viral promoters similar localization in rat fibroblasts and N. benthamiana leaves was observed, suggesting evolutionary conserved localization signals yet unknown. While SMP1 was found primarily in the ER, SMP2 was found primarily in membranes of different organelles. Similar localization of both genes was observed when expressed in N. benthamiana leaves under the control of their own promoters. Colocalization studies with GFP and RFP chimeras revealed that RFP-SMP1 colocalized with GFP-ER marker but in addition stained other organelles, not stained by the GFP-ER marker. These organelles where stained by SMP2 being either chloroplasts, other plastids with long stromules, peroxisomes and mitochodria. No colocalization of Golgi with SMP1 or 2 was observed. Further work to reveal the biological function of these proteins is carried out. ANTIOXIDANT ACTIVITY, POLYPHENOL CONTENT AND RELATED COMPOUNDS IN DIFFERENT FRUIT JUICES AND HOMOGENATES PREPARED FROM 29 DIFFERENT POMEGRANATE ACCESSIONS

R. Tzulker1, I. Glazer1, I. Bar-Ilan1, D. Holland2, M. Aviram3, R. Amir1

1Migal Galilee Technology Center, Kiryat Shmona, Israel, 2Institute of Plant Sciences, Agricultural Research Organization, Newe Ya’ar Research Center, Ramat Yishay, Israel, 3Lipid Research Laboratory, Technion-Israel Institute of Technology, Rappaport Faculty of Medicine, Haifa, Israel

The traditional importance of pomegranate as a medicinal plant is now backed by data obtained from modern science. Recent studies have indeed shown that secondary metabolites in the fruit exhibit anti-atherosclerotic, anti-carcinogenic activities, and possess antimicrobial and anti-viral properties. These attributed to its high level of antioxidant activity and total polyphenol content. Until today, most research studies have focused on a commercial pomegranate accession called Wonderful. Therefore considerable knowledge is still lacking about the similarities and differences in the various activities of diverse pomegranate accessions. In order to achieve this knowledge we screen 29 accessions differing in their arils and peels colors as well as in their taste. Next we search after the fruit fraction (arils and peels) that have the highest antioxidant activity and analyzed the content of polyphones, anthocyanins and four members of hydrolysable tannins to explore the roles of these compounds in the antioxidants activities. The results showed that the antioxidant activity in aril juice correlated significantly to the total polyphenol and anthocyanin contents. However, the homogenates prepared from the peels exhibited an approximate 40-fold higher antioxidant activity than the level found in the aril juice. Unlike the arils, the antioxidant level in the homogenates correlated significantly to the content of the four hydrolysable tannins in which punicalagin is predominant, while no correlation was found to the level of anthocaynins. The results also showed that accessions with high sugars contents have higher antioxidant activity, and among the 6 organic acids that can be found in arils juice, only the acetic acid content was correlated to the level of the antioxidant activity. In future, such information will enable breeders to select and breed genotypes having higher levels of health beneficial compounds, and also provide useful information for addressing consumer choices for healthier products. THE PLANT ACTIN-MYOSIN MOTILITY SYSTEM IN ORGANELLE TRAFFICKING AND VIRUS CELL TO CELL MOVEMENT

D. Avisar1,2, A.I. Prokhnevsky1, V.V. Peremyslov1, V.V. Dolja1

1Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA, 2Volcani Center, Bet-Dagan, Israel

Myosins are diverse molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, XI and VIII, whose specific functions are poorly understood. Here we present evidence for differential specific activities for myosins from the two groups. Six full-length myosin cDNAs from Nicotiana benthamiana (N.b.) were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana (A.t.) myosins XI-2, XI-F, XI-K, VIII-1, VIII-2, and VIII-B. We used dominant negative inhibition and RNA interference to identify myosins required for organelle movement and virus cell to cell movement. We found that myosin XI-K is involved in the movement of Golgi stacks, mitochondria and peroxisomes but not chloroplasts in both N.b. and A.t. Interference with myosin XI-K activity leads to slow down and/or arrest of the organelle movement. Our analysis of thousands of individual organelles revealed uncoordinated movement patterns and variable speeds for Golgi stacks, mitochondria, and peroxisomes. Thus we suggest that the active organelle movement is not directed by the motion of cytoplasmic streaming. Ectopic expression of the tail domains of each of the N.b class VIII, but not class XI myosins inhibited plasmodesmatal localization of Hsp70h, that functions in virion assembly and cell-to-cell movement of the Beet yellows virus (BYV). In contrast, overexpression of neither the motor domains, nor the entire myosins VIII affected Hsp70h targeting. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Our data implicate class VIII myosins into protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants. GTP BINDING PROTEIN TYPA AND REPRODUCTIVE ORGAN DEVELOPMENT IN CUCUMBER

M. Barak, T. Trebitsh

Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Cucumber (Cucumis sativus) is a monoecious plant that is extensively studied with respect to floral sex determination. While the genetic background, hormonal and environmental factors are well characterized, the molecular mechanisms regulating unisexual flower development are not well understood. A cucumber flower has the potential to develop into a male or female flower. To study the molecular basis of unisexual flower development we utilized near isogenic male (monoecious) and female (gynoecious) cucumber lines to isolate genes involved in female or male sex expression. Using differential cDNA-AFLP analysis between plant apices of male and female cucumber plants, we isolated a cDNA fragment that encodes a putative GTP binding protein tyrosine phosphorylated protein A (CsTypA1). Full length cDNA encoding CsTypA1 was isolated by 5' and 3' RACE. Bacterial TypA proteins are involved in protein translation and have been proposed to function as global regulators of different regulatory pathways. The role for TypA proteins in plants is unknown. This is the first report of a putative TypA gene in the plant kingdom that is differentially expressed during plant development. CsTypA1 is expressed in stamen primordia and its transcript is more abundant in monoecious plant apices compared with gynoecious ones, implicating the gene in the early stages of male reproductive organ development. However, at later stages of floral bud development CsTypA1 expression is high in stigmatic papilla, nectary and specifically in the ovule/ovary tissue of female flowers. Therefore, it is possible that CsTypA1 functions both in the early stages of flower development, possibly during sex determination, and at later floral developmental stages, after floral sex determination, in ovary/ovule development. IDENTIFICATION AND MAPPING OF POWDERY MILDEW RESISTANCE GENE DERIVED FROM WILD EMMER WHEAT ON THE DISTAL END OF CHROMOSOME 7AL

R. Ben-David1, Z. Peleg2, W. Xie1, Y. Saranga2, A. Dinoor2, A.B. Korol1, T. Fahima1

1Institute of Evolution, University of Haifa, Israel, 2Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel

Powdery mildew, caused by a biotrophic pathogen Blumeria graminis (DC.) E.O. Speer f. sp. tritici Em. Marchal (hereafter Bgt), is a foliar wheat disease resulting in severe yield losses worldwide. The wild emmer wheat, Triticum turgidum ssp. dicoccoides, genepool is known to harbor a rich allelic repertoire for numerous important traits including disease resistances. A large collection of wild emmer and cultivated wheat (T. durum, T. aestivum) genotypes was screened for powdery mildew resistance using a set of 42 Israeli Bgt isolates, revealing that wild emmer accession G18-16 was resistant for 29 isolates. Two populations, derived from a cross between wild emmer (acc. #G18-16) and durum wheat (cv. Langdon), were used to identify and map the powdery mildew resistance genes. At first, Ninety-three F3 families were tested for resistance with Israeli Bgt isolate #15 collected from T. durum wheat in Yavor, Israel. The segregation ratio for the powdery mildew reaction of the population indicated that the resistance in G18-16 was probably controlled by a single dominant gene. In a second stage, 152 Recombinant Inbreed Lines (RILs) from independent pedigree of the same cross were served for mapping. The segregation ratio for the powdery mildew reaction of the RILs population re-established the roll of a single gene, temporarily designated as PmG18-16. A linkage map consisting of 17 microsatellite and DArT markers was constructed (total chromosome length 144.5 cM). The PmG18-16 was mapped to the long arm of chromosome 7A, 6.4 cM away from marker Xgwm1061. The identification and mapping of new powdery mildew resistance gene, derived from wild emmer, will enhance the improvement of powdery mildew resistance in elite wheat cultivars by marker assisted breeding. RIBOSWITCH-DEPENDENT GENE REGULATION AND ITS EVOLUTION IN THE PLANT KINGDOM

S. Bocobza1,2, A. Adato2, T. Mandel2, M. Shapira1, E. Nudler3, A. Aharoni1

1Department of Natural Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel, 3Department of Biochemistry, New York Medical Center, NY, USA

Riboswitches are natural RNA sensors that affect gene control via their capacity to bind small molecules. Their prevalence in higher eukaryotes is unclear. We discovered a post-transcriptional mechanism in plants that employs a riboswitch to control a metabolic feedback loop through differential processing of precursor-RNA 3'-terminus. When cellular thiamin-pyrophosphate (TPP) levels rise, metabolite sensing by the riboswitch located in TPP biosynthesis genes directs formation of an unstable splicing product and consequently TPP levels drop. When transformed in plants, engineered TPP riboswitches can act autonomously to modulate gene expression. In an evolutionary perspective, a TPP riboswitch is also present in ancient plant taxa suggesting that this mechanism is active since vascular plants emerged 400 million years ago. COMPARATIVE INTERSPECIFIC PROMOTER ANALYSIS OF A MAJOR GENE (cwp) WHOSE EVOLUTIONARY SILENCING CONTRIBUTED TO THE DOMESTICATION OF THE FLESHY TOMATO

N. Chehanovsky1, A. Mett1, E. Belausov2, M. Moy1, S. Shen1, D. Gidoni1, A.A. Schaffer1

1Department of Vegetable Research, Institute of Plant Sciences, Volcani Center, Bet Dagan, Israel, 2Microscopy Unit, Institute of Plant Sciences, Volcani Center, Bet Dagan, Israel

Tomato fruit maintain high turgor even following harvest, due to a continuous, astomatous cuticular layer. Tomato genotypes characterized by dehydration during and following ripening were developed from the progeny of an interspecific cross between the cultivated Solanum lycopersicon and the primitive green-fruited wild specie Solanum habrochaites. The trait is determined by a single gene, termed cwp, and the wild species allele confers a microfissured cuticle which allows for water loss from the ripe fruit. The gene sequence was present in the genomes of all eight tomato species tested, but expressed only in fruit of the primitive green-fruited species (S. habrochaites, S. peruvianum, S. chmielewskii, S. penellii, S. lycopersicoides), not in the yellow/red-fruited species (S. lycopersicum, S. pimpinellifolium, S. cheesmaniae). Alignment and interspecific promoter comparisons of the upstream 800bp promoter sequences of these eight species revealed only two common differences which distinguish between the species expressing and not expressing cwp. A ten bp sequence (insert-I, 143bp upstream to start codon) and a nine bp sequence (insert-II, 261bp upstream to start codon) were present in the green-fruited species and were absent in the red/yellow-fruited species. In order to test the contribution of these sequences to gene expression/silencing we built promoter::GFP constructs harboring the two parental promoters as well as three additional artificial constructs containing the S. lycopersicon promoter with insert-I alone, insert-II alone and with both inserts. The constructs were electroporated into tobacco protoplasts and GFP fluorescence level was measured. The results show that in the absence of insert-II expression does not occur, insert-II alone allows for slightly reduced gene expression, and both inserts allow for wild-type fluorescence level. These results suggest that the deletion of the nine bp sequence may be a preadaptive molecular evolutionary event causal to cwp silencing, allowing for the subsequent domestication of the fleshy tomato berry. IN SEARCH OF THE MAJOR GENE DETERMINING ORGANIC ACID ACCUMULATION IN C. MELO- CLONING OF THE PATHWAY AND MAPPING OF THE GENES

S. Cohen1, N. Dai1, L. Yeselson1, S. Shen1, J. Burger2, U. Sahar2, G. Tzuri2, M. Lotan-Pomapan2, M. Harel-Beja2, V. Portnoy2, N. Katzir2, A.A. Schaffer1

1Department of Vegetable Research, Volcani Center, Bet Dagan, Israel, 2Department of Vegetable Crops and Plant Genetics, Newe Ya'ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel

All sweet melon fruits of the species C. melo are characterized by a low level of organic acid contents and sweet flesh pH values of over 6.0. Several wild accessions of the species accumulate higher levels of organic acids resulting in acidic pH values near 4.5. This striking trait is controlled by a single dominant gene, So, which when introgressed into sweet varieties imparts to the fruit a sweet-sour, exotic taste. In order to identify the function of this gene we adopted the strategy of cloning all the genes of the pathways that are postulated to contribute to organic acid metabolism and H+ transport into the flesh vacuole. We have succeeded in cloning 27 genes encompassing the complete organic acid metabolic pathway and 14 genes encoding for the numerous subunits of the vacuolar ATPase complexes. Included in this molecular toolbox are genes encoding for different compartmentalized isoforms and for individual subunits of enzyme complexes. Polymorphisms for each of the genes were identified between the acidic and non-acidic variants, and the genes were mapped in order to test for co-localization of the candidate genes with the So locus. The results clearly indicate that the genetic control of fruit pH does not reside in any of the cloned gene loci. SMALL RNA PRODUCTION BY TRANSGENE GIVES VIRUS RESISTANCE DEPENDING ON THE DEGREE OF HOMOLOGY BETWEEN THE TRANSGENE AND THE CHALLENGE VIRUS

V. Gaba, A. Gal-On, S. Singer, E. Kukurt, D. Leibman, L. Maslenin, Y.M. Shibolet, A. Rosner

Department of Plant Pathology, ARO Volcani Center, Bet Dagan, Israel

Tobacco plants (Nicotiana tabacum L.) were transformed with a binary vector containing a “hairpin” inverted repeat of 598 nucleotides derived from the Potato virus Y (PVY) NIb replicase gene. Such constructs are designed to produce dsRNA predicted to be processed to siRNA by DCL4, and thereby confer virus resistance by a post translational gene silencing mechanism. Homozygous plants were challenged with a number of PVY strains and resistance was measured by symptom expression and ELISA titer. We tested the resistance of the transgenic tobacco to five potato- infecting PVY strains and three other field strains from tomato and pepper. The nucleotide homology of the potato PVY strains to the transgene was 88.3-99.5%. A transgenic tobacco line was immune to the potato PVY strains, with which the transgene had the greatest homology. Infection with the tomato and pepper PVY isolates, that had a lower degree of homology with the transgene (86.3-86.8%), caused delayed symptom appearance in the transgenic tobacco compared with control non- transgenic plants. The accumulation of transgene-related siRNA was measured in non-inoculated plants using a custom-designed chip for the detection of small RNA molecules. Probes of 25 bases were printed, covering the transgene sense PVY, intron and antisense PVY sequences. The sequence of each probe was moved a base along the transgene from the previous probe, tiling the transgene, mapping siRNA accumulation of the whole transgene by 2873 probes, in both sense and anti-sense directions. Small RNA populations from transgenic and non-transgenic control plants were labeled differently, and applied to the chip. siRNA peaks were observed throughout the transgene, which, although generally coinciding with higher GC- content, were without pattern. Dicer apparently cut the dsRNA produced by the transgene at random. The resistance-breaking PVY strains bore additional mutations that correlated with peaks of siRNA accumulation. THE ROLE OF YABBY DERIVED SIGNALING IN PHYLLOTAXIS ESTABLISHMENT

A. Goldshmidt, J.P. Alvarez, Y. Eshed

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel

Plant architecture forms by a well-defined repeatable pattern for each and every plant species. All plant lateral organs such as leaves and floral organs are derived from apical meristems, suggesting that specification of where and when to produce a primordium occurs at the meristem. Previous studies have demonstrated that knockout mutations of YABBY family genes, filamentous flower (fil) and yabby-3 (yab3), can alter correct development, position and number of lateral organs. The YABBY genes mRNA is restricted to the abaxial side of all organ primordia. This shows that while YABBY genes are not expressed in the meristem they are important for correct positional information acquisition by meristem cells, presumably by a signaling process. We investigated the basis of YABBY-mediated signaling and found that miss-expression of YABBY genes under control of different vegetative and flower promoters resulted in non-cell autonomous phenotypes. We show that changes in YABBY expression domain lead to shifts in expression of inter-organ and meristem specific markers, suggesting that YABBY genes act as signaling molecules. EFFECT OF LYSINE ON METHIONINE METABOLISM

Y. Hacham1,2, G. Schuster2, L. Song3, R. Amir1,4

1Plant Science Laboratory, Migal, Kiryat-Shmona, Israel, 2Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel, 3Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel, 4Tel-Hai College, Upper Galilee, Israel

Lysine and methionine are two essential amino acids whose levels affect the nutritional quality of cereals and legume plants. Both amino acids are synthesized through the aspartate family biosynthesis pathway. Within this family, lysine and methionine are produced by two different branches, the lysine branch and the threonine–methionine branch, which compete for the same carbon/amino substrate. To elucidate the relationship between these biosynthetic branches, we crossed two lines of transgenic tobacco plants: one that overexpresses the feedback-insensitive bacterial enzyme dihydrodipicolinate synthase (DHPS) and contains a significantly higher level of lysine, and a second that overexpresses Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Significantly higher levels of methionine and its metabolite, S-methylmethionine (SMM), accumulated in the newly produced plants compared with plants overexpressing AtCGS alone, while the level of lysine remained the same as in those overexpressing DHPS alone. The increased levels of methionine and SMM were correlated with increases in the mRNA and protein levels of AtCGS and a reduced mRNA level for the genes encoding S-adnosylmethionine (SAM) synthase, which converts methionine to SAM. This decrease in SAMS level leads to lower amount of SAM. As SAM is a negative regulator of CGS, this reduction leads to higher expression of CGS and consequently to an increased level of methionine. The results disclosed a new regulatory point at which lysine affects methionine content. In addition, elucidating the relationship between lysine and methionine synthesis may lead to new ways of producing transgenic crop plants containing increased methionine and lysine levels, thus improving their nutritional quality. A STRUCTURE-FUNCTION STUDY OF THE COP9 SIGNALOSOME SUBUNIT 7

Y. Halimi1, M. Dasau2, N. Levanon1, E. Bachrach3, N. Ohad1, J. Hirsch2, D.A. Chamovitz1

1Department of Plant Sciences, Tel-Aviv University, Ramat Aviv, Israel, 2Department of Biochemistry, Tel-Aviv University, Ramat Aviv, Israel, 3Department of Cell Research and Immunology, Tel-Aviv University, Ramat Aviv, Israel

CSN7 is a component of the COP9 signalosome (CSN). csn7 (fus5) mutants show a constitutive photomorphogenic phenotype. A two-hybrid screen using CSN7 as bait identified a number of CSN7-interacting proteins, including other CSN subunits. The CSN subunits interacted with a central region of CSN7, in the predicted "PCI motif", while the other proteins interacted with either the N or C termini. To further define this "PCI motif", a series of stringent biochemical assays were carried out that identified a stable core domain in CSN7. One of the goals of this research is to elucidate the biological significance of this domain. Through the use of transgenic and biochemical studies, we demonstrate that while the PCI is sufficient to mediate in vitro interactions, in vivo, a larger protein is needed. One of the CSN7 interactors that does not interact with the "core" is the small subunit of the ribonucleotide reductase, RNR2a, a component of the RNR holoenzyme. This is the key enzyme for reducing NTPs to dNTPs, supplying building blocks for DNA after DNA damage or for cell cycle. This interaction was confirmed both in vitro and in planta. We are further characterizing basic RNR function in Arabidopsis to understand the biological significance of the CSN7-RNR2 interaction PEROXIDASE INVOLVEMENT IN THE PROCESS OF ANTHOCYANIN DEGRADATION IN BRUNFELSIA FLOWERS

R. Liberman1,2, A. Bar-Akiva1,2, L. Shahar1,2, A. Nissim-Levi1, H. Koltai1, D. Weiss2, V. Gaba1, M. Reuveni1, D. Evenor1, M. Oren-Shamir1

1Department of Ornamental Horticulture, ARO, The Volcani Center, Bet-Dagan, Israel, 2The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem, Rehovot, Israel

The color of fruits, flowers and leaves is of the utmost importance in a variety of agricultural products. In this study we focus on color change due to alteration in anthocyanin content, and in particular on their enzymatic degradation, an important yet poorly understood process. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, very little is known about the stability and catabolism of anthocyanins in plants. Previously we have shown that active in planta degradation of anthocyanins occurs in Brunfelsia calycina flowers, as they change color from purple to white. We have demonstrated that anthocyanin degradation in Brunfelsia is dependant on de-novo mRNA and protein synthesis, occurring at a specific flower developmental stage. We have determined that oxidative reactions are a crucial step in the process of anthocyanin degradation in Brunfelsia. One enzyme family that may be involved in the process is peroxidase, as we detected an increase in peroxidase activity in the flowers correlating with anthocyanin degradation. To test the role of peroxidases in anthocyanin degradation in Brunfelsia we have isolated an enzyme whose activity increased in correlation to the degradation. We also isolated a partial sequence of a peroxidase gene that showed increased steady-state transcript levels in parallel to anthocyanin degradation. For further testing of this gene and other closely related genes in the process, a relatively conserved region of the peroxidase gene was cloned for RNAi-mediated gene expression silencing in Brunfelsia flowers before the onset of anthocyanin degradation. A transient transformation system was developed for the functional testing of candidate genes and a regeneration system is being developed for future experiments involving stable transformation of Brunfelsia. HERITABILITY STUDIES AND DNA MARKERS FOR POWDERY MILDEW RESISTANCE IN STRAWBERRY FRAGARIA X ANANASSA DUCHESNE

C. Lifshitz1, N. Shalit1, S. Slotzky1, Z. Tanami1, Y. Elad2, N. Dai1

1Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel, 2Institute of Plant Protection, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel

Powdery mildew (PM) of strawberry caused by the obligate parasite Podosphaera aphanis, is one of the major fungal diseases of this crop. Marked variation for PM resistance among strawberry cultivars and high heritabilities for PM resistance have been reported in strawberry breeding populations, indicating that the negative economic impact of PM can be significantly reduced through the breeding of new cultivars with enhanced disease resistance. In this study, resistant and susceptible accessions were selected as parents and hybridized to develop seven PM segregating populations that were tested for PM disease severity. Broad sense heritability was assessed using phenotypic variance analysis and ranged between 0.46-0.56. Heritability over 50% was also estimated by the correlation between two sequential years of disease severity measurements. In order to develop genetic markers associated with the PM resistance two strategies were used. We screened for SSR markers which co-segregate with the PM resistance in different populations. 30 SSR markers from the 7 linkage groups were screened in five PM populations but none segregated with relation to PM resistance. In addition, we tested resistance gene analogs (RGAs) as candidate genes for PM disease resistance. Twenty different RGAs were isolated from genomic DNA and from cDNA using various degenerate primers designed to the conserved P-loop and Gly-Leu-Pro-Leu (GLPL) motifs within the nucleotide binding site (NBS) of the plant RGAs. Their predicted protein sequence alignments showed wide range of sequence identity (26.8 – 97.7%) and were classified to the Toll and interleukin-1 receptor (TIR) and nonTIR NBS-LRR classes. RGA expression patterns will be tested in susceptible and resistant varieties before and after PM inoculation by Real Time PCR using specific primers. The linkage of the selected R genes for mildew resistance will be further analyzed in the segregating populations using divergent markers that will be developed from linked polymorphic sequences. THE PRIMARY - SECONDARY METABOLISM INTERFACE: METABOLIC CHANGES IN ARABIDOPSIS PLANTS OVEREXPRESSING FIVE RELATED TRANSCRIPTION FACTORS DETECTED BY GC-MS AND LC- MS TECHNOLOGIES

S. Malitsky, E. Blum, I. Venger, Y. Eshed, A. Aharoni

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel

Secondary or specialized plant metabolites obtain their basic carbon skeletons from precursor pathways that are represented by primary metabolites including amino acids, lipids and carbohydrates. In order to study the interface between primary and secondary metabolism we conducted metabolite profiling of plants overexpressing a set of five transcription factors that activate the major glucosinolate (GS) biosynthesis pathways in Arabidopsis (Tryptophan and Methionine -derived GSs). Analyses were performed by the use of both GC/MS and UPLC-qTOF-MS-MS technologies in order to detect the largest number of metabolites. The mass chromatographic data were converted into matrix data sets and analyzed by various data-mining tools including hierarchical cluster analysis and principle component analysis. Metabolic profiles of samples derived from plants overexpressing the clade of genes activating the Tryptophan-GS pathway could be clearly distinguished from plants overexpressing factors activating the Methionine-GS pathway. In-depth data analysis including the identification of differential metabolites demonstrated that overexpression of the different transcription factors lead not only to the accumulation of GSs but also included activation of primary metabolic pathways supplying the bulk of sulfur, nitrogen and basic carbon skeletons required for GSs biosynthesis. The same profile of activation at the primary-secondary metabolism interface was also detected in GeneChips assays that determined whole-transcriptome expression in plants overexpressing these factors. Thus, switching on a metabolic pathway involved in synthesizing secondary metabolites is ought to be a well coordinated process in which activation occurs at multiple points, from the very primary pathways forming the basic structures to the last committed step in the formation of a specialized metabolite. THE INS AND OUTS OF TOMATO FRUIT PEEL METABOLOME

T. Mandel1, S. Mintz-Oron1, A. Adato1, M. Yativ1, R. Jetter2, I. Venger1, I. Rogachev1, A. Aharoni1

1Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel, 2Department of Botany and Chemistry, University of British Columbia, Vancouver, BC, Canada

The cuticle covering the surface of both vegetative and reproductive organs plays multiple roles in the interaction between plants and the environment. This layer is primarily composed of lipids produced by its neighboring epidermal cells. We are interested in understanding the genetic regulation of metabolic pathways constructing the surface of fleshy fruit and the relationship between cuticular structure and its biological function. Gene expression in tomato peel and flesh tissues, at five stages of fruit development, was monitored by microarrays. Approximately 300 unique transcripts showed significant up-regulation in the peel. A large number of these could be associated with metabolic pathways generating cuticular components such as fatty acids, wax, cutin and phenylpropanoids, mainly flavonoids. Expression profiles show that these genes are mostly active in early to middle stages of fruit development. Comparison between tomato peel-associated genes and up-regulated genes in epidermal tissues, derived from other plant species, revealed a set of epidermal transcripts that are common between different surface tissues. We also conducted experiments to identify metabolites accumulating in fruit peel during the tested developmental stages. A UPLC-Q-TOF-MS system was used to track semi-polar compounds while GC-MS was used to profile polar compounds and cuticular components, mainly wax and cutin. Overall, close to 200 different compounds were identified in tomato peel tissue and their expression was monitored in the course of fruit peel development. Metabolite and transcript profiling analyses were also conducted on a colorless peel tomato mutant named "y". Findings of these experiments demonstrate altered levels of many transcripts and metabolites associated with phenylpropanoid metabolism, suggesting that a lesion in a regulatory gene underlies the "y" phenotype. Several genes identified in the course of this study are currently subject to detailed characterization in order to shed new light on the biology of fleshy fruit surface. PLANTS EXPRESSING BACTERIAL AND MODIFIED ENZYMES OF THE METHIONINE BIOSYNTHESIS PATHWAY ACCUMULATE HIGH LEVELS OF METHIONINE AND THREONINE

I. Matityahu1, Y. Hacham1,2, R. Amir1,3, G. Schuster2

1Laboratory of Plant Science, Migal Galilee Technology Center, Kiryat Shmona, Israel, 2Technion-Israel Institute of Technology, Haifa, Israel, 3Tel-Hai College, Upper Galilee, Israel

Methionine and threonine are two essential amino acids that belong to the aspartate family biosynthesis pathway. These amino acids diverge from the same branch, and therefore they compete for the same carbon /amino substrate. To further study the relationship between the biosynthetic pathways leading to threonine and methionine, we crossed two lines of transgenic tobacco plants: one that overexpresses the feedback-insensitive bacterial enzyme aspartate kinase (AK) containing a significantly higher threonine level, and a second that overexpresses the full-length Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Plants co-expressing the both foreign genes have higher levels of methionine and threonine compared to levels found in wild-type plants. However, the methionine level does not increase beyond that found in plants expressing the AtCGS alone. These findings can be explained through the feedback inhibition regulation mediated by the methionine metabolite on the expression level of AtCGS, which does not enable methionine accumulation beyond a certain threshold. To test this possibility, the plants expressing bacterial AK were crossed with plants expressing the truncated and deleted forms of AtCGS. These two forms of AtCGS are not sensitive to the feedback inhibition caused by the high methionine level. Significantly higher methionine level accumulated in the newly produced plants compared to plants expressing each of these forms of AtCGS alone. It was also found that threonine significantly enhanced the expression level of the deleted form of CGS, suggesting that this form maintains a high methionine level under conditions when a high threonine level is produced. The results of this study suggest new ways of producing transgenic crop plants containing increased methionine and threonine levels, and consequently of improved nutritional quality THE EFFECT OF VERNALIZATION AND LONG DAY ON L. CANDIDUM FLOWERING AND ON THE EXPRESSION OF LcLFY, A LEAFY HOMOLOGUE

I. Mazor, M. Zaccai

Department of Life Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel

Madonna lily (Lilium candidum, Liliaceae) is a perennial plant belonging to the subclass of Monocotyledonae and naturally flowering in May. Cold exposure of Madonna lily bulbs considerably hasten flowering, but the molecular regulation of lily response to vernalization is still unknown. The gene LEAFY (LFY) from Arabidopsis and its homologues in other species are known to play important roles in the regulation of flower transition, but a direct link to the vernalization pathway has not been demonstrated. The Madonna lily LFY homologue (LcLFY) was isolated and its expression was assessed in the different plant tissues. The aims of this research were to characterize the response of Madonna lily to vernalization and to investigate LcLFY expression pattern in view of vernalization and floral transition. Vernalization at different physiological stages and day extension were carried out on lily plants . Vernalization induced faster floral transition and flowering, while day extension had only a slight effect on these parameters. LcLFY, expression pattern in the shoot apical meristem was examined by Real-Time-PCR. LcLFY was already expressed at a very early vegetative stages, before bolting, and its expression raised as a response to cold exposure. A sharp increase in LcLFY expression was obtained after 5 weeks of vernalization, which also particularly determinant in accelerating flowering. These results shows that (1) temperature is apparently the dominant factor effecting flowering time in Madonna lily,.while day extension acts a secondary factor (2) LcLFY may be involved in the vernalization pathway in lily and could, maybe, serve as a vernalization marker for the thresholds time of cold treatments. The investigation of the Madonna lily genes may serve as a basis for further research on the molecular regulation of the flowering process in other monocots geophytes. EMBRYO-SPECIFIC REGULATORY SEQUENCES ESSENTIAL FOR ARABIDOPSIS SEED DEVELOPMENT

D. Michaeli1, R.L. Fischer2, N. Ohad1

1Department of Plant Sciences, Tel-Aviv University, Ramat Aviv, Israel, 2Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA

The FIE (Fertilization Independent Endosperm) polycomb gene in Arabidopsis is crucial for endosperm and embryo development. Polycomb group proteins form complexes that silence gene transcription. When maternally inherited, a mutant fie allele initiates endosperm development in the absence of fertilization, and embryo abortion at the heart stage when fertilization occurs. We have identified an element from the FIE 5’ untranslated region that regulates expression specifically in heart- staged embryos, and delimited it to an 88 base pair sequence (“88-box”). We found that the 88-box controlled the transcription of FIE in embryos. The 88-box was sufficient to drive transcription of a minimal core promoter in embryos when arranged as a tetramer. When relocated adjacent to FIE‘s core promoter, the 88-box resulted in enhancement of expression in embryos. When the 88-box was specifically deleted from a functional FIE-reporter transgene, reporter expression in all seed tissues was diminished manyfold, further supporting the function of the 88-box as an enhancer. Thus, the 88-box is vital for regulation of FIE expression in developing seeds. We localized two major stretches within the 88-box that mostly affect reporter gene expression in embryos, and due to sequence similarity, hypothesized that these might be bound by a homeodomain-family protein. We are currently using a yeast one- hybrid screen to isolate factors that are binding these elements. The identification of embryo specific regulatory sequence(s) and the isolation of factors binding them, would decipher how FIE is being regulated and what additional genes are co- regulated with FIE in via similar sequences. Thus, this study would bring new insights in our understanding of processes controlling seed development in Arabidopsis. COMPARISON OF THE COMPLETE GENOMIC INVERTASE SEQUENCES OF SUCROSE AND HEXOSE ACCUMULATING SPECIES OF THE LYCOPERSICON CLADE OF SOLANUM

M. Moy, L. Yeselson, P. Marina, S. Shen, N. Dai, A.A. Schaffer

Department of Vegetable Research, Volcani Center, Bet Dagan, Israel

The Lycopersicon clade of the Solanum genus is divided into two subgenera, the green fruited wild species (Eriopersicon) and the red-yellow fruited species (Eulycopersicon) which include the cultivated L. esculentum. The Eriopersicon species accumulate sucrose and are characterized by a developmental loss of soluble acid invertase activity in the developing fruit. In contrast, the Eulycopersicon species accumulate hexose and activity of invertase increases in the mature fruit. We compared the complete genomic sequences of the soluble acid invertase gene (TIV) of three Eulycopersicon species (S. lycopersicon, S. pimpinellifolium and S. cheesmaniae) with those of three Eriopersicon species (S. habrochaites, S. peruvianum and S. chmielewskii). The genomic arrangement of the TIV gene downstream from the promoter was similar in all six species. The number and relative size of the 6 introns and 7 exons were similar. The genetic similarity was highest in the exon regions and the largest dissimilarity was in the intron regions. However, further upstream a common motif was observed distinguishing between the sucrose and hexose accumulators. The Eulycopersicon species contain a range of multiple repeats (2-7) of a 166 bp unit which is present in only a single copy in the Eriopersicon species. Our results show that the number of multiple repeats strongly impacts on the invertase activity in the mature fruit. Results from segregating genetic populations showed that two repeats caused significantly lower invertase activity than did 4 and 6 repeat units in the promoter region. These results suggest that the evolution of the hexose-accumulating Eulycopersicon from the sucrose-accumulating Eriopersicon species was accompanied by successive duplications of the166 bp unit and that the tandem repeat units play an important role in enhancing invertase gene expression and enzyme activity. FRUIT CAROTENOID DEFICIENCY (fcd) MUTANTS IN TOMATO AS A TOOL TO STUDY REGULATION OF CAROTENOID BIOSINTHESYS

I. Pankratov1, N. Galpaz1, D. Zamir2, J. Hirschberg1

1Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem, Rehovot, Israel

Fruit development is a complex genetically-programmed process unique to plants. One of the most important elements in this process is the activation of the carotenoid biosynthesis pathway. Carotenoid biosynthesis in fruit is tightly controlled at the transcriptional level of carotenoid biosynthesis genes. Although genes for the majority of the carotenoid biosynthesis enzymes have been cloned, the regulation of this pathway is poorly understood. Tomato is an excellent model for studying of carotenoid biosynthesis. Six FRUIT-CAROTENOID DEFICIENT (fcd) mutants have been identified among a collection of mutagenized tomato.The fcd mutants exhibit normal progression of fruit development, including characteristic ripening features such as chloroplast to chromoplast transition. Their only apparent phenotype is lack of carotenoids that is visible at the ripe stage. One of the mutants in this group, termed SUNRED, displays a unique phenotype: Its fruit are red at the side facing the sun whereas the shaded side is pale orange. HPLC analysis revealed differences in carotenoid composition of the two parts. FCD1 and SUNRED were crossed with the wild tomato species S. Pimpinellifolium in order to genetically map the mutations. Both mutations were mapped to a region defined by introgression line IL4-3-2 on chromosome 4. Fine mapping of this region is currently under way in order to clone the mutated loci. Characterization of these mutants will provide new insight on the regulation of the carotenoid biosynthesis pathway. ACTIVITY AND REGULATORY PROPERTIES OF THE SUBUNITS OF TOMATO FRUIT AGPase AS HOMO- AND HETERO-TETRAMERS HETEROLOGOUSLY EXPRESSED IN E.coli

M. Petreikov1, S. Shen1, Y. Yeselson1, C.M. Bejar2, A.A. Iglesias2, J. Preiss2, A.A. Schaffer1

1Department of Genetics and Vegetable Crops, Volcani Center, Bet Dagan, Israel, 2Department of Biochemistry and Microbiology, Michigan State University, East Lansing, MI, USA

ADP-Glucose pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glucose and functions as a limiting enzyme in starch synthesis. In tomato plants the enzyme functions as a heterotetramer of two large subunits (encoded by three possible AgpL genes) and one small subunit (encoded by a single AgpS1 gene). A set of near isogenic tomato plants was developed from an interspecific cross between the cultivated S. lycopersicum (E) and wild species tomato S. habrochaites (H), differing in the origin of the AgpL1 allele. The fruit carrying the AgpL1H allele is characterized by increased AGPase activity and increased immature fruit starch content, as well as higher total soluble solids (TSS) in the mature fruit, following the breakdown of the transient starch. In order to better understand the metabolic role of the alternate subunits involved in active enzyme formation we expressed all four subunits in appropriate expression vectors. AgpS1E gene was expressed in the pMON 17335 vector, while AgpL1H, AgpL2E, and AgpL3E were expressed in the pMON 17336 vector. For protein expression, E.coli mutant strain AC70R1-504 lacking endogenous AGPase activity was used to express the different subunits in the various heterotetrameric and homotetrameric combinations. Homotetramers, consisting of only one subunit (small and three different large) and heterotetramers, consisting of the small subunit and each of three large subunits) were expressed and analyzed. The differences in their enzyme activities, thermal stability, biochemical characteristics and physiological role will be discussed. THE ARABIDOPSIS THALIANA CO-CHAPERONE ROF1 AFFECTS TRANSCRIPTION PROFILING AND GERMINATION

O. Pisanty, E. Herzig, D. Meiri, A. Breiman

Department of Plant Sciences, Tel-Aviv University, Ramat Aviv, Israel

The Arabidopsis co-chaperons FKBPs are actively investigated due to their function in protein folding, signal transduction and chaperone activity. We have characterized two large FKBPs the 62kDa ROF1 and the 65kDa ROF2. Promoter analysis showed their developmental expression and organ specificity (Aviezer-Hagai., 2007) We have analysed the transcription profile of rof1 Knockout plants as compared to 35S::ROF1 overexpression transgenic plants by microarray chip analysis. Biological clustering using GO Browser identified genes that were affected by the absence/ presence of ROF1. One major family is composed of Trehalose biosynthesis genes (TPS); the other family is composed of genes involved in cell wall loosening needed in plant cell growth. Bioinformatics studies using selected microarrays from the Affymetrix whole genome ATH1 database (www.genevstigator.ethz.ch), showed high correlated expression of ROF1 and the TPS genes (r2 =0.917, P<0.001). Moreover no correlation was found between ROF1 and TPS expression pattern among 2507 microarrays of the genevestigator database (r2 =0.029, P<0.001). These data indicate that ROF1 and TPS have opposite transcriptional pattern possibly due to sharing a functional role as chaperones. In the absence of ROF1 all TPS transcripts were found to be upregulated as well as many of the cell wall loosening enzymes. Therefore we hypothesized that ROF1 is involved in germination repression. We are testing this hypothesis on a physiological level by germination bioassys on Glucose and ABA. High glucose/ABA concentrations are known to inhibit germination and prolong dormancy respectively. Hence rof1 knockout plants are expected to show higher and faster germination percentage on glucose and ABA. A LINK BETWEEN DEVELOPMENTAL REGULATION OF ROP GTPase AND HORMONAL RESPONSES

L. Poraty1, O. Hazak1, P. Zimmermann2, W. Gruissem2, S. Yalovsky1

1Department of Plant Sciences, Tel Aviv University, Ramat Aviv, Israel, 2Swiss Federal Institute of Technology, ETH Zurich, Switzerland

ROPs are plant specific family of Ras related small GTPases functioning as molecular switches in multitude of signaling cascades. ROPs comprise a protein family of eleven members in Arabidopsis. Direct roles of ROPs in differentiation processes have not been demonstrated. Furthermore, the redundancy between the different ROPs has hampered their analysis. An AtROP6 promoter-reporter-gene fusions and RNA in situ hybridizations demonstrate that AtROP6 is specifically expressed in the root cup the root apical and basal meristems and in developing lateral roots. Expression in pericycle lateral root founder cells is induced by auxin and detected before the onset of the first cell divisions. In lateral root initials, AtROP6 is localized in a polar fashion. First in the membrane separating the pericycle and the xylem pole and then in membranes marking the planes of cell divisions. Microarray analysis showed that expression of a constitutive active AtROP6 under regulation of its native promoter suppressed expression of the cytokinin type A response regulator ARR15 gene. RT-PCR on RNA extracted from roots showed that levels of ARR15 and another type A ARR, ARR5 are reduced in both the activated ROP and AtROP6 T- DNA knockout mutants. Transgenic plants expressing the activated AtROP6 showed decreased sensitivity of root elongation to cytokinin while AtROP6 T-DNA knockout seedlings showed increased sensitivity to the hormone. These data suggest that AtROP6 is part of an early auxin response and locally suppresses cytokinin response during root development. DIFFERENT SUBCELLULAR LOCALIZATIONS AND FUNCTIONS OF ARABIDOPSIS MYOSIN VIII

L. Golomb1, M. Abu-Abied1, E. Belausov1, E. Sadot1

Institute of Plant Sciences, Volcani Center, Bet-Dagan, Israel

Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB. In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncations, lacking the head domain), fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 distributed mainly in the cytoplasm, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the side of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies. In root hairs, GFP- ATM1(IQ-tail) formed dots organized into a network that ran along the cell into the clear zone at the tip. Using different markers and transient expression in Nicotiana benthamiana leaves, we showed that myosin VIII co-localizes to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlaps with the endosomal marker ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain) co- localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding. Taken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity. THE ROLE OF ACC SYNTHASE GENES DURING WATERMELON FLOWER DEVELOPMENT

A. Salman Minkov, T. Trebitsh

Department of Life Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel

Cultivated watermelon (Citrullus lanatus) is characterized by a repetitive pattern of 8- 11 staminate flowers followed by a single fruit-producing pistillate flower. Two modes of sexual phenotypes are exhibited in watermelon, andromonoecious plants that produce both perfect and male flowers and monoecious plants that produce both female and male flowers. Floral sex determination is genetically controlled and may also be regulated by hormonal factors. In the Cucurbitaceae family, the plant hormone ethylene is a major regulator of sex determination. However, its role in floral organ differentiation in watermelon is ambiguous: on one hand enhancement of ovary differentiation and on the other hand inhibition of pistillate flower development. To elucidate the role of ethylene during watermelon flower development we study the role of the key enzyme in ethylene biosynthesis, 1-aminocyclopropane 1-carboxylate synthase (ACC synthase, ACS), in the process. Four distinct ACS genes were isolated from watermelon (CitACS1-4). The CitACS genes are differentially expressed during flower development and in response to plant hormones. CitACS1 transcript is detected in vegetative tissue as well as during floral buds development and its expression is enhanced by ACC, auxin and gibberellin. The transcript level of the CitACS2-4 was non-detectable in vegetative tissue. However, CitACS2 expression is induced by auxin. Accumulation of CitACS3 transcript is regulated by ethylene and expressed in anther producing flowers but not in female flowers. CitACS3 transcript is mostly expressed in the anther filament of male flower buds. This gene may be involved in ethylene-regulated anther development. DIFFERENTIAL EXPRESSION ANALYSIS OF NEAR-ISOGENIC TOMATO LINES WITH DIFFERENT RATIOS OF FRUCTOSE TO GLUCOSE IN THE MATURE FRUIT

A. Shammai, Y. Yeselson, S. Shen, A.A. Schaffer

Department of Vegetable Research, Volcani Center, Bet-Dagan, Israel

Sugar content has a major effect on fruit flavor and quality in the cultivated tomato (Solanum lycopersicum), which is characterized by approximately equimolar levels of fructose and glucose. Since fructose is twice as sweet as glucose, increasing the fructose level is a goal of tomato breeding programs for improved fruit quality. Wild species of tomato are an important source for genetic variation. A genetic trait of increased fructose to glucose ratio (Fgr), originating in the wild species S. habrochaites, was introgressed into the cultivated tomato, imparting to the mature fruit fructose levels nearly twice that of glucose. In order to uncover the effects of the Fgr gene on soluble sugar metabolism in tomato fruit we compared the gene expression of near-isogenic fruit throughout development. Differential expression analyses were carried out using DNA microarray technology (TOM1). Based on the patterns of fructose and glucose accumulation, fruit from near-isogenic plants at three different developmental stages were examined (immature green, mature green and breaker stages). The results indicate that the expression of at least three genes involved in the hexose metabolic pathway is influenced by the allelic status of Fgr. A novel hexokinase of undefined function (HXK5), and a cytosolic phosphoglucomutase (PGM) are upregulated by the wild species allele, while a novel pyrophosphate-dependent phosphofructokinase (PPi-PFK) gene was downregulated in the fructose accumulating genotype. Polymorphisms (SNPs) were identified for these three genes which allowed for their mapping using advanced introgression lines. None of these three genes colocalized with the Fgr locus. These results, together with the mapping of other hexose metabolism genes, suggest that the Fgr gene does not encode for any of the metabolic steps of hexose metabolism. However, the Fgr gene does impact on the expression pattern of hexose metabolism genes, suggesting that their transcription is affected by the Fgr. STUDYING THE PHYSIOLOGICAL FUNCTION OF THE ARABIDOPSIS METAL/PROTON EXCHANGER AtMHX IN PLANTS THAT ECTOPICALLY OVEREXPRESS THIS TRANSPORTER

I. Berezin, E. Brook, K. Mizrahi, M. Elazar, R. Gaash, M. Avramov, T. Mizrachy-Dagry, O. Shaul

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

AtMHX is a vacuolar transporter encoded by a single gene in Arabidopsis thaliana. Electrophysiological analysis showed that it can exchange protons with magnesium, zinc, and ferrous ions. Despite an intensive search, no real null mutant of AtMHX could be identified thus far, suggesting that this transporter is essential for viability. The physiological impact of AtMHX was investigated thus far only in tissue-culture grown seedlings of tobacco plants overexpressing this transporter. These seedlings showed necrotic lesions only upon growth with high levels of magnesium or zinc ions, thus providing evidence for in planta transport of these two metals. Here we show that mature transgenic tobacco plants grown in hydroponics exhibit necrotic lesions and severe apical burnings not only upon exposure to magnesium and zinc ions, but also when grown in the presence of cadmium ions. This suggests that AtMHX can transport in planta cadmium ions as well. The current information about plant transporters that secrete cadmium ions into vacuoles is limited. Moreover, the physiological phenomena observed in transgenic plants that overexpress AtMHX are consistent with the assumption that this transporter can also affect the proton homeostasis of cells. AtMHX exchanges the metal ions with vacuolar protons. The transgenic tobacco plants showed necrotic lesions, apical burning, increased expression of the vacuolar H+-ATPase, and a transient reduction in plant size. The correlation between these phenomena and perturbations in proton homeostasis is discussed. Tomato plants in which AtMHX was overexpressed showed a much more pronounced reduction in plant size, due to decreased cell expansion, and had a lower potassium content. An AtMHX-mediated proton efflux from the vacuole is expected to lower the vacuolar proton-motive force, which drives potassium uptake into the vacuolar, and, consequently cell expansion. All these data agree with a postulated impact of AtMHX on cellular pH homeostasis. THE ARABIDOPSIS PRA FAMILY IS ESSENTIAL FOR PLANT DEVELOPMENT

K. Shichrur, S. Yalovsky

Department of Plant Sciences, Tel Aviv University, Ramat Aviv, Israel

Small GTPaes are known to affect cell polarity, vesicular trafficking and cytoskeleton organization, but the mechanism underlying those effects are not yet clear. In a yeast two-hybrid assay, dominant-negative ROP6 GTPase was used as a bait to find interactors that may potentially mediate small GTPases regulated functions. Arabidopsis thaliana prenylated Rab acceptor, AtPRA1, was identified as a novel binding partner. The mammalian and yeast PRA1 homologue are documented as interactors of small GTPases and are thought to play a role in protein targeting. However, only very little data exist on Pra1 and its function is mostly unknown. A bioinformatics analysis revealed a family of 14 PRA1-like genes in Arabidopsis. One member of the family, designated AtPRA1, interacts preferentially with GTP-bound type I ROP GTPases and with a lower affinity to a tomato LeRAB1 homolog. Co- localization and Biomolecolar Fluorescence Complementation (BiFC) analyses in plants demonstrated that AtPRA1 and AtROP6 interacted mostly in the ER. Silencing of AtPRA1 and its closest homolog AtPRA2 by RNAi resulted in a loss of apical dominance, delayed flowering and compromised cell division and growth. Ectopic expression of AtPRA1 led to reduction in the number of lateral roots and ABA hypersensitivity during germination. We propose that AtPRA1 and likely other members of the PRA family regulate ABA response and play an essential role in protein trafficking during developmental and stress responses. POST-TRANSCRIPTION AND POST-TRANSLATION REGULATION OF CCA1 AND LHY IN THE CIRCADIAN OSCILLATOR OF ARABIDOPSIS THALIANA

E. Yakir, D. Hillman, R.M. Green

Department of Plant and Environmental Sciences, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel

The circadian clock is an endogenous mechanism that generates rhythms with approximately 24-hour periods and enables organisms to predict and adapt to daily and seasonal changes in their environment. These rhythms are generated by oscillators comprised of transcription/translation feedback loops. The main oscillator of the model plant Arabidopsis thaliana has been shown to consist of at least three elements: CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLS (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1). CCA1 and LHY are transcription factors that are expressed early in the morning, bind to the TOC1 promoter and repress its expression. When their levels fall TOC1 is expressed and activates CCA1 and LHY expression by an unknown mechanism. However, this simple model cannot fully explain the generation of 24 hour rhythms. We are interested in understanding the post-transcriptional and post-translational regulation of CCA1 and LHY and how this affects the clock. At the post-transcriptional level, we have shown that CCA1 mRNA is stable in the dark and in far-red light but is destabilized by red and blue light. Furthermore, the instability determinants in CCA1 transcripts are probably located in the coding region. This change in CCA1 mRNA stability is likely to be an important entrainment point for the oscillator (Yakir et al., 2007). At the post-transcriptional level, in order to better understand how CCA1 and LHY interact and move within the cell to generate circadian rhythms, we have set up a system of tagged and florescent CCA1 and LHY expressed under the control of the endogenous CCA1 or LHY promoters in transgenic plants. We are using these transgenic lines to examine where and when CCA1 and LHY interact, how entrainment conditions affect their localization and how the oscillator operates in different tissues and in different cells.

Yakir E et al. (2007) Plant Physiol.: pp.107.103812. WHEAT-RICE COLINEARITY AT THE Yr15 STRIPE RUST RESISTANCE GENE REGION IN TETRAPLOID WHEAT

E. Yaniv1, L. Gelfarb1, A. Distelfeld2, A.B. Korol1, E. Nevo1, T. Fahima1

1Institute of Evolution, University of Haifa, Israel, 2Department of Plant Sciences, University of California, Davis, CA, USA

Colinearity between wheat and rice is well established and it can facilitate genetic mapping of the non-sequenced genome of wheat based on its similarity to the fully sequenced rice genome. The objective of the present study is to establish colinearity between the Yr15 gene region in chromosome arm 1BS of wild emmer wheat, Triticum dicoccoides and the parallel sequence of chromosome 5 of Oryza sativa. Yr15 is a single dominant R-gene conferring resistance to many races of Puccinia striiformis that causes the stripe rust disease of wheat. Yr15 is a target of positional cloning effort in our laboratory. A primary genetic map of the Yr15 gene region was developed using an F2 mapping population derived from a cross of a BC3F9 line containing a 1BS chromosome segment of T. dicoccoides carrying Yr15 with the recurrent parent Triticum durum cv. D447. In the present study we have developed a genetic map of the chromosome segment carrying Yr15 using markers Xgwm911, Xwmc406, Xbarc8, Xnor1B, Xgwm413, Xgwm18, Xuhw250, Xuhw252, Xuhw253 and Xuhw254. Based on these markers we were able to assign Yr15 to deletion bin Sat0.31. Yr15 flanking markers Xuhw250 and Xuhw252 are highly homologous to two rice BAC clones flanking a contig of 740kb on chromosome 5 of rice. Therefore, these results establish colinearity between the region containing Yr15, flanked by markers Xuhw250 and Xuhw252, and a 740kb rice contig. We are currently exploring this colinearity for saturation of the Yr15 containing region with additional markers and developing a physical map of this region. SMALL RNAs FACILITATE POLARITY AND LAMINAR GROWTH OF TOMATO LEAVES

T. Yifhar, I. Pekker, J.P. Alvarez, M. Sabban, D. Peled, G. Shahar, Z. Amsellem, Y. Eshed

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel

The classical tomato wiry mutants includes three non-allelic mutations; wiry1, wiry2 and wiry3. In all three, a partial or complete loss of organ polarity is evident in leaves and flowers, resulting in filamentous organs. We examined whether abnormal morphology of wiry mutants is correlated with changes in the expression levels of genes promoting adaxial/abaxial (top/bottom) organ identity. Increased expression of the abaxial promoting gene AUXIN RESPONSE FACTOR 4 (SlARF4) was evident in all three wiry mutants. An artificial microRNA targeting SlARF4 as well as SlARF3 results in tomato plants with misshapen and up-curled leaves. Strikingly, these effects were completely epistatic to the wiry mutant phenotype. This suggests that elevated levels of SlARF4 transcripts underlie the phenotypic defects of the wiry mutants. SlARF3/ETT, and SlARF4 are post-transcriptionally regulated by tans- acting siRNAs (ta-siRNA) derived from the TAS3 gene. The level of TAS3 tasiRNA was dramatically reduced in all wiry mutants, impling that the WIRY genes are involved in ta-siRNA biogenesis in tomato. To further examine whether compromised ta-siRNA regulation underlied the wiry leaf phenotype, we disrupted ta-siRNA regulation of the genes by constructing ta-siRNA-resistant versions of SlARF3/ETT, and SlARF4. While missexpression of the sensitive SlARF3 or SlARF4 versions resulted in wild type looking plants, over expression of the ta-siRNA-resistant versions yielded plants with filamentous organs, indistinguishable from wiry plants. Consistent with this, the genes SlAGO7, SlRDR6 and SlSGS3, whose Arabidopsis orthologs are involved in tasiRNA biogenesis, co-mapped with the wiry loci, and sequence analyses identified lesions in all mutant alleles. It is therefore argued, that fine tuning of SlARF3 and SlARF4 by ta-siRNA is pivotal for tomato organ polarity. Strikingly, the same pathway is conserved in all plants, but only in some it is critical for normal formation of bifacial organs. CHARACTERIZATION OF A NOVEL PLASTIDIAL THIOREDOXIN-LIKE FAMILY IN ARABIDOPSIS

I. Dangoor, A. Levitan, H. Peled-Zehavi, A. Danon

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel

The reduction and oxidation of regulatory disulfide bonds serve as key signaling elements in chloroplasts. This type of regulation is widespread in spite of the highly reducing environment of the chloroplast. We have characterized a new family of plant specific thioredoxin-like proteins in Arabidopsis thaliana, denoted AtTots (Thioredoxins with Oxidative Tendency). The AtTots contain a variable atypical redox active-site. Assays of subcellular localization demonstrated that all of the AtTots are localized to the chloroplast. The pattern of expression of the AtTots differs from one another and from classic thioredoxins, suggesting different roles for the family members. Activity assays show that members of the family are indeed active oxidoreductases. More importantly, we found that the redox midpoint potential of three selected AtTots; AtTotA, AtTotB2 and AtTotD is significantly less reducing than that of classical thioredoxins. The intermediate redox midpoint potential value of these proteins might suggest adaptability to function in reactions of reversible disulfide formation, such as those catalyzing regulatory disulfides. In order to better understand the physiological role of the AtTot proteins we generated transgenic plants over-expressing an active-site mutant protein. This strategy enabled us to trap in vivo complexes that contain the AtTot protein in a mixed-disulfide bond with interacting proteins. Characterizing these complexes will allow us to identify AtTot cohort proteins and thereby shed light on the biological function of the oxidative type thioredoxins. Dab2 IS A CARGO SPECIFIC ENDOCYTIC ADAPTOR INVOLVED IN THE REGULATION OF CELL MATRIX INTERACTIONS

D. Chetrit, M. Ehrlich

Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, Israel

Cell matrix interactions are a complex and highly regulated dynamic process. As such, the cell integrates various sources of input, and responds by modulating, in a coordinated manner, the structure of the cytoskeleton, intracellular traffic, and the turnover of signaling/adhesion complexes. Disabled 2 (Dab2) is a tumor suppressor. The loss of its expression has been implicated in ovarian, breast, prostate, esophageal , lung and colon cancer. Dab2 directly interacts with and modulates the activity of: Smad 2 and 3, c-src, beta integrin receptors, Transforming Growth Factor beta (TGF- beta) receptor I, as well as with structural and regulatory elements of the intracellular trafficking machinery such as Clathrin and Myosin VI. Due to its structure and function, the characterization of the spatio-temporal dynamics of membrane association of Dab2 in different molecular, cellular and physiological contexts holds the potential of further elucidating its biological role. We have recently generated GFP-fusion proteins and structural mutants of the p82 and p59 isoforms of Dab2 in order to be able to follow the protein in live intact cells. Furthermore, we have begun to characterize the effect of Dab2 overexpression on cell-matrix interactions and the internalization of signaling receptors. Dab2 overexpression enhances the spreading of cells on fibronectin. This enhancement of cell spreading is directly correlated to the ability of Dab2 to modify the structure and distribution of clathrin assemblies at the ventral membrane of the cell. We propose that in addition to being governed by differing levels of expression, the role of Dab2 in regulating cell-matrix interactions is sensitive to phosphorylation. ROLE OF THE GTPase-ACTIVATING PROTEIN ArfGAP1-3 IN COPI MECHANISM

L. Kliouchnikov, A. Parnis, N. Goldfeder, D. Cassel

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

The Small GTPase Arf1 initiates the budding of vesicles carrying transport from the Golgi to the ER or to endosomes/lysosomes. The retrograde Golgi to ER transport is mediated by the COPI system and is regulated by Arf GTPase-activating proteins ArfGAP1-3. While the ArfGAP1 protein has been studied extensively, only little information about ArfGAPs2/3 is available. These two closely-related proteins are localized at the Golgi apparatus, and are known to interact with the heptameric COPI coat (coatomer). Here we present the results of experiments in which Golgi targeting motifs in ArfGAP3 were investigated using GFP fusions, and COPI interaction motifs were mapped by a new approach in which we follow the diversion to the ER of the plasma membrane protein CD4 by its fusion to ArfGAP3 fragments, which is probably the result of recruitment of the COPI system. We find that 1) Golgi localization of ArfGAP3 is mediated by its non-catalytic part, with a critical role for a carboxy terminal amphipathic motif; 2) There are multiple COPI interaction motifs along the ArfGAP3 protein; 3) In vitro binding experiments indicate that coatomer- binding elements are present in the amino part of ArfGAP3 but not at the carboxy part; 4) a 6-residue motif that follows the amphipathic helix plays a role in Golgi localization and appears to be involved in COPI interaction. Experiments are underway to further define the Golgi localization and COPI interaction motifs on ArfGAP3. TOPOLOGY OF AMPHIPATHIC MOTIFS MEDIATING GOLGI LOCALIZATION IN ArfGAP1 AND ITS SPLICE ISOFORMS

S. Levi, M. Rawet, L. Kliouchnikov, A. Parnis, D. Cassel

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

ArfGAP1 is a GTPase activating protein that cycles between the Golgi and the cytosol and participates in COPI vesicle-mediated transport in the ER-Golgi shuttle. COPI vesicle formation is initiated by Arf, a small GTPase that interacts with the Golgi membrane in its GTP-bound form and recruits COPI coat components. ArfGAP1 is required for the subsequent vesicle uncoating by promoting GTP hydrolysis on Arf1, returning it to its cytosolic GDP-bound form. The interaction of ArfGAP1 with the Golgi apparatus depends on motifs in its non-catalytic part that are unstructured in solution but can fold in vitro into amphipathic helices upon binding to liposomes containing poorly packed lipids. In previous work a few hydrophobic residues that are critical for lipid binding and Golgi localization were identified, but the precise topology of the amphipathic motifs has not been determined. Here we present a detailed analysis of the Golgi targeting and folding features of the region encompassing the amphipathic motifs. Our data show that the ALPS1 motif is considerably longer then initially thought and the entire 100-residue stretch can be assigned to two amphipathic helices separated by a very short break. We also show that the first amphipathic helix is truncated by a 10-residue insertion that is present in tissue-specific splice isoforms. Finally, we provide data indicating that Golgi targeting of ArfGAP1 is not a result of binding to highly curved membrane domains as previously suggested based on studies with liposomes. A ROLE FOR THE ERYTHROPOIETIN RECEPTOR DOMAINS IN RECEPTOR MATURATION

O. Ravid, T. Nahari, N. Ben-Califa, E. Barzilay, D. Neumann

Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Israel

Erythropoietin (EPO), the major hormone regulating erythropoiesis, functions via activation of its surface receptor (EPO-R). The majority of newly-made EPO-R molecules remain sequestered intracellularly, presumably in association with the endoplasmic reticulum (ER). Our ongoing research is designed to elucidate the roles of the EPO-R intracellular and extracellular domains in exit from the ER and in cell surface expression of the receptor. Led by the idea that coping with hypoxia may involve mechanisms that increase the extracellular expression of EPO-R, we studied the Subterranean blind mole rat (Spalax) as a unique animal model of adaptation to hypoxia underground. Our working hypothesis was that the EPO-R from Spalax (sEPO-R) is expressed at higher levels on the cell surface, conferred by its molecular structure. Analysis of sEPO-R compared to mouse EPO-R showed that the unique sequence features of sEPO-R contribute to its glycan maturation and transport to the cell membrane. The extracellular region of sEPO-R containing the N-glycosylation site was found to contribute to its maturation. The role of the EPO-R cytosolic region was analyzed using a fusion protein between the thermo-reversible folding mutant of vesicular stomatitis virus G protein (VSVGtsO45) and the EPO-R cytosolic domain (VSVG-WT). VSVG-WT displayed delayed intracellular trafficking and surface expression as compared to the parental VSVGtsO45 molecule; suggesting that the EPO-R cytosolic domain can hamper ER exit. VSVG-WT containing an NPVY insert (VSVG-NPVY) displayed enhanced glycan maturation and surface expression as compared to VSVG-WT. Notably, the NPVY sequence also conferred improved maturation and cell surface expression to the EPO-R. Our findings suggest that: (a) The EPO-R cytosolic and extracellular domains are involved in ER exit of the receptor. (b) Sequence motifs that participate in endocytosis can also modulate transport along the secretory pathway. (c) VSVG-fusion proteins may be employed to screen for intracellular sequences that regulate transport.

Acknowledgement: Part of the studies were carried out in collaboration with the group of Nevo and Avivi Institute of Evolution, University of Haifa, Mt.Carmel, Haifa, Israel. A NEW APPROACH FOR STUDYING THE ROLE OF CYTOSOLIC TRAFFICKING PROTEINS

M. Rawet, A. Parnis, D. Cassel

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

The selective transfer of material between membrane-delimited organelles is mediated by protein-coated vesicles. Members of the Arf family of small GTPases are central regulators of coated-vesicle formation. There are six members of the Arf family in mammalian cells, of which Arf1 and Arf6 are the best studied; Arf1 regulates the budding of COPI and clathrin coated vesicles, whereas Arf6 is thought to regulate both clathrin-dependent and clathrin-independent traffic in the endocytic system. ArfGAP1 is a GTPase-activating protein that promotes GTP hydrolysis on Arf1 and is required for the uncoating of vesicles in the COPI system. In addition, ArfGAP1 is proposed to function in the processes of cargo sorting and vesicle formation. We have initiated a new approach for studying the role of cytosolic trafficking proteins by fusing them to a transmembrane reporter protein (CD4), which normally localizes to the plasma membrane, and following the cellular localization of the fusion proteins. Signals on a trafficking protein that are involved in its interaction with components of pathways other than that leading to transport to the plasma membrane are expected to divert CD4 to a new cellular location. Our results show that CD4 is diverted to the ER when fused to Arf1 or to ArfGAP1, whereas CD4 fused Arf6 shows a cell-surface distribution. This diversion of the CD4-Arf1/ArfGAP1 fusions to the ER is probably due to their incorporation into COPI vesicles. Initial mutagenesis analysis revealed determinants in both the catalytic and non-catalytic domains of ArfGAP1 that are required for efficient ER localization of the CD4 fusion proteins. Further dissection of the COPI interaction motifs on Arf1 and ArfGAP1 are underway. DISCERNING BETWEEN RETENTION SIGNALS AND DEGRONS ACTING IN ERAD

I. Shapira, K. Hirschberg, S. Bar-Nun

Department of Biochemistry, Tel-Aviv University, Ramat Aviv, Israel

Endoplasmic reticulum-associated degradation (ERAD) eliminates aberrant proteins from the secretory pathway. Such proteins are retained in the endoplasmic reticulum and targeted to degradation by the ubiquitin-proteasome system. Cis-acting motifs function in ERAD as retention signals, preventing vesicular export from the endoplasmic reticulum, or as degrons, targeting proteins to degradation. Here we show that mu-stp, the C-terminal 20 residues tailpiece of the secretory IgM mu-s heavy chain, serves as a proteasome-targeting motif that directs a cytosolic version of yellow fluorescent protein to proteasomal degradation. Moreover, the mu-stp functions also in ERAD, serving a dual role of a portable retention signal and an ERAD degron. It confers retention to the endoplasmic reticulum onto a secreted version of the yellow fluorescent protein. Retention requires either the mu-stp penultimate cysteine or the absence of the mu-stp N-glycan. However, degradation does not rely on the penultimate cysteine and is accelerated in the absence of the mu- stp N-glycan. Hence, the dual role played by the µstp-Cys as a retention signal and as a degron can be attributed to distinct elements within this sequence. PARACOCCUS MARCUSII – A CAROTENOGENIC BACTERIUM HARBORING A NOVEL SECRETION MECHANISM

O. Weisshaus, V. Mann, J. Hirschberg

Hebrew University of Jerusalem, Israel

Paracoccus marcusii is a bright orange Gram-negative alpha-proteobacterium isolated in our laboratory. Pigment analysis at stationary phase of growth indicated robust synthesis of keto-carotenoids and their secretion into the medium. Transmission electron micrographs demonstrated that carotenoids were secreted by a novel mechanism: Membrane-free particles containing carotenoid are accumulating inside the cell. These particles then migrate to the periplasm and emerge outside the outer membrane. The particles accumulate around the cell surface and are passively washed off into the medium or actively released by an unknown mechanism. Investigation of this novel secretion mechanism was held back due to lack of genetic tools for this species. This work presents genetic transformation of P. marcusii and creation of transposon-tagged mutants that impair carotenoid secretion. Genetic transformation of P. marcusii was obtained by conjugation with E.coli donor strain and a suicide vector carrying Tn5 transposon. The effectiveness of transposon-tagged mutagenesis was demonstrated by creating color-mutants and screening colonies for a visible phenotype, followed by cloning and sequencing of the interrupted locus of the carotenoid biosynthesis pathway. A larger screen was then carried out in order to generate a collection of mutants that are impaired in carotenoid secretion. Characterization of the interrupted loci in the secretion-impaired mutants yielded genetic elements possibly involved in the secretion system. These genes are currently investigated, along with the further development of genetic tools for P. marcusii. CELL-SCAFFOLD TRANSPLANT OF HYDROGEL SEEDED WITH RAT BONE MARROW PROGENITORS FOR BONE REGENERATION

T. Kizhner1, D. Ben David1, T. Koheler2, R. Mueller2, E. Livne1, S. Sruji1,3

1Department of Anatomy and Cell Biology, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel, 2Institute for Biomechanics, ETH Zurich, Switzerland, 3Department of Oral and Maxillofacial Surgery, Carmel Medical Center, Haifa, Israel

Bone is the second most frequently transplanted tissue in humans. Extensive research efforts are focused on the development of ideal transplant, which is cell-scaffold construct. The objective of this study was to identify the osteogenic commitment stages of rat bone marrow derived progenitors (RBMP) seeded on the gelatin-based hydrogel scaffold and to assess the in vivo potential of the obtained transplant. Expanded RBMP were seeded on the hydrogel scaffold for implantation in nude mice cranial model or in vitro follow up. Osteoprogenitors were able to proliferate on the scaffold in a cell density-dependent manner. The cultures expressed classic osteogenic markers, and were positive to Alizarin Red S and osteocalcin staining. Light microscopy and scanning electron microscopy (SEM) analysis demonstrated characteristic osteogenic condensation and cell cluster formation followed by changes in cell morphology. Energy dispersive spectroscopy (EDS) analysis proved the occurrence of extensive matrix mineralization. In vivo results in cranial defect animal model indicated enhanced cranial defect closure following osteogenic cell-construct implantation. Conclusion: our results demonstrated the high potential of 3-D gelatin based hydrogel scaffold not only to serve as an effective delivery tool but also as a supportive carrier for bone marrow derived osteoprogenitors for bone reconstruction. IMMUNOGENICITY OF ACELLULARIZED EXTRACELLULAR MATRIX FOR ARTERIAL TISSUE ENGINEERING

N. Dahan, M. Machluf

Faculty of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel

Cardio vascular disease is the main cause of death in the industrial world. The accomplishment of coronary artery bypass grafting is contingent upon low or non immunogenic response. Isolated extracellular matrixes (ECM) have been studies as scaffold for arterial tissue engineering. Immunogenicity of the ECM could lead to chronic rejection. Therefore, suitable ECM scaffold must exhibit appropriate characteristics, including low immunogenicity, high biocompatibility, thromboresistance and with appropriate mechanical and physiological properties. Our study focuses on the isolation procedure with emphasis on the immunogenicity of the ECM for arterial tissue engineering Acellularization protocol is based on trypsin digestion, detergent solution and washing. The acellularized xenograft artery (AXA) immunogenicity was studied at the cellular level by H&E and Masson-Trichrome staining and at the nuclear level by DAPI. Ex-Vivo immunogenicity of Mouse-Bone- Marrow-Macrophages reaction to the AXA versus synthetic polymer (PLGA/PLAA) was examined by the expression of pro-inflammatory factor IL1-beta by RT-PCR and by nitric-oxided (NO) production. In-Vivo immunogenicity was studied by subcutaneous implantation of 0.5cm long pieces from the AXA to C57 black male mice. No traces of cellular or nuclear components could be detected in the AXA by the histological staining. In the Ex-Vivo assays, no significant finding was detected between the AXA and the synthetic polymer. The AXA was biocompatible compared to native xenoartery, which caused severe immunogenic response and increased by 150 % the NO production and by 10 fold the IL1-beta expression. In-vivo histological staining revealed that, the host cells infiltrated and resided inside the synthetic matrix in contrast with the AXA, which remained clean. RT-PCR for the pro-inflammatory factors performed on lymph nodes indicated for a minor response of the immune system at the first week, which declined over time. These findings suggest that the AXA is a biocompatible scaffold for arterial tissue engineering exhibiting no significant immunogenic response thus promoting its application in the clinical setting. PULSATILE INTERSTITIAL FLUID FLOW PROMOTES ERK1/2 CASCADE LEADING TO CARDIAC TISSUE REGENERATION

T. Dvir1, L. Oren2, M. Shachar1, Y. Granot2, S. Cohen1

1Department of Biotechnology Engineering , Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Deciphering the cellular signals leading to cardiac muscle assembly is a major challenge in ex vivo tissue regeneration. In this study, we demonstrate that pulsatile interstitial fluid flow in 3-dimensional alginate matrices, seeded with neonatal cardiac cells, can activate ERK1/2 6-fold, as compared to static-cultivated constructs. Activation of ERK1/2 was attained without activating the p38 cell death signal above its basic level, and under physiological conditions. ERK1/2 signaling cascade induced high levels of cardiac protein synthesis, while inhibition of this cascade diminished the inducing effects of pulsatile flow. The pulsed medium-induced cardiac cell constructs showed improved cellularity and viability, while the regenerated cardiac tissue demonstrated ultra-structural features resembling, in many respects, those of adult myocardium. The cardiomyocytes had elongated morphology and they were aligned into myofibers with defined Z-lines and multiple high-ordered sarcomeres. Numerous intercalated disks were positioned between adjacent cardiomyocytes and deposits of collagen fibers surrounded the myofibrils. The regenerated cardiac tissue exhibited high density of connexin 43, a major protein involved in electrical cellular connections. Our research thus demonstrates that by judiciously applying fluid shear stress, cell signaling cascades can be augmented with subsequent profound effects on cardiac tissue regeneration. SULFATION OF ALGINATE HYDROGELS ENABLES SPECIFIC BINDING AND SUSTAINED RELEASE OF HEPARIN-BINDING PEPTIDES

I. Freeman, S. Cohen

Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel

In recent years, engineering efforts have been directed at developing hydrogels with affinity sites, enabling the specific binding of growth factors and their release in a controlled manner. Taking an example from nature, most growth factors, chemokines and cell adhesion molecules, collectively known as heparin-binding peptides, bind the proteoglycans heparin and heparan sulfate via high affinity, specific electrostatic interactions. Such interactions are mediated by low- and high-sulfated sequences in these glycosaminoglycans (GAG). To mimic the specific binding of heparin-binding peptides to heparin/heparan sulfate, the uronic acids in non-sulfated alginate were sulfated, and hydrogels of mixed alginate/alginate-sulfate were fabricated. Surface Plasmon Resonance analysis probed the interactions of 13 peptides with alginate- sulfate. Of these, the 10 heparin-binding peptides revealed strong binding to alginate- sulfate and heparin, but not to alginate. The equilibrium binding constants to alginate- sulfate were comparable or one-order of magnitude higher than those obtained between the peptides and heparin. Only the fibroblast growth factors (FGFs) revealed higher affinity for heparin than to alginate-sulfate. Mixed hydrogels of alginate/alginate-sulfate sustained basic FGF release, with the release rate being dependent on the percentage of alginate-sulfate incorporated into the microspheres. Our results thus describe engineering of animal-origin free alginate hydrogels for the temporally and spatially controlled delivery of heparin-binding peptides. OSTEOCLAST RESORPTION APPARATUS: STRUCTURE DYNAMICS AND ACTIVITY

D. Geblinger1,2, C. Luxenburg1,2, B. Geiger2, L. Addadi1

1Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel, 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

Osteoclasts are multinucleated cells that degrade bone. Bone is composed of collagen fibrils, mineralized by carbonated apatite crystals. With maturation, the mineral to collagen ratio increases, leading to a change in the chemical composition and the mechanical properties of bone. Osteoclast activity is regulated by diverse signals from the extracellular matrix. Yet the nature of substrate properties that are sensed by osteoclasts and the mechanism whereby these properties affect cell behavior are still poorly understood. We examined the structure, dynamics and activity of the resorption apparatus of - osteoclasts, attached to bone, calcite crystals and glass. Examination of the general architecture of the adhesion apparatus, known as the sealing zone, revealed similarity between the structures formed on the three substrates(1). In contrast, the structure of the membranous resorption organelle, the ruffled border, differs on the three substrates. On glass the ruffled border does not develop, on calcite the intrusions of the membrane are mainly sustained by the surface topography while on bone the highly convoluted membrane is supported by cytoskeletal fibers. These differences suggest the existence of surface dependent regulation of this organelle. Both on calcite and on bone the cells create resorption pits, indicating that the altered structure of the ruffled border does not influence the capability to degrade the mineral substrate. Time lapse movies of osteoclasts expressing GFP-actin indicated that cells plated on bone or calcite are less motile, forming intact sealing zone rings with longer life span than on glass, where fragmented rings were commonly observed. Determining the manner in which artificial substrate properties influence osteoclast behavior can lead to a better understanding of osteoclast function and activity under physiological and pathological conditions.

1. Luxenburg, C., Geblinger, D., Klein, E., Anderson, K., Hanein, D., Geiger, B., and Addadi, L., The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly, PLoS ONE, 2, e179 (2007). BONE-MARROW STROMAL CELLS EXERT IMMUNOSUPPRESSIVE EFFECTS ON IMMUNE CELLS CO-CULTIVATED IN 3-D ALGINATE SCAFFOLDS

Y. Kaminer1, A. Monsonego2, S. Cohen1

1Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Shraga Segal Department of Microbiology and Immunology, National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Bone Marrow-derived mesenchymal stem cells (MSCs) and stromal cells have been shown to exert a potent immunosuppressive effect on immune cells, when co- cultivated on flat two-dimensional (2-D) cultures. Yet, this type of cell cultivation may not reliably represent the function of the cells in a physiological setting. In this work, we aim to investigate whether stromal cells would have similar immunosuppressive effects on dendritic cells (DC) and T cells, when co-cultured within porous alginate scaffolds. Alginate scaffolds herein are replacing the extracellular matrix (ECM), providing both a physical and biological support for the seeded cells and closing the gap to in vivo conditions. Our results demonstrate that 3- D co-cultures of stromal cells and DCs promoted the formation of mixed clusters distributed along the scaffold. Lipopolysaccharide-induced DC maturation was attenuated in the presence of stromal cells as indicated by the lower expression of TNF-ƒׁƒnand the co-stimulatory molecules CD86 and CD80. Furthermore, upon their co-culture with stromal cells, DCs induced limited T-cell activation, secreting lower ,levels of proinflammatory cytokines such as IL-2 and TNF-ƒׁ. Therefore, our data thus, suggest that 3-D cultivated stromal cells can exert an immunomodulating effect presumably via inhibiting DCs maturation. This modulation effect may have a therapeutic potential for T cell-dependent tissue damage caused in autoimmune diseases. ALGINATE SCAFFOLD-CULTIVATED HUMAN HEPATOCYTE CELL LINE REVEALS SUPERIOR HEPATOCELLULAR FUNCTIONS COMPARED TO COLLAGEN SCAFFOLD-SEEDED CELLS

O. Lesman, T. Elkayam, S. Cohen

Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Introduction: Three-dimensional (3D) cell cultures are replacing the "flat biology" of the Petri dishes, thus providing a wealth of information on cellular behavior in a physiologically relevant context. The 3-D cultivation is frequently achieved by seeding cells within polymeric scaffolds, which play a critical role in determining the quality of the culture. The main goal of this work was to compare hepatocyte behavior in alginate vs. collagen 3-D porous scaffolds. The scaffolds had similar physical characteristics, but differ in the extent of cell adhesion to the matrix. Materials and Methods: We tested cell morphology (histology, fluorescence microscope) viability and proliferation (MTT, PCNA immunostaining, AST biochemistry) albumin secretion (ELISA, immunostaining) and pre- and post-induction gene expression (real-time PCR). Results and Discussion: Cultivation of C3A, a human hepatocytes cell line, in alginate scaffolds induced the formation of multi-cellular spheroids, while in the collagen scaffolds the cells were attached and generally adopted a spread-out morphology. AST enzymatic assay revealed a smaller amount released in the alginate- based constructs compared with the collagen-based constructs. By MTT viability analysis, both constructs maintained nearly constant metabolic activity during cultivation. PCNA immunostaining, as indicative for cell proliferation, was generally negative in the collagen-based cell construct, while in the alginate it decreased with time. Albumin secretion and ECM component production were higher in the alginate cell-constructs compared to the collagen cell-constructs. There was no significant difference in the basal gene expression of albumin, alpha-fetoprotein, CYP1A2 and UGT2B7 between the two cultivation methods. However, post induction mRNA expression of CYP1A2 was significantly higher (9-folds) in the alginate cell constructs. These results further substantiate the superiority of alginate scaffolds as 3- D matrix for culturing hepatocytes. The preferred formation of hepatocyte spheroids in alginate scaffolds enhanced and maximized cell-cell interactions and elevated liver functions. ALGINATE-BASED COTROLLED DELIVERY OF HGF AND IGF-1 IMPROVES SELF-REPAIR AND HEALING AFTER EXTENSIVE MYOCARDIAL INFARCTION

E. Ruvinov1, I. Freeman1, R. Holbova2, N. Landa2, J. Leor2, S. Cohen1

1Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Neufeld Cardiac Research Institute, Sheba Medical Center, Tel Hashomer and Tel-Aviv University, Ramat Aviv, Israel

Purpose: We aimed to test the hypothesis that local control delivery of hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-1) via injectable alginate hydrogel will enhance repair and endogenous regeneration after acute myocardial infarction (AMI). Methods and Results: Sulfated alginate has been used to produce injectable HGF and IGF-1-bioconjugates. In vitro characterization and validation by ELISA revealed controlled release of biologically active growth factors (GFs) over a period of 10 days under physiological sink conditions. Western blot analysis showed that the released GFs induced activation and phosphorylation of AKT and ERK1/2 in isolated rat neonatal cardiomyocytes. In vivo, the GF-alginate bioconjugates were injected immediately post-AMI in male Sprague-Dawley rats. After 1-week follow- up, vascularization and morphometric analyses showed that GF-bioconjugate injection significantly increased blood vessel density and prevented infarct expansion. PCNA staining pattern of the infarcted hearts treated with the combined HGF/IGF-1 bioconjugates or the soluble form of GFs revealed sites of possible myocardial regeneration. Conclusions: The present work suggests that the sustained delivery of cardio-protective growth factors, HGF and IGF-1, from injectable alginate-based bioconjugates can enhance self-repair and tissue healing. PANCREAS TISSUE ENGINEERING FOR LONG TERM FUNCTION OF BETA CELLS

A. Soroker, E. Mitrany

Department of Cell and Animal Biology, Hebrew University of Jerusalem, Israel

The main objective of our work is to obtain long term in vivo-like function and viability of beta cells. The rational used is based on the premise that in order to have proper function of parenchymal cells it is necessary to provide an appropriate stroma. We have developed an in vivo-like culture system based on micro-organs (MOs). MOs are organ fragments of macroscopic thickness that preserve the natural epithelial/mesenchymal interactions and that ensure appropriate diffusion of nutrients and gases to all cells. MOs maintain the basic organ microstructure and transcribe tissue specific genes even when cultured for long periods on serum-free medium. Based on the concept of micro-organs we have prepared MO-derived matrices which preserve the architecture and the basic composition of the stroma of the organ of origin and ensure that no cell is more than 150 microns from a source of nutrients and gases. In the present work, pancreas-derived micro-organ matrices (p-MOMs) constitute the stromal bases in which beta cells are cultured. We here report that: 1. We have been able to prepare p-MOMs which display normal basement membrane pattern as indicated by laminin staining. 2. Insulinoma cells seeded onto p-MOMs populate the matrix but only some cell clusters stain positive for insulin. 3. Whole islets seeded onto p-MOMs remain viable with some cell outgrowth, spreading into the p-MOMs. 4. Within the outgrowth a significant proportion of cells stain positive for insulin. 5. Single cells derived from islets from transgenic mouse which express GFP driven by the Pdx1 promoter and seeded onto pancreas-derived MOMs spread into the p-MOMs and express GFP. The approach presented here, in which beta cells are incorporated into an engineered tissue together with their natural micro- environment, that preserves the natural interactions between parenchyma, and the normal pancreas stroma is a unique and novel approach to obtain long-term in vivo- like beta cell function. DNA COMPACTION DURING IN VITRO ASSEMBLY OF SV40 CAPSIDS AROUND SUPERCOILED PLASMID DNA

M. Abd El-Latif, A. Oppenheim

Department of Hematology, Hebrew University-Hadassah Medical School, Jerusalem, Israel

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm, encompassing its 5.2 kb double stranded circular DNA genome. The DNA is assembled in nucleosomes in a chromatin-like structure and is termed ‘minichromosome’. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. We have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA. The reaction is performed in a cell-free system and the packaged plasmid carries a reporter gene. The assembled nanoparticles were demonstrated to be efficient in transduction of target cells and may be used for gene delivery experiments. In the assembly reaction DNA bound to VP1 within minutes, while VP1 assembly around the DNA was slow (1-3 hrs). DNA isolated from packaged nanoparticles appeared to be more highly condensed than the input supercoiled plasmid, as it migrated in electrophoresis more rapidly. It appeared as a single band, suggesting that complete molecules were packaged. We used chloroquine gels to analyze whether there is a change in the linking number of the supercoiled DNA. However the DNA did not resolve at chloroquine concentrations 10-100 µg/ml. Our present explanation is that the DNA is either knotted or denatured. In the latter case, the two complimentary strands most likely remain circular and intertwined, as the DNA appears to regain transcription and replication activities when delivered to recipient cells. We plan to distinguish between these two possibilities using topoisomerases I and II, which are specific for single and double stranded DNA respectively. THE ROLE OF A PENTAPEPTIDE HINGE OF THE SV40 MAJOR CAPSID PROTEIN IN ASSEMBLY AND CAPSID STABILITY

O. Ben-Nun-Shaul, H. Bronfeld, O. Schueler-Furman, A. Oppenheim

Hebrew University - Hadassah Medical School, Jerusalem, Israel

The SV40 outer shell is composed of 72 pentamers of VP1. The core of the VP1 monomer is a beta-barrel with jelly-roll topology, with extending N and C-terminal arms. A pentapeptide hinge, KNPYP, tethers the C-arm to the VP1 beta-barrel core. The five C-arms that extend from each pentamer insert into the neighboring pentamers, tying them together through different types of interactions. In the mature virion, this element adopts either of 6 distinct conformations, according to their location in the capsid, suggesting that the hinge directs the invading C-arm to a neighboring pentamer (1). We found that the hinge is conserved among 16 members of the polyomaviridae. We have used site-directed mutagenesis in order to gain an understanding into the structural requirements of this element: Y299 was changed to A, F and T and P300 to A and G. The mutants showed reduction in viability to varying degrees. Diminished viability may be caused by defect in assembly, in stability or in cell recognition and entry. Assembly was reduced in all the mutants, but only to a small extent, not accounting for the large reduction in viability. However the mutants were very unstable. The largest effect was observed for mutations of P300, especially for the P300G mutation. This rigid hinge could therefore be responsible for "fixing" the angle of emergence of each C-arm. Y299T and Y299A were more defective in viability than Y299F, highlighting the importance of an aromatic ring at this position. Structural inspection showed that this aromatic ring contacts C-arms of neighboring pentamers, suggesting a more complex role for the pentapeptide hinge for capsid formation, stability and presumably for cell recognition and entry. STUDY OF THE INTERACTION BETWEEN THE ADENOVIRUS E4orf4 PROTEIN AND A PROTEIN INVOLVED IN CHROMATIN REMODELING

A. Brestovitsky, R. Sharf, T. Kleinberger

Unit of Microbiology, The B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Adenoviruses are nonenveloped icosahedral viruses containing linear double-stranded DNA. The viral genome contains five distinct early regions E1A, E1B, E2, E3, and E4, that encode several different mRNAs as a result of alternative splising. The adenovirus E4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator encoded by the adenovirus early region 4 transcription unit. It was shown that E4orf4 protein down-regulates expression of cellular and viral genes, induces hypo-phosphorylation of various viral and cellular proteins, regulates alternative splicing of adenovirus mRNAs, regulates protein translation, and induces apoptosis in transformed cells. Early in infection, the adenovirus E1A protein induces cellular and viral gene transcription. When E4orf4 starts to accumulate, it down regulates most of the early viral promoters, including E2 and E4 itself. Therefore, E4orf4 is part of a negative feedback loop, which contributes to the temporal regulation of early viral gene expression. Various E4orf4 partners are involved in the signaling induced by E4orf4 during viral infection and apoptosis. E4orf4 interacts directly with the heterotrimeric protein phosphatase 2A (PP2A), causing hypo-phosphorylation of various proteins. The E4orf4-PP2A interaction is required for down-regulation of gene expression and apoptosis induced by E4orf4. E4orf4 also associates with members of the Src kinase family leading to its Tyr phosphorylation and to deregulation of Src signaling, further contributing to induction of apoptosis. To find other proteins that interact with E4orf4 we applied the yeast Ras Recruitment System (RRS) using a HeLa cDNA library. One of the proteins found in the screen was a protein that is a part of a chromatin remodeling complex. The purpose of this work is to understand the significance of this interaction to E4orf4-regulated events, including down regulation of transcription and induction of apoptosis. HOW VIRUSES ENSURE dNTP POOL IN NONDIVIDING CELLS: THE HBV MODEL

D. Cohen, Y. Shaul

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

The replication of both cellular and DNA virus genomes requires a balanced and adequate supply of deoxyribonucleotides triphosphates (dNTPs). Since nucleotide synthesis is tightly linked to DNA synthesis, resting cells have very low dNTP pool in comparison to S-phase cells. Hepatitis B virus (HBV) is a partially double stranded DNA virus with tissue and species specificity to human hepatocytes, which are quiescent cells. During replication the viral polymerase reverse transcribes the pregenomic RNA to DNA using dNTPs. It has been shown that the viral release into the periphery of acute infected individuals is ~10(7) to 10(11) virus particles per day. Since nucleotide pool in quiescent hepatocytes is very limited, the virus must have developed a mechanism to increase this pool. The key enzyme responsible for dNTP synthesis is ribonucleotide reductase (RNR), which reduces NTPs to dNTPs and composed of two subunits, R1 and R2. Both proteins are required for RNR activity, which is regulated by S-phase specific de novo synthesis and degradation of the R2 subunit. While R1 expression level is stable during the cell cycle, R2 expression is hardly detectable in quiescent cells (G0/G1). We examined the possibility that HBV induces unscheduled RNR expression in resting hepatocytes, a process termed deregulation. We report here that R2 expression is elevated in HBV-expressing cells,whereas other cell proliferating genes remained silent. This was the case in quiescent hepatocytes, in tissue culture and mice models. In fact the induction of R2 is so efficient that an HBV producing cell line secretes high amounts of thymidine. This previously unseen selective activation of R2 gene expression is directly linked to HBV gene expression. We propose a viral mechanism for efficient HBV reproduction in quiescent hepatoma cells through RNR and nucleotide synthesis activation. MOLECULAR EVOLUTION OF VP1 FOR IMPROVED ASSEMBLY OF SV40-BASED VECTORS

D. Fradkin1, S. Koby2, A. Oppenheim1

1Hebrew University-Hadassah Medical School, Hematology, Jerusalem, Israel, 2Hebrew University-Hadassah Medical School, Molecular Genetics and Biotechnology, Jerusalem, Israel

Simian Virus 40 (SV40) infects a wide variety of cell types, is non-pathogenic for humans and has very low mmunogenicity. SV40-based vectors were shown to be effective in gene delivery to both in vitro and in vivo targets. The viral capsid is composed of three proteins VP1, VP2 and VP3. VP1 forms the outer shell and alone is sufficient for capsid formation. SV40 vectors can be prepared in vitro from recombinant VP1expressed in insect Sf9 cells and supercoiled plasmid DNA propagated in E.coli. Production of recombinant VP1 in E.coli has a major biotechnological advantage over expression in insect cells. However, assembly of wt VP1 produced in E.coli is very poor. The objective of the project is to isolate VP1 variants with improved assembly. I develop molecular evolution strategy taking advantage of the ability to express VP1 in E.coli and the strong selection for plasmids packaged in SV40 capsids provided by infection and propagation in mammalian cells. At present I establish methods to express VP1 in E.coli using a small plasmid. The rationale is that the plasmid used for VP1 expression will be packaged by the SV40 capsid assembled in the bacterial cell, and will be able to infect and propagate in primate cells. This enrichment procedure will be combined with mutagenesis to obtain VP1 variants with improved assembly. ENGINEERING OF ANTI-INFLUENZA scFv, WHICH INHIBIT MULTI STRAIN INFLUENZA REPLICATION

G. Gubi, L. Lobel

Department of Virology and Developmental Genetics, Ben Gurion University of the Negev, Beer Sheva, Israel

Background: The highly contagious, acute respiratory illness known as Influenza appears to have afflicted humans since ancient times. The genome of Influenza A virus consists of eight segments of negative-sense, single-stranded RNA molecules. In the viral particle, the RNA segments are complexes with the major viral structural protein, the nucleoprotein (NP), to form ribonucleoprotein (RNP) complexes. Three virus-encoded polypeptides: PB1, PB2, and PA, which constitute the viral RNA dependent RNA polymerase (RDRP), are associated with biochemical analyses have shown specific interactions between PB1 and PA as well as PB2, indicating that PB1 is the backbone of the complex. Twenty-five amino acids at the N terminus of PB1 were sufficient for binding PA in vivo, with the same efficiency as the complete PB1. Aim: Engineering of anti-Influenza scFv, which inhibit multi strain Influenza replication. Results and Conclusions: 1. We isolated six scFvs that specifically bind to the Twenty-five amino acids at the N-terminus of PB1 protein of Influenza A. 2. We cloned the scFv gene (antisence copy) into a plasmid where the scFv is flanked by the human RNA polymerase I promoter with a 5’ untranscribed region and the RNA polymerase I terminator with a 3’ UTR. Intracellular transcription of that construct by RNA polymerase I in 293T cells generated an scFv vRNA that could be packaged into Influenza VLPs or transcribed by the w.t Influenza RDRP and inhibit the replication of the virus. 3. Our results demonstrate that scFv against N-terminal 25 amino acids of PB1 clearly inhibit the replication of multi strain Influenza virus, specifically if the transfection with the scFv construct was 24 hours before the infection. DEVELOPMENT OF A PLANT VIRUS AS A UNIQUE PLATFORM FOR ORAL VACCINATION OF POULTRY

R. Hochman1, E. Bar-Shira1, A. Friedman1, D. Leibman2, T. Arazi3, A. Gal-On2

1Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Sciences, Hebrew University of Jerusalem, Israel, 2Department of Plant Pathology, ARO, Volcani Center, Bet Dagan, Israel, 3Department of Ornamental Horticulture, ARO, Volcani Center, Bet Dagan, Israel

Oral vaccines offer the hope of more convenient immunization strategies in livestock and poultry. As plant viruses accumulate to high levels and are harmless to animals, they could be used as carriers for immunogenic epitopes by fusing them to the exposed coat protein. Such recombinant virions display numerous copies of the epitope on their surface, thus enhancing their immunogenicity, providing potentially effective, inexpensive and safe vaccine. We developed a novel attenuated Zucchini yellow mosaic potyvirus (ZYMV-AGII) as a non-pathogenic virus-vector in cucurbits and have demonstrated that AGII can express foreign epitopes on its viral surface without impairing squash development or crop yield. We determined the optimal epitope integration site on AGII coat protein by comparing the effects of different fusion sites on epitope exposure and viral accumulation. To test the immunogenic potential of this vector we created a Human cMyc expressing recombinant virus, AGII-cMyc. Chicks were either fed with purified virus, or voluntarily consumed fresh or desiccated infected zucchini leaves expressing this virus. Specific anti-cMyc antibody responses were determined and compared to those induced following intramuscular injection of recombinant virus in adjuvant. Our observations show AGII-cMyc to be immunogenic in all instances. After we established the model with AGII-cMyc, we extended our vaccine system to include epitopes of a well known avian pathogen: epitopes HN and F of the Newcastle disease virus (NDV). We successfully constructed epitope expressing AGII-NDV-HN and AGII-NDV-F and demonstrated the ability of these AGII chimeric particles to induce anti-epitope specific antibody responses following injection or voluntary intake of fresh and dry infected zucchini leaves. PHOSPHORYLATION OF SV40 CAPSID PROTEINS IS REQUIRED FOR DNA PACKAGING

S. Kler, A. Oppenheim

Department of Hematology, Hadassah Medical School, Hebrew University of Jerusalem, Israel

SV40 is non-enveloped virus with a capsid consisting of three proteins, VP1, VP2 and VP3. VP1 is present, as pentamers, at the outer surface, whereas VP2 and VP3 form the bridge between the VP1 shell and viral DNA. VP3 is translated from the second in-frame initiation codon within VP2 and is identical to the C-terminus of VP2. VP2/3 have been implicated in nuclear entry of viral genome. The capsid proteins are known to be phosphorylated, but the phosphorylation sites, apart from one, are not identified, and the functions of phosphorylation are unknown. I found that VP2/3 are phosphorylated at serine. The phosphorylated site has been mapped to the C-terminus, that contains the DNA binding domain. My studies showed that VP2/3 are phosphorylated only in "full" SV40 virions, containing viral DNA, while in "empty" virus particles they are not phosphorylated. My working hypothesis is that phosphorylation of VP2/3 is critical for DNA packaging, and may be also needed for carrying the viral genome to the nucleus of infected cells. In our laboratory an experimental system of SV40 assembly in vitro has been established, using viral recombinant proteins, produced in insect cells (Sf9). It was found, however, that recombinant VP2/3 do not enhance infectivity of in vitro packaged particles. I found that recombinant VP2/3 is serine-phosphorylated at a much high level than VP2/3 of wt SV40 propagated in CV-1 cells. I propose that overphosphorylation prevents the proper binding of VP2/3 protein to DNA. It could explain the fact that recombinant VP2/3 do not facilitate in vitro assembly, which is assayed as infectivity. I plan to identify the precise site(s) of VP2/3 phosphorylation in wt SV40 and in recombinant viral proteins by mass spectrometry, and to investigate the functions of the phosphorylation in viral life cycle using in vitro mutagenesis. ATTENUATION OF VERY VIRULENT INFECTIOUS BURSAL DISEASE VIRUS AND COMPARISON OF FULL SEQUENCES OF VIRULENT AND ATTENUATED STRAINS

S. Krispel1, D. Lazarus1, M. Pasmanik-Chor2, B. Gutter3, M. Barbakov3, J. Pitcovski1

1Department of Immunology, MIGAL, Kiryat Shmona, Israel, 2Department of 3 Bioinformatics, Tel-Aviv University, Ramat Aviv, Israel, Abic Biological Laboratories Teva, Beit Shemesh, Israel

Infectious Bursal Disease (IBD) in poultry is caused by IBD viruses (IBDV) of the family Birnaviridae. Infected chickens suffer from diarrhea, and many other severe complications. Mortality rate is high and the surviving chickens exhibit immunosupression, growth retardation and high sensitivity to other diseases. Protection against the disease is achieved by vaccinating the birds with non- pathogenic IBDV vaccines. Since 1986, new very virulent (vv) pathogenic strains had appeared independently all over the world and the existing live attenuated vaccines do not protect against these new strains. The new strains isolated in Europe have been found to be closely related to one another, but to differ from the previous "classic" strains. "Variant" strains isolated in the US also differ from the "classic" strains. In recent years, sequences of virulent and attenuated strains of IBDV have been analyzed and the contribution to virulence has been identified in viral protein 2 (VP2) on segment A, viral protein 1 (VP1) on segment B, or both. Following attenuation processes the antigenicity and neutralization abilities were conserved. An attenuated vaccine strain (IBDVmb) was developed by Abic Biological laboratories Teva in 1989 by serial passages of the virulent strain (IBDks) in embryonated Specific Pathogen Free (SPF) eggs. In the present work the full sequences of IBDVks and IBDVmb were compared to each other as well as to published sequences, in order to contribute to our understanding of the nature of IBDV attenuation at the molecular level. The comparison revealed seven nucleotides that were different, four of them leading to changes in the amino-acid sequence. None of the changes interfered with the neutralization site identified in VP2. This study offers novel sites for virus attenuation and strengthens the possibility that more than one protein is involved in IBDV attenuation. REGULATION OF THE HIV-1 INTEGRASE ACTIVITY BY THE VIRAL REV PROTEIN: THE EFFECT OF CELL PERMEABLE PEPTIDES DERIVED FROM THE INTEGRASE AND Rev PROTEINS.

A. Levin1, J. Rosenbluh1, Z. Hayouka2, A. Friedler2, A. Loyter1

1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Department of Organic Chemistry, Institute of Chemistry, Hebrew University of Jerusalem, Israel

The Human Immunodeficiency Virus (HIV) has caused the death of over 25 million people, worldwide, since 1981. Two types of HIV are known to infect humans; HIV-1 and HIV-2. HIV-1 is the more common of the two and the one that causes the larger number of fatalities. An essential step in the replication cycle of all retroviruses is the integration of the viral DNA (after reverse transcription) into the host genome. The research set forth here deals with the molecular mechanisms of the integrase (IN) activity and its regulation. The HIV-1 IN mediates the integration of the viral genome into the host cell DNA. In HIV-1 infected cells there are only one or two integrated viral genomes (proviruses) per cell while in other retroviruses the number of proviruses per cell is much higher. The mechanism which prevents multiple integrations is specific to HIV-1 and is yet unknown. Thus elucidation of the detailed mechanism of the regulation of IN activity may lead not only to a better understanding of the viral replication cycle but also to the development of new anti- HIV-1 drugs. We suggest that one of the HIV-1 early translated proteins, namely the Rev protein, may be responsible for this regulation. The viral Rev protein, mediates nuclear export of viral un-spliced and partially spliced RNA. We have observed that Rev binds to IN and furthermore it inhibits the IN activity in-vitro. These results may indicate that the Rev protein may regulate the enzymatic activity of the IN and consequently the viral DNA integration process. Support of this view obtained from results showing that Rev derived peptides are able to inhibit the IN activity in-vitro and in-vivo while peptides derived from the IN, which binds to the Rev, abrogate those inhibitions and increase the IN activity in cell cultures. NOVEL APPROACHES FOR TREATMENT OF AVIAN INFLUENZA INFECTION

R. Oren Benaroya2, R. Azar1, D. Katz2, E. Mendelson1, M. Mandelboim1

1Central Virology Laboratory, Chaim Sheba Medical Center, Tel Hashomer, Israel, 2Bio Vent Ltd., Rehovot, Israel

Background: Influenza virus undergoes frequent genetic and antigenic changes resulting in the occasional emergence of new types of viruses which may posses a threat to the entire population. We are currently facing such a possible threat by the deadly avian H5N1 influenza virus. Vaccine development against avian influenza infection is slow and potent antiviral drugs are not available. Therefore, development of new tools for the treatment of avian flu infection is a critical issue. Working hypothesis and aims: We developed a new treatment for avian flu infection which is based on using a sequence of around 70 nucleotides, known as an aptemer, which generates a unique secondary structure capable of interacting with the hemagglutinin of various influenza viruses. Methods and results: We demonstrate that the aptamer is stable in plasma and is able to inhibit various influenza infections including avian flu (H5N1), H3N2 and H1N1. We plan to further study the mechanisms of the aptamer inhibition and we suggest that aptamer treatment might be used in the future against various flu infections. Importance and Probable implications to Medicine: We are facing an extremely deadly virus that might cause the next influenza pandemic. Current avian vaccination is absent and mutant strains of avian flu viruses that are resistant to Tamiflu (the only efficient medicine against avian flu infections) were identified in Egypt. Thus, developing new medicine to treat avian flu infection is mandatory. TWO DISTINCT PORTALS IN THE GIANT ACANTHAMOEBA POLYPHAGA MIMIVIRUS

N. Zauberman1, Y. Mutsafi1, E. Klein2, E. Shimoni2, A. Minsky1

1Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Chemical Services, Weizmann Institute of Science, Rehovot, Israel

With a diameter of ~650nm and a 1.2Mbs genome, the recently discovered nucleocytoplasmic large DNA virus Acanthamoeba Polyphaga Mimivirus, is the largest virus ever identified. The size of the Mimivirus particle as well as the size of its genome render this organism into a uniquely suitable candidate for addressing fundamental, yet poorly understood, structural aspects of viral replication cycle. Accordingly, a combination of advanced microscopy techniques are being exploited, including electron tomography of sections of infected host cells obtained at various post-infection times, immuno-labeling, specific DNA staining for TEM preparations, SEM studies, and confocal microscopy analyses, in conjunction with genetic tools, to analyze specific events along the viral life cycle. All icosahedral DNA viruses for which structures have been solved appear to utilize a single portal for both delivering and packaging their genome. Here we present the first exception to this paradigm by demonstrating that genome delivery and packaging in the giant virus Acanthamoeba Polyphaga Mimivirus occur through two distinct portals. We show that Mimivirus genome release entails a major conformational change of the capsid, whereby five triangular icosahedral faces open up to form a massive tube-like conduit. A large non- vertex orifice located at the center of a triangular face distal to the DNA delivery site provides a portal for DNA packaging. Results exposed here guarantee such techniques to be useful in shedding light on important viral processes as well as in the deepening of our comprehension of the viral world. SV40 ASSEMBLY IN VIVO AND IN VITRO

A. Oppenheim, A. Gordon-Shaag, S. Mukherjee, O. Ben-Nun-Shaul, M. Abd El-Latif, H. Bronfeld, S. Kler

Department of Hematology, Hebrew University - Hadassah Medical School, Jerusalem, Israel

The SV40 capsid is a T=7d icosahedral lattice ~45 nm in diameter surrounding the ~5 kb circular minichromosome. The outer shell is composed of 360 monomers of the major capsid protein VP1, tightly bound in 72 pentamers. VP1 is a jellyroll ?-barrel, with extending N- and C-terminal arms. The N-terminal arms bind DNA and face the interior of the capsid. The flexible C-arms tie together the 72 pentamers in three distinct kinds of interactions, thus facilitating the formation of a T=7 icosahedron from identical pentameric building blocks. Assembly in vivo was shown to occur by addition of capsomers around the DNA. We apply a combination of biochemical and genetic approaches to study SV40 assembly. Our in vivo and in vitro studies suggest the following model: One or two capsomers bind at a high affinity to ses, the viral DNA encapsidation signal, forming the nucleation center for assembly. Next, multiple capsomers attach concomitantly, at lower affinity, around the minichromosome. This increases their local concentration facilitating rapid, cooperative assembly reaction. Formation of the icosahedron proceeds either by gradual addition of single pentamers to the growing shell or by concerted assembly of pentamer clusters. A NOVEL APPROACH FOR TREATMENT OF ALZHEIMER`S DISEASE: ANTIBODIES AGAINST BETA-SECRETASE CLEAVAGE SITE OF APP

M. Arbel, B. Solomon

Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

Although the extracellular A-beta plaques are the main hallmark and criteria for an ultimate post mortem diagnosis of Alzheimer's disease (AD), it remains to be determined whether extracellular or intracellular A-beta accumulation initiates the disease process. Intraneuronal A-beta accumulation is increasingly linked with early preplaque synaptic and pathological abnormalities occur in brain of AD patients and transgenic animals. Understanding the mechanisms that underline A-beta accumulation in neurons and its relation with the extracellular A-beta pool, may be beneficial in developing therapeutic tools for AD treatment. We developed a novel approach to inhibit A-beta production via antibodies against the beta-secretase cleavage site of APP. This approach limits APP processing by beta -secretase, mainly through the endocytic pathway and overcomes some of the limitations of BACE1 (beta-secretase) inhibition methodologies. Anti beta-site antibodies bind human APP expressed by AD cellular model and internalize into the cells after APP binding at the plasma membrane. Antibodies mediated inhibition of beta-secretase cleavage led to a 50% reduction of intracellular A-beta levels. Intracerebroventricular injection of these antibodies to non transgenic mice in which neprilysin activity was inhibited led to 60% and 80% reduction in intracellular and extracellular brain A-beta levels, respectively, highlighting the therapeutic potential of these antibodies. Moreover, systemic administration of anti beta-site antibody to tg2576 mice facilitated a beneficial effect on the mice cognitive function and did not lead to any adverse effects. To the best of our knowledge this is the first attempt to inhibit beta-secretase cleavage of APP by blocking the cleavage site upon the substrate rather then a total inhibition the enzyme. This approach overcomes some of the limitations presented by both BACE1 inhibition methodologies and A-beta based immunotherapies. IL-6/IL-6R AXIS PLAYS A CRITICAL ROLE IN ACUTE KIDNEY INJURY

Y. Nechemia-Arbely1, D. Barkan1, G. Pizov2, A. Shriki1, S. Rose-John3, E. Galun1, J.H. Axelrod1

1The Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel, 2Department of Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel, 3Institut fuer Biochemie, Christian-Albrechts-Universitaet zu Kiel, Germany

Tissue injury constitutes a major biological and medical problem involving the coordination of both inflammatory and repair processes. In acute kidney injury (AKI), IL-6 expression correlates with the onset and severity of injury; however, its role in AKI remains unclear. In this study, we have established a critical role for IL-6 in the inflammatory response to, and the resolution of AKI. IL-6-deficient mice displayed resistance to HgCl2-induced AKI and reduced peritubular neutrophil accumulation. Neutrophil depletion in wild-type mice significantly reduced HgCl2-induced injury, demonstrating the critical role of IL-6 signaling in the inflammatory process that exacerbates AKI. Renal IL-6 expression and STAT3 activation in renal tubular epithelial cells strongly increased during injury development, indicating the presence of IL-6 signaling in the renal parenchyma. However, a lack of renal IL-6 receptor (IL- 6R) precludes signaling via the classical-signaling pathway. IL-6R also exists in a soluble form (sIL-6R) that together with IL-6 stimulates target cells in a process termed trans-signaling. During injury development, serum sIL-6R levels, derived in part through receptor shedding from neutrophils, increased 3-fold, suggesting that IL- 6 trans-signaling may also be an intrinsic response to injury. Stimulation of IL-6 trans-signaling using an IL-6/sIL-6R fusion protein activated STAT3 in renal tubular epithelium and dramatically prevented AKI. The IL-6/sIL-6R-mediated reduction of lipid peroxidation levels following injury suggests that the protective effect of IL- 6/sIL-6R is largely mediated through amelioration of oxidative stress. Thus, IL-6 simultaneously supports an inflammatory response that exacerbates injury and, through a mechanism of trans-signaling, also protects the kidney from further injury. CCR5-CONJUGATED STEALTH-LIPOSOMES. A NEW DRUG DELIVERY SYSTEM TO HIV RESERVOIR CELLS AND FREE VIRION ENTRAPMENT

T. Bronshtein1, S. Pollack2, M. Machluf1

1Faculty of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel, 2Department of Immunology, Bruce Rappaport Medical School, Technion-Israel Institute of Technology, Haifa, Israel

Most existing anti-retroviral therapies focus on prolonging the latent phase of HIV/AIDS by the inhibition of viral growth; unfortunately, none of them are completely capable of destroying viral reservoirs. Our goal is to develop a drug delivery system that would specifically target all HIV infected cells and deploy agents that are toxic only when introduced into the cell-cytoplasm. The liposomal system is targeted by conjugating CCR5, the human receptor for HIV-gp120 that is found on the surface of virions and infected cells. Such a system can specifically destroy infected cells, without promoting the evolution of drug-resistance. CCR5-liposomes were composed of DYNAL™-beads surrounded by lipid bi-layers containing native CCR5 that was anchored to the beads and held in correct orientation via binding to 1D4-Mab's. For the studies of CCR5-liposomes/gp120 interactions, a model BHK/gp120 cell-line was engineered. The bio-activity of BHK/gp120 was confirmed by co-culturing with Jurkat/CXCR4+/CD4+ cells. EDTA was selected as an ideal liposomal-encapsulate following a bioinformatic analysis. Purified CCR5 was incorporated into EDTA-containing stealth-liposomes. SDS-PAGE and immuno- blotting has demonstrated that the CCR5-liposomes' protein composition was of 1D4- Mab's and CCR5. Confocal microscopy has demonstrated a correct spatial distribution of the lipid bi-layers surrounding the beads. BHK/gp120 bioactivity assay exhibited a 30% reduction of Jurkat/CXCR4+/CD4+ cells viability following co-culture. FACS analysis has demonstrated specific binding of CD4-FITC-beads to BHK/gp120 cells. Binding of CCR5-liposomes to BHK/gp120 cells was demonstrated using FACS, light and confocal microscopy and was found to be 3-fold higher then the binding to control cells. EDTA was found to have significant in-cell toxicity when introduced by mild sonication. EDTA-containing CCR5-liposomes were found to have a mean diameter of 70[nm]. EDTA-containing stealth-liposomes significantly differed in their binding and affect over cell-viability relative to non-stealth-liposomes. The results imply that a drug delivery system based on CCR5-liposomes may serve as a new therapy. A 3D PERSPECTIVE ON GAUCHER DISEASE

B. Brumshtein1, T.H. Futerman2, I. Silman3, J.L. Sussman1

1Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel, 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 3Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

Gaucher disease is the most common lysosomal storage disease, and is associated with mutations in the gene coding for the enzyme acid-beta-glucosidase (GCase, E.C. 3.2.1.45). There are ~200 known mutations, mostly missense mutations which result in substitution of amino acids in the protein. Some mutations cause complete deactivation of the enzyme; others impair its stability, and some affect both activity and stability. There are three known phenotypes of the disease: a mild, severe and acute. The acute phenotype is neuropathic, while severe and mild symptoms are caused by accumulation of GlcCer in macrophage cells resulting in bone atrophy, spleen enlargement, etc. The enzyme is a lysosomal, membrane-associated glycoprotein, and its 3D structure revealed that its catalytic domain is a TIM barrel. It catalyzes hydrolysis of the sphingolipid, glucosylceramide (GlcCer), to glucose and ceramide at the acidic pH prevailing within the lysosome. We have solved structures of recombinant GCase expressed in both mammalian and plant cell cultures. as well as of complexes with deoxynojirimycin (DNJ) based inhibitors, which have been shown to inhibit GlcCer synthetase as well as GCase. These molecules were shown to stabilize several mutant forms of the enzyme, hence prolonging its effective therapeutic span. We have shown that the molecular basis for the stabilization of GCase by these inhibitors is by binding to the active site and contributing additional non-covalent bonds that stabilize the enzyme’s overall structure. TECHNOLOGIES TO INCREASE THE CONTENT OF SECONDARY PHENOLIC METABOLITES (SPhMs) IN ALOE SPECIES

E. Chauser-Volfson, Y. Gutterman

Ben-Gurion University of the Negev, Campus Sde-Boker, Beer-Sheva, Israel

More than 100 Aloe species originating from the desert of South Africa we have introduced to the Botanical Garden of the Jacob Blaustein Institute for Desert Reseach at Sade Boker in the Negev Desert of Israel. The Aloe plants have flourished in the desert conditions and are planted in loose soil. In addition to the average annual 100 mm of rain in this area in winter, they are irrigated with about 300-400 mm water. Leaf exudates from Aloe species are used to great extent in traditional medicines. The succulent leaves of Aloe species have been used for medicinal purposes, cosmetic and food supplement, and also in gardening to save water as ornamental plants. Among the succulent leaves of Aloe plant species the SPhMs were analyzed, including: barbaloin, homonataloin, nataloin, aloeresins and aloenins. SPhMs have been found the strong inhibitory effect, antiinflamatory and catharactic effect in vivo. SPhMs are very important defending compounds against leaf eater in order to keep the natural balance in the green world. The SPhMs of Aloe plant are also involved in the protection of the plants from UV irradiation damage. The aim of thi study is to present different methods, which were used in our experience for increase content of SPhMs in some Aloe species. HIGH THROUGHPUT SCREENING OF SYNTHETIC LIBRARIES FOR DISCOVERY OF POTENTIAL DRUG CANDIDATES FOR TREATMENT OF HUMAN GENETIC DISEASES

M. Cherniavsky, T. Baasov

Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel

A large number of human genetic disorders result from nonsense mutations, single point alterations in the DNA, where one of the three stop codons replaces an amino acid-coding codon, leading to a premature termination of the translation and eventually to a nonfunctioning protein. Many serious illnesses, such as cystic fibrosis, Duchenne muscular dystrophy and more occur from such mutations. For all of them there is no effective treatment to date. There are several substances that are known to promote stop codon readthrough, allowing synthesis of full-length, functioning proteins. Majority of them belong to a subclass of aminoglycoside antibiotics, which exert their activity by binding to the ribosome and promoting mistranslation of mRNA. In this study, we describe an entirely new class of compounds, which promote stop codon readthrough and enable to obtain full-length proteins. In order to perform a rapid evaluation of the readthrough activity of a large number of substances we have developed a high-throughput screening assay. The system consists of two plasmids each carrying two luciferase genes (renilla and firefly). One of the plasmids includes a stop codon between the two genes (mutant) and thus in the lack of its suppression only the first gene product is being translated (renilla luciferase). The other plasmid does not include a stop codon (wild type), and thus both gene products are being translated. The firefly to renilla luciferase activities ratio of the mutant and the wild type represents the readthrough level. The results of this pilot study lead to the discovery of a new class of compounds that carry high potential as stop codon mutation suppressors. Using this developed high throughput, functional screening assay we plan to scan large synthetic libraries in order to discover other substances promoting stop codon readthrough and to perform a systematic structure activity relationship studies. CHARACTERIZATION OF THE INTERACTING DOMAIN OF THE HIV-1 FUSION PEPTIDE WITH THE TRANSMEMBRANE DOMAIN OF THE T- CELL RECEPTOR

T. Cohen1, M. Pevsner-Fischer2, N. Cohen2, I.R. Cohen2, Y. Shai1

1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

HIV infection is initiated by the fusion of the viral membrane with the target T-cell membrane. The HIV envelope glycoprotein, gp41, contains a fusion peptide (FP) in the N terminus that functions together with other gp41 domains to fuse the virion with the host cell membrane. We recently reported that FP co-localizes with CD4 and T- cell receptor (TCR) molecules, co-precipitates with TCR, and inhibits antigen-specific T-cell proliferation and pro-inflammatory cytokine secretion. Molecular dynamic simulation implicated an interaction between an alpha-helical transmembrane domain (TM) of the TCRalpha-chain and the beta-sheet 5-13 region of the 16 N-terminal aa of FP (FP1-16). To correlate between the theoretical prediction and experimental data, we synthesized a series of mutants derived from the interacting motif GALFLGFLG stretch (FP5-13) and investigated them structurally and functionally. The data reveal a direct correlation between the beta-sheet structure of FP5-13 and its mutants and their ability to interact with TCRalpha-TM and to induce immunosuppressive activity; the phenylalanines play an important role. Furthermore, studies with fluorescently labeled peptides revealed that this interaction leads to penetration of the N-terminus of FP and its active analogs into the hydrophobic core of the membrane. A detailed understanding of the molecular interactions mediating the immunosuppressive activity of the FP5-13 motif should facilitate evaluating its contribution to HIV pathology and its exploitation as an immunotherapeutic tool. MODULATION OF INTRACELLULAR AGGREGATES THROUGH FILAMENTOUS PHAGES IN A CELLULAR MODEL OF PD

H. Dimant, N. Sharon, B. Solomon

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv Universit, Ramat Aviv, Israel

Bacteriophages have several potential applications in the field of biotechnology, such as phage display systems and gene therapy delivery systems. Previous study conducted in our lab had demonstrated that the filamentous phage fd, display anti aggregating properties including prevention and disaggregation of already formed A- beta aggregates. Here we demonstrate that filamentous phages affect alpha synuclein filaments formation. Alpha synuclein aggregates, termed Lewy Bodies, are cytoplasmic aggregates which constitute a hallmark in the pathogenesis of Parkinson’s disease (PD). Since alpha synuclein is an intracellular antigen we generated a filamentous phage which presents on its coat protein a peptide containing a cyclic RGD motif enabling phage internalization to mammalian cells. We employed dopaminergic SH-SY5Y neurons overexpressing ƒׁ-synuclein as a model of PD. The cells were differentiated in order to enhance alpha syn aggregation in the cytoplasm. RGD presenting phages were analyzed for internalization as well as their effect on alpha syn aggregation. Here we demonstrate the internalization of the RGD phages into SH-SY5Y cells as visualized both in immunocytochemistry staining and in phage infectivity assay. In order to determine the effect of the phages on alpha syn aggregation we have analyzed the amount of alpha syn in the triton soluble and triton insoluble fraction following treatment with the RGD phages and determined the soluble/insoluble ratio of alpha syn. In treated cells we observed a shift in the alpha syn soluble/insoluble ratio, towards lower amounts in the insoluble fraction demonstrating the antiaggregating properties of filamentous phages. Taken together, the results demonstrate the unique properties of filamentous phages in reducing intracellular aggregates towards treatment of PD. INTRANASAL DELIVERY OF FILAMENTOUS PHAGE DISPLAYING MYELIN IMMUNODOMINANT EPITOPES TOWARD MULTIPLE SCLEROSIS TREATMENT

I. Rakover, K. Elsner, B. Solomon

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel

Multiple Sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS) associated with primary destruction of myelin sheaths. The cause of inflammation remains unclear, but an autoimmune response directed against CNS antigens is suspected. Although the exact pathogenesis of MS is not fully understood, it involves direct cytotoxicity mediated by T-lymphocytes, specific antibodies and complement as well as toxic products of macrophages aimed to the myelin components. The experimental autoimmune encephalomyelitis (EAE) model is used worldwide as the most common animal model for human MS. To induce EAE, C57BL/6 mice were immunized with fragments of myelin oligodendrocyte glycoprotein (MOG) and chronic disease developed approximately two weeks later. We engineered a filamentous phage in which its major coat protein was fused to myelin oligodendrocyte glycoprotein (MOG37-44) for delivery of an immunodominant epitope. Nasal administration of phages presenting MOG37-44 to EAE C57BL/6 mice was able to suppress the immune response against myelin and induced tolerance to EAE disease as measured by improved neuronal dysfunctions, reduced proinflammatory cytokine and without change in anti inflammation cytokine levels. Based on this data, we suggest a new approach using phages displaying immunodominant epitopes to treat MS. This peptide delivery system via filamentous phages enables direct brain delivery through the limbic system and stimulation of immune reactive mechanisms towards induction of tolerance in the periphery. JX401, A P38ALPHA INHIBITOR CONTAINING A 4-BENZYLPIPERIDINE MOTIF, IDENTIFIED VIA A NOVEL SCREENING SYSTEM IN YEAST

Y. Friedmann1, A. Shriki1, E.R. Bennett1, S. Golos2, R. Diskin3, I. Marbach3, E. Bengal2, D. Engelberg1

1Jexys Pharmaceuticals Ltd., Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel, 3Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel

In vivo screening of compounds for potential pharmacological activity is more advantageous than in vitro screening. In vivo screens eliminate the isolation of compounds that cannot cross biological membranes, are cytotoxic, or are not specific to the target. However, animal-based or even cell-based systems are usually expensive, time-consuming, and laborious. Here we describe the identification of inhibitors of the mitogen-activated protein kinase p38alpha via a high throughput screen using yeast cells. p38alpha is hyperactive in inflammatory diseases, and various indications suggest that its inhibition would reverse inflammation. However, there are currently no p38alpha inhibitors in clinical use. Because the human p38alpha imposes severe growth retardation when expressed in yeast, we screened a library of 40,000 randomly selected small molecules for compounds that would restore a normal growth rate. We identified two compounds; both share a structural motif of 4- benzylpiperidine, and both were shown to be efficient and selective p38alpha inhibitors in vitro. They were also active in mammalian cells, as manifested by their ability to reversibly inhibit myoblast differentiation. Thus, the yeast screen identified efficient and specific p38alpha inhibitors that are capable of crossing biological membranes, are not toxic, and function in mammalian cells. The rapid and cost- efficient high-throughput screening used here could be applied for isolation of inhibitors of various targets. A NOVEL BETA-BREAKER PEPTIDE EMERGES AS POTENTIAL AMYLOID-BETA FIBRILS ARREST

A. Frydman-Marom1, M. Recther1, I. Shefler1, Y. Bram1, D.E. Shalev2, E. Gazit1

1Department of Molecular Biology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel, 2Wolfson Centre for Applied Structural Biology, Hebrew University of Jerusalem, Israel

Self-assembly of pathological nanostructures is the hallmark of multiple degenerative syndromes. One of the most devastating neurodegenerative disorders of the 21 century is Alzheimer’s disease (AD). In the past several years a large body of evidences has established a contributory role for amyloid beta peptide in AD. In contrary to the initial hypothesis that mature aggregated forms of amyloid-beta are the most toxic, recent studies indicate that the globular amyloid-beta oligomers of about 56kDa are the major toxic specie. Therefore, an attractive therapeutic strategy for AD will be to block the early stage of misfolding and aggregation of soluble amyloid-beta peptide. Towards this end, we have rationally designed a dipeptide inhibitor, D-Trp- Aib, which contains an aromatic residue and a beta-breaker element. The inhibitor was designed to interact with the peptide through aromatic interactions and to interfere with the assembly into beta-sheet rich fibrillar structures. We demonstrated that the D-Trp-Aib has a highly valuable pharmaceutical profile such as low molecular weight, high stability in aqueous solution, serum stability and trans- membrane transport. It has an efficient oral bioavailability and penetration through the blood-brain barrier and it was proven to be safe both in vitro and in vivo. We showed the potency of the peptide in inhibiting amyloid aggregation, and in preventing its cytotoxicity. NMR analysis shows indications of involvement of D-Trp-Aib with the aromatic core of the amyloid-beta. Moreover, we observed prevention of cognitive decline in AD model transgenic (tg) mice that were treated with D-Trp-Aib. This was in agreement with a significant reduction of amyloid plaques in the transgenic mice's brain. Thus, we suggest that D-Trp-Aib interferes with the initial molecular recognition processes that are involved in the early stages of the amyloid-beta fibrillization, and consequently blocks the transition of the assemblies into the toxic beta -sheet globulomers structure. UNCOVERING NATURE’S ROUTE FOR AMYLOID PREVENTION: MOLECULAR MAPPING OF THE RECOGNITION INTERFACE BETWEEN THE ISLET AMYLOID POLYPEPTIDE AND INSULIN

S. Gilead, E. Gazit

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

The self-assembly of endogenous proteins into amyloid fibrils is the hallmark of a group of diverse human disorders including Alzheimer's disease, Parkinson's disease, and type II diabetes. Many inhibition strategies have been developed in the attempt to prevent this key process. A leading approach is the search for natural entities that stabilize the native protein conformation and prevent amyloid formation in healthy individuals. Such a stabilizing agent might be insulin, in the case of the highly amyloidogenic islet amyloid polypeptide (IAPP), which forms amyloid fibrils in the pancreas of type II diabetes patients. Insulin was shown to be an exceptionally potent inhibitor of IAPP fibrilization, and is thought to form a complex with IAPP within the pancreatic beta-cells. Here, we investigated the molecular mechanism underlying the IAPP-insulin interaction. First, we compared the inhibition abilities of the A and B chains of insulin. Using Thioflavin T fluorescence and circular dichroism measurements, we found that the B chain, but not the A chain, is an excellent inhibitor of IAPP amyloid formation. Next, we mapped the entire B chain using consecutive overlapping peptide arrays and located a region that inhibits IAPP amyloid formation. We propose that this domain mediates recognition between IAPP and insulin. Moreover, by using a reciprocal assay, we located the interaction domain within IAPP as well. Interestingly, we found it to correspond to the IAPP self-recognition site. The molecular mapping of this interaction is of major importance in elucidating the mechanism underlying amyloid formation in type II diabetes. Moreover, it may be utilized for the design of peptidomimetic inhibitors of amyloid formation. INHIBITING HIV-1 INTEGRASE BY SHIFTING ITS OLIGOMERIZATION EQUILIBRIUM

Z. Hayouka1,2, A. Levin2, A. Loyter2, A. Friedler1

1Department of Organic Chemistry, Hebrew University of Jerusalem, Israel, 2Department of Biological Chemistry, Hebrew University of Jerusalem, Israel

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome, and requires binding of the cellular protein LEDGF/p75 for its activity. We have recently described a new approach for inhibiting proteins by “shiftides”: ligands that specifically bind to an inactive oligomeric state of a disease-related protein and modulate its activity by shifting the oligomerization equilibrium of the protein towards it. We demonstrate the feasibility of our approach for the inhibition of the HIV-1 integrase (IN) protein using peptides derived from its cellular binding protein LEDGF/p75 in a non competitive mechanism. We showed that the peptides inhibit the DNA-binding of IN by shifting the IN oligomerization equilibrium from the active dimer towards the inactive tetramer, which is unable to catalyze the first integration step of 3’-end processing. Our results show that the LEDGF/p75-derived peptides inhibit the enzymatic activity of IN in vitro and consequently block HIV-1 replication in cells due to the lack of integration. The LEDGF - derived peptides either inhibit or activate IN catalytic activity in vitro, depending on their molar ratio compared to IN and their order of addition compared to the DNA. Those findings support the shiftides mechanism .These peptides are promising anti-HIV lead compounds that modulate oligomerization of IN via a novel mechanism. Hayouka Z, et al. Proc Natl Acad Sci U S A. 104(20): 8316-21, 2007 ENDOMORPHIN-1 AND ENDOMORPHIN-2 AS A PROMISING ENDOGENOUS AMYLOID-BETA INHIBITORS

A. Lampel, A. Marom-Frydman, E. Gazit

Department of Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

It is widely believed that the assembly of amyloid fibrils is a fundamental factor in the generation of various diseases, among them is Alzheimer's disease (AD). This neurodegenerative disease is becoming a great concern over the years mainly because of the increasing in life expectancy. The early study of AD as amyloid disease focused on the mature fibrils of amyloid beta peptide as the pathological agents. However, contemporary studies provide strong evidence that the soluble oligomers of amyloid-beta are the main toxic factor which causes apoptotic death of neural cells. According to this novel study, the therapeutic target should be the inhibition of the soluble oligomers. Previous work in the lab has demonstrated the importance of small peptide containing aromatic amino acid and beta-breaking element as a potential inhibitor for the amyloid fibril formation. The endogenous tetrapeptides endomorphin-1 (Tyr-Pro-Trp-Phe) and endomorphin-2 (Tyr-Pro-Phe-Phe) appear to be appropriate potential inhibitors agents. Endomorphins are found in the central and peripheral nervous system and have an important role in pain modulation. Both endomorphins possess Pro that has the beta-breaking element, and aromatic amino acids that can interact with the aromatic core of the amyloid-beta peptide. Based on the properties of the endomorphins structural units and on the important fact that they are endogenous tetrapeptides that found in the CNS, our hypothesis is that endomorphin-1 and endomorphin-2 can act as amyloid beta oligomers inhibitors and fibrils formation blockers. In this study we will reveal the endogenous inhibition potential of the endomorphins and its derivative peptide using spectroscopic, microscopic and cells methods. THE USE OF GENE SILENCING RNAI TECHNOLOGY FOR ANTI APOPTOTIC GENES AS A POSSIBLE TREATMENT FOR PSORIASIS

G. Lerman, D. Avni, Y. Sidi

Laboratory of Molecular Cell Biology, Department of Medicine C, Sheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel

Gene Silencing by siRNA is beginning to be applied as a therapy of human diseases. Skin disorders are an attractive target for the application of Gene Silencing RNAi Technology due to the ability to apply effective agents by local, selective, application. My research focuses on non-malignant proliferative disease; psoriasis. Psoriasis is a very common chronic inflammatory skin disorder affecting 2-3% of the world’s population. The characteristic of this multigenic disorder are abnormally increased cell proliferation, inflammation and formation of chronic erythematous and scaly lesions that are usually distributed symmetrically on the skin. Psoriasis is presently without a permanent cure .It is a complex disease at the cellular, genomic and genetic level. Despite its accessibility, frequency and persistence, many puzzling questions about psoriasis remain unanswered. In psoriasis, as in most malignant tumors, many anti apoptotic genes are over expressed. Apoptosis is utilized as an intrinsic mechanism that maintains homeostasis in normal human epidermis. Apoptosis in the skin is a common and essential process. Keratinocytes derived from psoriatic plaques are resistant to apoptosis induction relative to keratinocytes derived from normal skin. Moreover, the expression of proteins involved in apoptosis differs between normal and psoriatic skin. The goal of the present study is to further understand the pathology of psoriasis and to elucidate the role of apoptosis in the progression of the disease. Our preliminary results indicate that we are able to suppress the expression of anti apoptotic genes in primary keratinocytes cultures. We are investigating the effect of these changes on cell cycle progression and sensitivity to apoptosis of treated cells. This may enable us to design specific siRNA against anti apoptotic genes as active novel medications against psoriasis. Acknowledgments: Chaim Sheba research authority. EXPLORING THE INFLUENCE OF CHEMICAL CHAPERONES ON LIVING ORGANISM USING FLIES MUTATED IN SERPIN NECROTIC GENE

M. Levy, E. Gazit, D. Segal

Department of Molecular Biology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

Chemical chaperones are low-molecular-weight compounds that can stabilize proteins in their native form and to restore activity of mutated misfolded proteins. A large number of human maladies are now known as misfolding-diseases, such as cystic fibrosis. Chemical chaperones offer a promising strategy for treating such diseases. The majority of studies on chemical chaperones are performed using in-vitro systems. We are currently developing a unique in-vivo system to evaluate the efficacy of diverse chemical chaperones, using Drosophila as a model system. Proteins from the serpin (serine proteinase inhibitor) superfamily are very sensitive to mutations since their native form is not the most stable. Common mutations that play a role in diverse human serpinopathies diseases facilitate highly stable serpin polymers formation. Drosophila necrotic mutations are homologues to diseases-associated variants of human serpins. The mutated flies are temperature-sensitive and have a visible phenotype. It is possible to monitor the polymers formed by the mutants by electrophoresis. Using flies is very attractive due to their short life-span and the ability of exposing them to chemical chaperones by simply feeding them. These characteristics together make the Drosophila necrotic mutations an ideal system model to explore the influence of chemical chaperones in a whole living organism. THE INTESTINAL AND BBB PERMEABILITY OF THE ORALLY ACTIVE MC4 AGONIST BL-3020-1 IS SEQUENCE AND CONFORMATIONAL DEPENDENT

Y. Linde1, S. Hess2, O. Ovadia2, T. Lapidot3, I. Winkler3, A. Faier3, D. Yarden3, E. Safrai4, D.E. Shalev4, C. Haskell-Luevano5, A. Hoffman2, C. Gilon1

1Department of Organic Chemistry, Hebrew University of Jerusalem, Israel, 2Department of Pharmaceutics, Hebrew University of Jerusalem, Israel, 3Bioline Innovations, Jerusalem, Israel, 4Wolfson Center of Applied structural Biology, Hebrew University of Jerusalem, Israel, 5Department of Medicinal Chemistry, University of Florida, Gainesville, FL, USA

BL-3020-1 is a backbone cyclic peptide selected from the BL-3020 library with conformational diversity. The library was based on the linear minimal sequence of MSH that activates the MC4R (Phe6-D-Phe-Arg-Trp-Gly10). All the members of the library had the same sequence, but they differ in ring size and ring chemistry. BL- 3020-1 is an MC4R selective agonist. It is metabolically stable to intestinal enzymes. It is highly intestinally permeable by the passive transcellular mechanism. Bioanalysis reveals the BL-3020-1 is also BBB permeable after oral administration. Given orally to mice (0.5 mg/Kg daily administration 3 weeks) it inhibits weight gain (40%). Another member of the backbone cyclic library BL-3020-12, with a ring size of 23 atoms compared to BL-3020-1 that have 20 atoms, show marginal intestinal permeability. Ala scan of BL-3020-1 revealed that all the side chains are essential for intestinal permeability and bioactivity. The members of the Ala scan library had very low intestinal permeability and were devoid of biological activity. Conformational analysis of BL-3020-1 by NMR spectroscopy indicated that addition of DPC caused conformational change that resulted in a unique conformation (called the intestinally permeable conformation) stabilized by internal hydrogen bonding. BL-3020-12 did not show the intestinally permeable conformation upon the addition of DPC. We conclude that the intestinal permeability of BL-3020-1 is sequence and conformational dependent. THE QUINAZOLINE DERIVATIVE 9a AS A NEUROPROTECTIVE AGENT

I. Maniv1, L. Veenman1, S. Leschiner1, I. Spanier1, I. Marek2, A. Shterenberg2, E. Hadad2, M. Gavish1

1Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel, 2Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel

Secondary brain damage often occurs after traumatic brain injury (TBI). TBI can result from explosions, including terrorist attacks, traffic and household accidents, and other forms of violence. Neuronal death due to secondary brain damage and neurodegeneration have in common an excitotoxic process leading from overexcitation of glutamate receptors to mitochondrial damage. The mitochondrial damage can lead to neuronal death, including apoptosis. Our studies suggest that the mitochondrial Translocator Protein (TSPO) plays an important role in this process. Recently, we have developed compounds that bind with high affinity to the TSPO and apparently block its apoptotic function. These novel compounds reduced basal apoptotic levels in neuronal cells. Some of them also reduced apoptosis induced by glutamate in the SH-SY5Y cell line by more than 50%. In our in vivo experiments we found that one of our ligands (9a) (15mg/kg) reduces the adverse effects of kainic acid (9-10mg/kg) in rats. For example, seizures induced by kainic acid are indicative for neurodegeneration in the hippocampus. Severe seizure activity was reduced by 9a by more than 67%. We envision that secondary brain injury due to TBI may be prevented by soldiers and paramedics carrying with them one of the anti-apoptotic drugs we have developed and use it on site. This may reduce the incidence of disabilities presently occurring in the aftermath of TBI. In addition, such a drug might also find application in the treatment of neurodegenerative diseases. CHARACTERIZING THE CARBOHYDRATE STRUCTURE OF NKp46 RECEPTOR FOR DESIGNING GLYCOPOLYMERS WITH INHIBITORY ACTIVITIES TOWARDS INFLUENZA VIRUS INFECTION

M. Mendelson1,2, A. Porgador1, A. David2

1Department of Microbiology and Immunology, Ben Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Pharmacology, Ben Gurion University of the Negev, Beer-Sheva, Israel

Influenza virus H1N1 (A/PR/8/34) infects host cells through the binding of viral hemagglutinins (HAs) to sialic acid (Sia) residues on the host cells surface. Sialic acid terminates the oligosaccharide chains, found on sialoglycoproteins and sialoglycolipids, and plays a key role in the carbohydrate-protein recognition events. Natural killer (NK) cells serve as a crucial first line of defense against virus-infected cells, without the need of prior antigen stimulation. The NKp46 and NKp44 NCRs (natural cytotoxicity receptors), but not NKp30, interact with viral HAs and activate the NK cell, which causes lysis of the target cell. We previously have shown that the interaction of NKp46 with viral HAs depends on a sialyl group on T225 amino acid of the NKp46 receptor. In this study we have characterized the type of the Sia linkage (alpha 2-3 or alpha 2-6) on NKp46 receptor which play a central role in the recognition of H1N1 (A/PR/8/34). We found that: 1) Removal of 2,3- sialic acid linkage reduces the binding of NKp44 and NKp46 to A/PR/8/34, while addition of 2,3- sialic acid linkage increases the binding of the control protein asialofetuin to A/PR/8/34. 2) Recognition between glycopolymers based polyacrylamide (PAA) containing 2-3 sialylation by A/PR/8/34 was observed in linear and branched conformations, while recognition of 2-6 sialylation by A/PR/8/34 was found in alpha chain and in linear conformations only. By blocking the initial interaction of the viral HAs with these host cell receptors, viral infection could be prevented. This interaction may be inhibited by the use of an extracellular therapeutic agent that resembles the surface-binding component of the host cells. Currently, N-(2-hydroxypropyl) methacrylamide (HPMA) based glycopolymer carrying multivalent carbohydrate residues that will target the viral HA are being synthesized and characterized. NMR STRUCTURAL STUDIES OF THE HUMAN INTERFERON alpha2 COMPLEX WITH THE HUMAN TYPE I INTERFERON RECEPTOR

I. Nudelman1, S.R. Akabayov2, J.H. Chill3, R. Levy1, N. Kessler1, J. Anglister1

1Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel, 2Department of Biochemistry, MS 009, Brandeis University, Waltham, MA, USA, 3Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel

Type I Interferons (IFNs) are a family of homologous helical cytokines initiating strong antiviral and antiproliferative activity. Since IFNs are at the forefront of defense against viral infection and promote a variety of biological effects, they are essential for the survival of higher vertebrates. Not surprisingly, IFNs are the human proteins most widely used as therapeutics for the treatment of several kinds of cancer and viral diseases. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. Our group has been conducting studies on the structure and dynamics of the 24kDa extra cellular domain of IFNAR2 and the 44 kDa complex of IFN-alpha2 with IFNAR2 by multidimensional NMR techniques. A model of the IFN-alpha2/IFNAR2 complex based on NMR and Double Mutant Cycle (DMC) data has been derived recently. The structures of the larger IFNAR1 subunit and of the binary IFN-alpha2/IFNAR2 and ternary IFNAR1/IFN-alpha2/IFNAR2 complexes have not been solved yet. Structural information will give valuable insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN-like molecules. Although NMR has contributed significantly to the study of this complex, at 44 kDa structure determination by NMR of IFNAR2/IFN-alpha2 presents considerable challenges. The goal of our project is to determine the three-dimensional structure of the IFNAR2/IFN-alpha2 complex and to study the interactions in the multimeric signaling complex IFNAR2/IFN-alpha2/IFNAR1. A3 ADENOSINE RECEPTOR IS OVER-EXPRESSED IN AUTO-IMMUNE INFLAMMATORY DISEASES: A NOVEL TARGET TO COMBAT INFLAMMATION

A. Ochaion1, S. Cohen1, F. Barer1, R. Patoka1, S. Bar Yehuda1, L. Del Valle2, G. Perez Liz2, J. Ophir3, R. Musberg-Galili4, T. Reitblat4, H. Amital5, P. Fishman1

1Can-Fite BioPharma Ltd., Kiryat-Matalon, Petah-Tikva, Israel, 2Department of Neuroscience, Neuropathology Core and Center for NeuroVirology, Temple University School of Medicine, Philadelphia, PA, USA, 3Dermatology Unit, Edith Wolfson Medical Center, Holon, Israel, 4Department of Neurology, Rabin Medical Center, Petah-Tikva, Israel, 5Internal Department D, Meir Medical Center, Kfar Saba, Israel

The A3 adenosine receptor (A3AR), a Gi protein associated cell surface receptor is highly expressed in the inflammatory tissues. Interestingly, A3AR was found to be over expressed in peripheral blood mononuclear cells (PBMCs) derived from adjuvant induced arthritis (AIA) rats, reflecting receptor status in the remote tumor or inflammatory cells. Activation of the A3AR with synthetic highly selective agonists, such as CF101 or CF102 (generically known as IB-MECA and Cl-IB-MECA, respectively) induces anti-inflammatory effects in mitogen activated PBMCs and in experimental animal models of collagen- and adjuvant-induced arthritis. The molecular mechanism involves modulation of the NF-kappaB signal transduction pathway, resulting in decreased levels of the pro-inflammatory cytokine TNF-alpha and apoptosis of the inflammatory cells. In this study, a survey of the A3AR expression levels in inflammatory cells and PBMCs from Rheumatoid Arthritis (RA), Psoriasis and Multiple Sclerosis (MS) patients was conducted. A3AR over-expression was found in Synovial tissue from RA patients, in reactive astrocytes within plaques of demyelination in brain samples from MS patients, and in skin lesions from Psoriasis patients in comparison to relevant adjacent tissues. In PBMCs derived from the above diseases, a statistically significant up-regulation of A3AR expression level was recorded in comparison to that of PBMCs from healthy subjects. A3AR up- regulation in the PBMCs was found to be attributed to an increased expression level of TNF-alpha, PKB/Akt, IKK and NF-kappaB, the latter being a transcription factor of A3AR. Taken together, A3AR was found to be highly expressed in the inflamed tissues and in PBMC of patients with various auto-immune inflammatory diseases. Receptor over-expression is linked to up-regulation of the transcription factor NF- kappaB. These findings and our previous data suggest A3AR as a potential target to combat inflammation. USING CHIMERIC PROTEIN TO TARGET AND TREAT BACTERIAL INFECTIONS

O. Sabag, H. Lorberboum-Galski

Department of Cellular Biochemistry and Human Genetics, Hebrew University of Jerusalem, Israel

In recent years there has been a marked increase in the number of the bacterial strains resistant to any antibiotic treatments known today, due to the frequent use of antibiotic agents. For this reason, looking for new strategies for developing antibiotics has become of a major interest. We are developing a new approach for designing antibiotic reagents using chimeric proteins, fusing two moieties: one containing an immune evoking domain and the other containing a human antibacterial peptide. As the immune triggering domain we used flagellin, a bacterial protein that is highly conserved and can recognized by the innate immune system upon binding to its receptor the Toll –Like-Receptor (TLR5). As the active domain we used a human antibacterial peptide. Antibacterial peptides are an efficient part of the first line of host defense because of their ability to recognize and neutralize invading microorganisms quickly and specifically. By fusing these two moieties we anticipate getting a protein that can directly kill bacteria by the antibacterial peptide and at the same time and locally trigger the immune system to kill remaining microorganisms that escape from the peptides' action. To test our hypothesis we have choosen the gene filc coding for salmonella typhimurium flagellin and the gene coding for beta- defensin-3 as the antibacterial peptide. Three proteins were designed flagellin alone, beta-defensin-3 alone, and flagellin-beta-defensin-3. All the peptide/proteins were expressed and purified. We demonstrated that the native peptide and antibacterial peptide moiety in the chimera can kill different strains of bacteria in a dose and time depending manner, and that the chimera can be safely given to mice without having toxic effects. NOVEL QUINONE BASED MOLECULES AS INHIBITORS OF Abeta OLIGOMER FORMATION IN ALZHEIMER'S DISEASE

R. Scherzer, A. Frydman-Marom, E. Gazit

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Alzheimer's disease (AD), a progressive neurodegenerative disease for which there is no cure or effective treatment, is the leading cause of dementia in aged humans. In the past several years a large body of evidences has established a pathological role for beta-amyloid polypeptide (Abeta) in AD. Recent studies had indicated that in contrary to the initial hypothesis that mature aggregated forms of Abeta are the most toxic, recent studies indicate that the globular Abeta oligomers of about 50kDa (globulomers) may be the major toxic specie. Therefore, an attractive therapeutic strategy for AD will be to block the early stage of misfolding and aggregation of soluble Abeta polypeptide. Our recent studies show that a rationally designed inhibitor, EG30, which contains an aromatic tryptophan residue and a beta-breaker element, interacts with the peptide through aromatic interactions and interferes with the assembly into b-sheet rich fibrillar structures. A variety of quinones are known to act as inhibitors of various metabolic paths in the cell. Quinones are known to serve as antibacterial, anti-viral, and also anti-cancer agents. Furthermore, it has been established that danthron (1,8-dihydroxyanthraquinone) reduces neurotoxicity related to beta-amyloid proteins. Based on the resemblance of EG30 and the aromatic naphthoquinones, and the found effect of quinones on beta-amyloids, we have analyzed the inhibition potential of several synthetic naphthoquionones. SY-83 and SY-81, tryptophan substituted naphthoquinones, and a naphthoquinone attached to a beta-breaker element, Aib, showed the most significant inhibition of the globular Abeta oligomers. Notably, inhibition was still observed at a concentration as low as 26.6 micro M, indicating the profound potential of these molecules. INHIBITION OF ALLERGIC ASTHMA IN AN ANIMAL MODEL BY OXIDIZED CELLULOSE MICROPARTICLES

N. Shani1, Z. Shani2, O. Shoseyov1, D. Shoseyov3

1The Robert Smith Institute of Plant Sciences and Genetics, Faculty of Agriculture, The Hebrew University of Jerusalem, Israel, 2Private Residence, Mazkeret Batia, Israel, 3Department of Pediatrics, Hadassah University Hospital, Mount Scopus, Jerusalem, Israel

Grass pollen is one of the major causes of type I allergy that is induced by airborne allergens affecting as many as 20% of the general population and resulting in a range of disease including allergic rhinitis and seasonal asthma. An allergic reaction is induced, in atopic individuals, by the interaction between IgE antibodies and an inhaled allergen. The prevalence of type I allergy and asthma has increased dramatically in recent years and is becoming a major health problem world wide. Both group-I grass allergens (beta-expansin), the most potent grass allergens, and groups II/III grass allergens (expansin like) contain a cellulose binding module (CBM) that was shown by us to be the minimal structure required for plant cell-wall expansion and disruption of cellulose fiber-to fiber interactions. Thus, the CBM shared by pollen allergen families I/II/III may act as a common antigen, a notion that is supported by findings indicating that the IgE binding epitops are located specifically on the cellulose binding cleft of the CBM. We show here that oxidized cellulose, amorphous and biocompatible cellulose, has high binding capacity for rye grass allergens, specifically to group III (expansin like proteins). This allergen binding capacity of oxidized cellulose is immensely superior to that of crystalline cellulose. We further show that oxidized cellulose is able to block an allergic lung inflammation in a rat model of rye grass allergy. We hypothesize that the expansin allergens bind to oxidized cellulose microparticles via their CBM. That way the cellulose can block the interaction between the IgE and the allergen and may also increase allergens clearance from the airways by the mucociliary transport. This reaction may prevent the allergic reaction. In conclusion: we propose the use of oxidized cellulose microparticles as a broad-spectrum allergen blocker for effective prevention of allergic reactions. THE ISLET AMYLOID POLYPEPTIDE CRYSTALLIZATION

A. Shusterovitch, S. Gilead, E. Gazit

Department of Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Amyloid fibrillation is a hallmark of more than 25 cell degenerative diseases. The most prominent list includes type II diabetes, Alzheimer’s disease, and prion–protein related encephalopathy. There is more and more evidence supporting the presumption that the major factor of the pathological symptoms of the mentioned diseases is the assembly of amyloid fibrils. These amyloid deposits are considered a significant factor in the development of the disease and derived from misfolded islet amyloid polypeptide (IAPP), a 37 residue hormone. The hIAPP concentration inside the storing secretory granules is 100-fold higher than the concentration of hIAPP needed to rapidly accumulate amyloid fibrils in vitro. It was suggested that the stabilizer of hIAPP in the beta-cells is insulin, which is co-localized within the secretory vesicles. Furthermore, hIAPP is normally secreted from these vesicles with insulin. Additional studies showed that insulin and proinsulin inhibits IAPP amyloid fibrillation at physiological molar ratios. For better understanding of the hIAPP structure and the relations with insulin, we aimed to solve the three dimensional structure of hIAPP. Due to the capability of hIAPP to form amyloid fibrils at high concentrations, the method of GST as a fusion protein was applicated. The fusion of hIAPP to GST protein contributes to the stabilization of soluble form of hIAPP and future structure determination by molecular replacement. The results promoted few crystal generations in the plates of GST-hIAPP, thus a trial for crystallization of GST-hIAPP with its inhibitor - insulin was performed. The results were quite unexpected; while the ratio of insulin ascended (1:1, 1:2), the amount of the crystals rose respectively. Analyzing the X-Ray diffraction data revealed that the crystals contained insulin crystallized in hexameric form with Zinc and potentially with another metal. EFFICIENT CLEARANCE OF ASPERGILLUS FUMIGATUS BY ULTRASHORT ANTI-MICROBIAL LIPOPEPTIDES IN MURINE LUNGS

A. Vallon-Eberhard*1, A. Makovitzki*2, S. Jung1, Y. Shai2

1Department of Immunology, Weizmann Institute of Science, Rehovot, Israel, 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis (IA), a severe life-threatening infection. Both mucociliary clearance and phagocytic defense prevent disease development in immuno-competent individuals despite constant inhalation of A. fumigatus conidia. The absence of these protective mechanisms in immuno-compromised individuals allows conidia to germinate and invade the lung tissue. The inefficiency of antifungal agents and resulting high IA- associated mortality rate remain major clinical concerns. Recently, we have reported on a new family of ultrashort cationic lipopeptides that in vitro specifically killed fungi structures. Studies on their plausible mode of action support a membranolytic or detergent-like mechanisms (Makovitzki et al. 2006, PNAS 103(43):16001). Here we screened several lipopeptides in vitro for their anti-A. fumigatus activity based on their minimal inhibitory concentrations and their hemolytic activity. To investigate the clearance properties of the selected peptides in vivo, we challenged immuno- suppressed C57BL/6 wt mice intra-nasally with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was further tested in a survival assay and was found to significantly prolong life and reduce mortality of the treated animals, whereas no mice survived after the current standard treatment with amphotericin B. Moreover, histology of peptide-treated lungs did not show any toxicological effect of this peptide. Our results highlight the potential of this family of lipopeptides in the treatment of pulmonary aspergillosis.

*equal contribution 4-METHYLTHIOBUTYLISOTHIOCYANATE (MTBI) AS A POTENTIAL ANTI-PSORIATIC AGENT

H. Yehuda1, S. Khatib2,3, I. Sussan4, R. Musa2, J. Vaya2,3, S. Tamir1,3

1Laboratory of Human Health and Nutrition Sciences, MIGAL - Galilee Technology Center, Kiryat-Shmona, Israel, 2Laboratory of Natural Medicinal Compounds, MIGAL - Galilee Technology Center, Kiryat-Shmona, Israel, 3Tel-Hai Academic College, Upper Galilee, Israel, 4Pharmacy, Kfar Vradim, Israel

The isothiocyanate, 4-methylthiobutylisothiocyanate (MTBI), isolated from the seeds of the edible cruciferous vegetable, rocket (Eruca sativa), was evaluated as a potential bio-agent for psoriasis, an autoimmune skin disease, characterized by hyperproliferation and aberrant keratinocyte cell differentiation, inflammation and angiogenesis. Isothiocyanates form a group of organosulfur compounds, which have been demonstrated to have anti-carcinogenic, anti-inflammatory and anti-proliferative activities. Since such biological activities could possibly be advantageous in psoriatic treatment, MTBI was tested on the proliferation of cells related to psoriasis, represented by HaCaT keratinocytes, Jurkat lymphocytes and THP-1 monocytes, as well as for its potential effect on inflammation. A crude extract of MTBI (which contained MTBI and sulforaphane, the oxidized form of MTBI) was isolated from rocket seeds and further purified by flash chromatography to obtain pure MTBI. The crude MTBI extract inhibited cell proliferation of monocytes more than that of keratinocytes. Results indicated that the sulforaphane in the extract was responsible for the differential inhibition. In addition, the crude MTBI extract caused cell cycle arrest of the HaCaT keratinocytes in the S stage and of the immune cells in the S and G2 stages. The purified MTBI inhibited cell growth of the LPS-activated monocytes (which are found in psoriatic tissue) more than that of non-activated monocytes, revealing its potentially specific effect on inflammation, characteristic of psoriasis. Moreover, MTBI inhibited the activation of THP-1 monocytes, as revealed by the inhibition of the proinflammatory cytokine mRNA expression of IL-1beta, IL-12p40 and TNF-alpha, as well as by the prevention of IL-12p40 and TNF-alpha up- regulation in the LPS-treated THP-1 monocytes, demonstrating that MTBI, may be a bio-agent capable of taking part in the prevention, and/or possibly the alleviation of psoriasis. However, further efforts must be made to understand its mechanisms of action. SANDFLY VIRUSES ARE STILL ENDEMIC IN ISRAEL

H. Bin1, L. Shulman1, U. Shalom2, I. Groto3,10, N. Davidovitch3,10, R. Balicer3,10, T. Meir4, L. Weiss1, K. Shilon1, M. Niedrig5, L. Nicoletti6, T. Kato7

1Central Virology Laboratory, Chaim Sheba Medical Center, Tel Hashomer, Israel, 2Ministry of Environment Protection, Pest Control Unit, Jerusalem, Israel, 3Israel Defense Forces, Israel, 4Nature and Gardens Protection Agency, Israel, 5Robert Koch Institute, Berlin, Germany, 6Instituto Superiore di Sanita, Rome, Italy, 7Kibbutz Cabri, Israel, 8Aristotelian University of Thessaloniki, Greece, 9Chaim Sheba Medical Center, Tel-Hashomer, Israel, 10Ben Gurion University, Beer-Sheva, Israel

Sandfly viruses (SFV) belong to Phleboviruses, and are transmitted to humans by sandflies (SF). The virus is endemic in Mediterranean countries, with recorded outbreaks in Israel half a century ago. Sicily, Naples, and Cyprus viruses are transmitted by Phlebotomus papatasi, also endemic in Israel, causing a Flu-like syndrome. Recently, meningitis/encephalitis epidemics occurred in Italy and Portugal caused by Toscana virus. In order to evaluate whether SFV is still active in Israel, we aimed to find out the seroprevalence to SFV in humans, and their location. IgG tests against SFV were performed with sera of patients negative to other arboviruses, residing in regions of SF habitats (n=84). 8% were positive, and 33% were borderlines, with a total of 35 people exposed to SFV. Tveria, Eilot region, Michmoret and Jerusalem were mostly implicated as places of endemicity. In parallel, SF were collected from different habitats. Isolation attempts were performed on Vero cells. Universal Primers for SFV so far haven't been successful suggesting different variation in local strains. We also conducted a retrospective investigation of a febrile syndrome outbreak among soldiers camping near Gaza strip (n=23). IFA IgM and IgG were conducted with 4 different antigens. Half were found to be recently infected. Cyprus and Sicily viruses were the main agents, but Toscana and Naples were also involved. 82% were exposed to at least one genotype. Additional places of activity were sought, with an IgG serosurvey (n=48) in Kibutz Cabri where SF were found. 39% were exposed to at least 1 out of 4 viruses. We'll present an imported case of Toscana. The most prevailing virus was Cyprus then Sicily. In conclusion: SFV are highly endemic in Israel, with pathogenicity still unrevealed, with local species probably somewhat different from strains present in neighboring countries, and the emergence of Toscana virus to be considered. AS101 ANTIVIRAL EFFECT ON WEST NILE VIRUS IN VITRO AND IN VIVO

V. Indenbaum1, H. Bin2, D. Makarovsky1, M. Albeck3, B. Sredni1, E. Mendelson2

1Safdie Institute for AIDS and Immunology Research, Faculty of Life Sciences, Bar- Ilan University, Ramat Gan, Israel, 2Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Tel Hashomer, Israel, 3Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel

West Nile virus is a flavivirus which may cause outbreaks and serious CNS infections which may lead to death. One of its reported cellular receptors is integrin alpha v beta 3. We have studied the antiviral effect of The immunomodulator AS101 [ammonium trichloro (dioxyethylene 0-0')tellurate] against WNV in vitro and in vivo, and its mechanism. Cell viability assays showed that treatment with AS101 before WNV infection conferred up to 90% cell survival, while virus titer determined by a plaque assay fell by 83%. Addition of AS101 to cultures immediately following WNV inoculation at high MOI (5) reduced virus yield by nearly two logs, while the intracellular expression of the viral envelope protein E decreased by up to 60%. Inhibition by AS101 was dose-dependent and effective mostly when AS101 was added either before or during infection. To elucidate the inhibitory mechanism, we examined the interaction of AS101 with the WNV receptor integrin alpha v beta 3. We found that AS101 blocked the attachment of Vero cells to human integrin alpha v beta 3 specific antibody as well as the attachment of integrin alpha v beta 3 to WNV coated ELISA plates in a dose dependent manner. Injection of AS101 to Balb/c mice one day before WNV infection increased animal’s survival by 50%. Our results show that AS101 has an antiviral effect against WNV in vivo and in vitro, and suggest a mechanism of prevention of WNV cell entry by interfering with its binding to integrin alpha v beta 3, a novel AS101 cellular target. SSR BASED TYPING AND EPIDEMIOLOGICAL STUDY OF ENVIRONMENTAL VIBRIO VULNIFICUS

Y.Y. Broza1, Y. Danin-Poleg1, L. Lerner2, Y. Kashi1

1Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel, 2Government Central Laboratories, Ministry of Health, Jerusalem, Israel

Vibrio vulnificus is a major etiological agent of severe human infection acquired through skin wounds or consumption of contaminated seafood. Its natural habitat is aquatic environment, mainly estuarine ecosystems. Most natural isolates are pathogenic and are divided into three different biotypes. Biotype 3 was found until now only in Israel and is connected to fish grown in artificial ponds. Simple sequence repeats (SSR), also termed VNTR, have been widely used for genotyping of bacteria in the last several years. In this study, twelve SSR loci chosen in silico were initially analyzed in a set of 32, well characterized, isolates representing the three biotypes and both clinical and environmental source. High polymorphism was found, with up to 16 alleles and high corresponding genetic diversity. Strains of biotype 3 showed conserved values and were distinctly separated from strains of biotype 1 & 2. Subsequently, 330 environmental V. vulnificus isolates from fish specimens were successfully collected from artificial fish ponds and fish stores in northern Israel during 2004-2006. The first 135 isolates together with clinical strains from parallel years were analyzed using the SSR markers. New alleles were found in most of the tested SSR loci, thus contributing to the separation ability. Results revealed that 75 of the environmental V. vulnificus isolates clustered together with strains of the highly clonal biotype 3. Thus, we were able to isolate environmental biotype 3 in relatively high numbers compared to only few isolates found until today. Furthermore, similar SSR fingerprints patterns were found for clinical an environmental isolates from nearby regional areas and related dates. Consequently, this SSR-based method is a promising tool for typing and epidemiology study of V. vulnificus. OPTICAL FIBER IMMUNOSENSOR FOR THE DETECTION OF IgG ANTIBODY TO RIFT VALLEY FEVER VIRUS

A. Sobarzo1, J.T. Paweska2, S. Herrmann3, T. Amir1, R.S. Marks3,4, L. Lobel1

1Department of Virology, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Special Pathogens Unit, National Institute for Communicable Diseases, Johannesburg, South Africa, 3Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 4The National Institute of Biotechnology in the Negev, Beer-Sheva, Israel

Introduction: Rift Valley fever virus (RVFV) causes severe outbreaks in livestock and humans and is considered as a potential biothreat agent. Recent and first outbreaks of RVF outside Africa and effects of global warming have the implication that the virus might spread further into non-endemic RVF areas since it utilizes a wide range of mosquito vectors. Aims/Rationale: There is an increased international demand for rapid and accurate assays for confirmation and monitoring of natural outbreaks and/or bioterrorism events involving RVFV. Our aim was to develop and evaluate an optical fiber immunosensor (OFIS) for the detection of anti-RVFV IgG antibody in human sera. Methods: The OFIS procedure was based on a sandwich ELISA. Mouse anti- RVFV antibody was immobilised on a silica fiber-glass surface activated by an argon silanization which allows for covalent binding of bio-compounds. Validation data sets derived from testing field-collected sera from Africa (n = 261) were categorized according to the results of a virus neutralization test (VNT). OFIS raw data were normalized by conversion to test sample/negative control ratios (S/N). A cut-off value at 95% accuracy level was optimized using the two-graph receiver operating characteristic analysis. Results: At the cut-off of 2.66 S/N, of 70 VNT positive sera, all tested positive in OFIS (100% sensitivity) and of 191 VNT negative sera, all but two tested negative (specificity 98.95%). The assay was highly reproducible within and between routine runs. Conclusions: The OFIS assay for IgG serodiagnosis of RVF is a low cost, safe, robust and highly accurate system which has the potential to be used in a portable format and thus aid early diagnosis, disease surveillance and bio- defence field monitoring. PREVENTION OF HAIR CELL DEATH IN TRANSCRIPTION FACTORS Pou4f3 AND Gfi1 KNOCKOUT MICE USING ANTI-APOPTOTIC FACTORS

O. Atar, D. Alaluf, K.B. Avraham

Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel

Treatment today for hearing impairment is limited to hearing aids and cochlear implants. A large effort is being made to try different therapeutic approaches for treatment of hearing loss. Some of these include prevention of cell death, manipulation of expressed genes by gene therapy methods, inhibition of negative regulators and stem cell therapy. These therapeutic strategies for hearing impairment are attractive and promising for restoring hearing loss of genetic origin. Research from our laboratory and others have found that the transcription factors Pou4f3 and Gfi1 are both genes associated with hearing impairment. Pou4f3 and Gfi1 knockout mice have no cochlear hair cells, resulting in complete deafness. The hair cells in these mice progressively degenerate via apoptosis. In order to rescue the hair cells in the mutant mice, we produced explant cultures from mouse cochleae at an early embryonic stage. Two different anti-apoptotic agents were used in order to inhibit cell death. We treated the cells for six days in vitro with z-VAD-FMK or retinoic acid, after which time the cultures were immunostained with myosin VI. We found that both anti-apoptotic factors partially rescued hair cells from Pou4f3 and Gfi1 KO mice. Efforts are now underway to find a combination of factors that will completely inhibit hair cell death, maintain structural integrity, decipher the mechanisms leading to this rescue, and transfer this system to an in vivo model. Research supported by the European Commission FP6 Integrated Project EUROHEAR and US-Israel Binational Science Foundation. THE ROLE OF GLP-1 IN PROMOTING Pdx-1 INDUCED LIVER TO PANCREAS TRANSDIFFERENTIATION

V. Aviv1,2, I. Meivar-Levy1, S. Ferber1,2

1Endocrine Institute, Sheba Medical Center, Tel-Hashomer, Israel, 2Sackler School of Medicine, Tel-Aviv University, Ramat Aviv, Israel

Beta-cell replacement is considered to be the most promising approach for the treatment of type 1 diabetes. However, the shortage in tissue availability from cadaveric donors and the need for life long immunosuppression, severely restrict its large scale application. We have suggested a novel source of tissue to replace the beta-cell mass: transdifferentiation of liver cells toward beta cell phenotype and function. We have shown that ectopic expression of the pancreatic transcription factor Pdx-1 in primary culture of human liver cells, induced transdifferentiation in a subpopulation of liver cells into pancreatic-like insulin-producing cells. Here, we have examined whether GLP-1 enhances the extent of the transdifferentiation process induced by Pdx-1 using its agonist Exendin-4. Our data suggested that Exendin-4 indeed augmented the effect of Pdx-1 on pancreatic hormone gene expression, improved insulin production, its processing and its glucose regulated secretion. Furthermore, we found an increase in the expression of factors participating in insulin processing and granules assembly, such as SGNE-1, SCG-2, PC1/3 and PC-2. In addition, an increase was observed in the expression of pancreatic transcription factors such as isl1, NeuroD1 and the endogenous Pdx-1. Thus, Exendin-4 is a potent factor in promoting liver to pancreas transdifferentiation as the cells exhibit a more mature beta cell phenotype. HES1 IS INVOLVED IN ADAPTATION OF ADULT HUMAN BETA CELLS TO PROLIFERATION IN VITRO

Y. Bar, H.A. Russ, S. Knoller, S. Efrat

Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel

In vitro expansion of insulin-producing beta cells from adult human islets could solve the tissue shortage for cell-replacement therapy of diabetes. Propagation in culture of adult human islets from cadaveric donors results in <16 cell doublings and loss of insulin expression. Using cell-lineage tracing we recently demonstrated that the expanded cell population included cells derived from beta cells, which underwent dedifferentiation and were induced to replicate (see accompanying abstract by Russ HA et al.). Understanding the molecular mechanisms involved in beta-cell dedifferentiation and replication in vitro is crucial for optimizing expansion and redifferentiation of these cells. In the developing pancreas, important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split (HES) 1 transcriptional regulator. However, this pathway is not normally active in adult pancreatic islets. Here we report that beta-cell dedifferentiation and entrance into the cell cycle in vitro involve activation of the NOTCH pathway, including a 5-fold upregulation in HES1 transcripts. These changes correlate with downregulation of the cell cycle inhibitor p57 and loss of insulin expression. Inhibition of HES1 expression using small hairpin RNA completely prevented p57 downregulation and significantly reduced beta-cell replication. In addition, HES1 inhibition reduced beta-cell dedifferentiation, as manifested by a higher level of insulin and beta-cell-specific transcription factor transcripts, as well as a higher fraction of cells immunostained for insulin. These findings suggest possible molecular targets for prevention of beta-cell dedifferentiation in culture, and induction of cell redifferentiation following in vitro expansion. THE ROLE OF Pax4 AND MafA IN THE TRANSDIFFERENTIATION PROCESS OF LIVER CELLS TOWARDS PANCREATIC BETA CELLS

D. Berneman1,2, I. Meivar-Levy1, S. Ferber1,2

1Endocrine Institute, Sheba Medical Center, Tel-Hashomer, Israel, 2Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel

Recent advances in pancreatic islet transplantation emphasize the potential of this approach for the long-term control of blood glucose levels in diabetic patients. However, tissue-replacement therapy will became widely available as a treatment for diabetes only when new sources of islets and insulin-producing cells are found. We have suggested transdifferentiation of liver cells toward beta-cell phenotype and function as a novel source of tissue to replace the beta-cell mass. The role of the pancreatic transcription factor, PDX-1 in inducing a transdifferentiation process of liver along the pancreatic lineage has been demonstrated in mice and human. Recently, our lab demonstrated that PDX-1 has a double role in the transdifferentiation process; while promoting pancreatic differentiation, it serves also as a hepatic dedifferentiation factor. Here, we analyze the capacity of additional pancreatic transcription factors; Pax4 and MafA to induce pancreatic transdifferentiation and hepatic dedifferentiation in adult human liver cells. Our results suggest that both Pax4 and MafA may activate the pancreatic lineage in the liver. Ectopic expression of Pax4 or MafA induced expression of the pancreatic hormone genes, insulin processing and secretion in a glucose regulated manner. Furthermore, both Pax4 and MafA induced hepatic dedifferentiation, as indicated by reduced levels of adult hepatic marker ADH1b and enhanced expression of the fetal marker AFP. Taken together, our data suggest that Pax4 and MafA can induce pancreatic transdifferentiation associated with hepatic dedifferentiation in adult human liver cells. In this study we unravel fundamental aspects of the mechanism underlying liver to pancreas transdifferentiation process. The identification of key events in the transdifferentiation process will allow us to increase the therapeutic merit of using liver as a source of pancreatic tissue for treating diabetes. SV40 EMPTY CAPSIDS PROTECT FROM ORGAN FAILURE BY ELICITING CELLULAR SIGNALS AND HOST DEFENCE

V. Butin-Israeli1, D. Uzi1, M. Abd El-Latif1, G. Pizov3, A. Eden4, Y.S. Haviv2, A. Oppenheim1

1Department of Hematology, Hebrew University of Jerusalem, Israel, 2Division of Nephrology, Hadassah Medical Center, Jerusalem, Israel, 3Department of Pathology, Hadassah Medical Center, Jerusalem, Israel, 4Department of Anesthesiology and Critical Care Medicine, Carmel Medical Center, Haifa, Israel

Simian virus 40 (SV40) infects dividing and non-dividing cells. Empty capsids (Virus-Like Particles, or VLPs), composed of the recombinant major capsid protein VP1, can be produced in insect cells. We study signaling events that allow nuclear entry of the viral genome in non-dividing cells, by SV40 and by VLPs. We found that both rapidly activated PARP-1, within one hour. Apoptosis was arrested by upregulation of stress response protein Hsp/c70 and by activation of Akt-1 survival pathway. The key player of this signaling network was PLC. These findings suggested that VLPs might have a protective effect against apoptosis that leads to organ failure, such as acute kidney injury (AKI). AKI is a critical prevalent disorder in humans, caused by nephrotoxic agents, sepsis and ischemia. We found that VLPs target the kidney after systemic administration. Experiments in a mercury-induced AKI mouse model demonstrated that pretreatment with VLPs prior to mercury insult increased survival rate from 12% to >60%. Analyses of kidneys and sera indicated that the VLPs activated Akt-1 survival pathway and upregulated Hsp/c70, leading to attenuation of renal tubular apoptosis, oxidative stress and toxic renal markers. This study demonstrates that SV40 VLPs, devoid of nucleic acids, may potentially be used as a prophylactic agent for AKI. THE ROLE OF THE GROWTH FACTOR- ACTIVIN A ON PDX-1 INDUCED LIVER TO PANCREAS TRANSDIFFERENTIATION PROCESS

Y. de Rooy1,2, K. Molakandov1,2, I. Meivar-Levy1, S. Ferber1,2

1Institute of Endocrinology, Sheba Medical Center, Tel-Hashomer, Israel, 2Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel

Liver was suggested as progenitor tissue for the generation of insulin producing cells for treating type-1 diabetes. Ectopic expression of PDX-1 (Pancreatic and Duodenal homeoboX gene 1) in liver cells induced pancreatic phenotype and function, both in- vivo and in-vitro, leading to insulin production, secretion and to the amelioration of hyperglycemia in STZ (Streptozotocin) induced diabetic mice. The goal of my study is to analyze the effects of the growth factor activin A on the efficiency of PDX-1 induced liver to pancreas transdifferentiation in-vitro and in-vivo, while gaining knowledge on its mechanism of action. Both activin A and PDX-1 are ectopically expressed using recombinant adenoviral vectors, in primary cultured adult human liver cells (in-vitro) or in mice (in-vivo). The effects of activin A on PDX-1 induced transdifferentiation is examined at the molecular and functional levels. The results demonstrate that activin A augments functional insulin production and secretion in liver cells, while improving glucose sensing ability, both in-vitro and in -vivo. Gene expression of pancreatic hormones and transcription factors is enhanced. The combined PDX-1 / activin A treatment leads to an increase in the number of liver cells capable of activating the insulin promoter. The study will allow us to evaluate whether activin A promotes the maturation of transdifferentiated liver cells towards pancreatic beta-cell phenotype and function. Using a growth factor may promote liver to pancreas transdifferentiation process thus; increase the therapeutic merit of using liver as a source of pancreatic tissue for treating diabetes. LIGAND INTERACTION SCAN: ENGINEERING A LIGAND-MODULATED PROTEIN

O. Erster1, M. Eisenstein2, M. Liscovitch1

1Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel, 2Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

Chemical genetic methods that harness the power of small molecule pharmacology for evaluating the cellular functions of proteins were recently developed. Here we describe a novel, general and simple procedure for engineering small molecule ligand- regulated forms of virtually any protein. The ligand interaction scan method involves insertion of a chemical-genetic ‘switch’, comprising of a genetically-encoded peptide module that binds with high affinity to a small molecule ligand, into a given protein in a position(s) selected empirically to confer ligand-dependent modulation of activity. To demonstrate the feasibility of the new method we applied it to the TEM-1 beta- lactamase antibiotics resistance gene, utilizing a tetracysteine (4C) hexapeptide and a cell-permeable biarsenical fluorescein derivative (FlAsH) as ligand binding insert and ligand, respectively. We generated several ligand-sensitive mutants of TEM-1, and screened them for FlAsH-modulated activity. Two mutants were inhibited by FlAsH, whereas a third was stimulated by it. The effect of FlAsH on one of the inhibited mutants and on the stimulated mutant was found to be dose dependent and partially reversible. In-silico modeling of the stimulated mutant suggests that FlAsH may act by stabilizing an open conformation in which the enzyme’s active site is exposed. Our results demonstrate that drug-sensitive alleles of TEM-1 can be generated by ligand interaction scan and suggest that the method may be applied to any protein, given an appropriate activity assay. Ligand-regulated mutants may then be expressed in cells, wherein they can be regulated by ligand administration. Because of its simplicity and generality the ligand interaction scan method may complement other genetic and chemical genetic methods for analysis of protein function and drug target validation. DEVELOPMENT OF THYROID CANCER-SPECIFIC GENE THERAPY VECTOR(S)

M. Grafi-Cohen1, G. Weisinger1, R. Limor1, E. Izkhakov1, Z. Kraiem2, N. Stern1

1Department of Endocrinology, Metabolism and Hypertension, Tel Aviv Sourasky Medical Center and the Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 2Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Background: Thyroid cancer is the most common endocrine malignancy, accounting for the majority of deaths from endocrine cancers. As a step towards the development of gene therapy vectors directed toward human thyroid carcinoma, we set upon defining a minimal thyroid cancer-specific enhancer/promoter. Thyroglobulin (Tg) is a glycoprotein precursor for thyroid hormone biosynthesis; while the Tg promoter has been employed in gene therapy strategies for thyroid cancer, further substantial tissue- specific enhancement with a minimal Tg enhancer has yet to be established. Aims and Methods: To enhance Tg promoter transcriptional activity by deletion analysis of a human Tg upstream enhancer (size 1.4 kbp, localized between -3.6 and -2.2 kbp from the transcription start site) in order to characterize a minimal fragment which will provide maximal, thyroid-specific, transcriptional activity in thyroid cells. The CAT (chloramphenicol acetyltransferase) reporter gene was used to measure Tg enhancer/promoter transcriptional activity in human thyroid carcinoma cell lines (NPA-papillary, MRO-follicular and ARO-anaplastic) as well as primary human thyroid and nonthyroid cells. Results: The full length of the upstream enhancer (1.4 kbp) gave 24.5 and 3.2% CAT converted in follicular and papillary thyroid carcinoma cells, respectively. Deletion analysis of the 1.4 kbp enhancer revealed a Tg enhancer/promoter fragment (624 bp) which is specific to follicular and papillary thyroid carcinoma cells and yields maximal transcriptional activity (49.6 and 28.9% CAT converted, respectively, as compared to <1% CAT converted for anaplastic and primary human thyroid cells). Conclusions: The Tg enhancer/promoter construct we are developing may serve as a template for future thyroid-specific gene therapy. Such a model could be used in exploring target gene manipulations aimed at thyroid cancer therapy. CORRECTION OF GLYCOGEN STORAGE DISEASE 1a IN A MURINE MODEL USING A NON-PRIMATE LENTIVIRAL VECTOR

R. Condiotti1, A. Grinshpun1, S. Peretz2, J. Chou3, E. Galun1

1Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem, Israel, 2Harlan Biotech Israel Ltd., Rehovot, Israel, 3Institute of Child Health and Human Development, NIH, Bethesda, MD, USA

Glycogen storage disease type 1a (GSD-1a) is an autosomal recessive metabolic disorder that is caused by a defective form of glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis. This disorder causes life-threatening hypoglycemia, growth retardation, and severe long-term consequences. GSD-1a is a model candidate for gene therapy since the affected gene is known and the resulting protein is mainly expressed in one tissue, the liver. Transgenes delivered by feline immunodeficiency virus (FIV)-based lentiviral vectors undergo integration into the host cell genome resulting in long-term expression. We have previously utilized FIV-based vectors to efficiently deliver reporter genes to liver cells in vitro and in vivo [Condiotti, et al (2004) BBRC 320:998]. In the present work we constructed a FIV vector carrying the hG6Pase gene and evaluated its efficacy for treatment of GSD1a in a neonatal murine model. Neonates were chosen since (i) their liver has not yet reached its final volume (ii) to increase the effective viral moi and (iii) this model closely mimics the human patients. GSD1a-/- neonates were transduced twice via venous administration at one day and one week of age with FIV viral particles containing the hG6Pase gene driven by a liver-specific promoter (hAAT). Following transduction, significant improvement in blood glucose levels was observed and normal levels were obtained within 30 days. Body weight of treated homozygotes was comparable to healthy siblings at 8 weeks of age. In addition, the treated animals exhibited normal levels of serum cholesterol and triglycerides. The hepatic G6Pase activity has also improved significantly after the virotherapy. Current studies are being performed to elucidate changes in hyperlipidemia, renal damage, and enzyme function. These results demonstrate that FIV vectors have a therapeutic potential for correction of GSD-Ia metabolic imbalances. Thus, successful treatment of these animals may serve as a paradigm for treatment of other disorders. DIFFERENTIATION OF MESENCHYMAL STEM CELLS FROM ADULT HUMAN BONE MARROW INTO PANCRETIC ENDOCRINE CELLS INDUCED BY NEUROGENIN3 AND NEUROD1

Y. Izhar-Prato1, O. Karnieli1, S. Bulvik2, S. Efrat1

1Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel, 2Laniado Medical Center, Netanya, Israel

Beta-cell replacement is a promising approach for treatment of type 1 diabetes, however it is limited by shortage of pancreas donors. The pluripotent mesenchymal stem cells (MSC) in adult bone marrow (BM) offer an attractive source of cells for generation of surrogate beta cells. BM-MSC can be obtained with relative ease from each patient, allowing potential circumvention of allograft rejection. In the developing pancreas, the basic helix-loop-helix (bHLH) transcription factor Neurogenin3 (NGN3) initiates the islet differentiation program in pancreatic progenitor cells. NEUROD1, a closely related bHLH protein and a downstream target of NGN3, is involved in maintaining this differentiation. In addition to its role in development of endocrine islet cells, NEUROD1 is also involved in direct activation of the insulin gene in beta cells. We evaluated the effect of NGN3 or NEUROD1 expression in human BM-MSC. Our results showed that NGN3 activated the islet differentiation program in these cells and induced expression of the islet hormones glucagon, somatostatin, and pancreatic polypeptide, however it did not activate insulin gene transcription in our culture conditions. Likewise, NEUROD1 activated expression of all islet hormones but insulin. In addition, NEUROD1 upregulated its own transcription. The lack of expression of the insulin gene and some of the major beta- cell transcription factors in the manipulated cells may be overcome by transfer of combinations of transcription factor genes, or following transplantation in vivo. The latter may provide the cells with a suitable environment for further differentiation towards the beta-cell phenotype. CELL-REPLACMENT THERAPY FOR DIABETES; THE USE OF HUMAN SKIN CELLS AS A SOURCE OF AUTOLOGOUS INSULIN PRODUCING CELLS

M. Mauda-Havakuk1,2, O. Nakar1,2, I. Meivar-Levy1, A. Liran3, S. Ferber1,2

1Endocrine Institute, Sheba Medical Center, Tel-Hashomer, Israel, 2Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel, 3Department of Plastic Surgery, Sheba Medical Center, Tel-Hashomer, Israel

The use of surrogate beta-cells engineered to mimic pancreatic islet function has been suggested as a mean to overcome the severe shortage in donor tissues available for transplantation in type I diabetes. Our group has suggested a novel source of tissue to replace the beta-cell mass; developmental redirection of liver cells toward beta-cell phenotype and function using pancreatic transcription factors. Here we propose the use of an accessible source of tissue, the skin to replace the lost pancreatic function. This study aims to analyze whether ectodermal originated cells as skin can be converted into cells of endodermal origin. Our in-vitro studies demonstrate that skin cells are capable of producing all three major pancreatic hormones (insulin, glucagons, somatostatin) in response to ectopic expression of the pancreatic transcription factor PDX-1. Insulin and C-peptide secretion from PDX-1 treated human skin cells were regulated by glucose. In parallel, PDX-1 induces the expression of factors involved in glucose sensing, pro-hormone processing and vesicle formation. Moreover, PDX-1 induces the expression of numerous pancreatic transcription factors genes, including the expression of endogenous human PDX-1 gene. Thus, our data suggest that skin cells may serve as a pancreatic progenitor tissue for cell replacement therapy. Pdx1-vp16: A POTENET AGENT FOR LIVER TO PANCREAS TRANSDIFFERENTIATION

K. Molakandov1, D.T. Curiel3, S. Ferber2

1Department of Human Molecular Genetics and Biochemistry, Tel Aviv University, Ramat Aviv, Israel, 2Endocrine Institute, Sheba Medical Center, Tel-Hashomer, Israel, 3Division of Human Gene Therapy, Departments of Medicine, Surgery, and Pathology, Gene Therapy Center, University of Alabama at Birmingham, AL, USA

Background: Inducing developmental redirection of adult liver cells offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue. Pdx1-vp16, an active form of Pdx1 (Pancreatic and duodenal homeobox 1 gene), has been suggested of being more efficient than the wild-type Pdx1 in inducing transdifferentiation of liver to pancreas in several models. The role of Pdx1-vp16 in activating the pancreatic lineage in adult human liver cells has been never analyzed. Here, we compare the efficiency of the Pdx1-vp16 fusion protein to that of Pdx-1 in activating the pancreatic lineage and function in primary cultures of adult human liver cells. Methods: The differential effects of pdx1-vp16 versus wild-type Pdx1 were studied in a primary culture of adult human liver cells. Gene delivery was performed by recombinant adenoviruses. The relative effects of Pdx1-vp16 fusion protein vs. wt-Pdx-1, was analyzed at the levels of insulin promoter gene expression and pancreatic beta-cell function. Results: Pdx1- vp16 fusion protein demonstrated an improved activation of the insulin promoter and increased the number of cells capable of activating the insulin promoter (60% vs. 7% of Pdx1). Ectopic expression of this fusion protein in human liver cells led to the induction of critical pancreatic transcription factors like NeuroD1 (FOI of 500 vs. 30) and Pax4 (FOI of 150 vs. 10) and the endocrine hormones Glucagon (FOI of 105 vs. 102) and Somatostatin (FOI of 30 vs. 1). This novel transdifferentiation agent possesses a higher efficiency in converting primary cultures of human liver cells towards the endocrine phenotype than Pdx1 alone. THE ROLE OF HGF AND Nkx6.1 IN Pdx1 INDUCED LIVER TO PANCREAS TRANSDIFFERNTIATION

I. Rachmut1,2, S. Gefen-Halevi1,2, I. Meivar-Levy1, S. Ferber1

1The Endocrine Institute, Chaim Sheba Medical Center, Tel Hashomer, Israel, 2The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

Cell replacement therapy for diabetic patients will be widely used only when new sources of tissues or insulin producing cells will be found. Our lab demonstrated that liver cells can be used as pancreatic progenitors and that Pdx1 (pancreas and duodenal homeobox gene-1) can activate the pancreatic lineage and function in these cells. Trans-differentiated liver cells acquire many beta-cells functional characteristics including production, processing and secretion of insulin in a glucose regulated manner. Aim: analyze the role of HGF (Hepatocytes Growth Factor) in promoting, the Pdx-1 induced, trans-differentiation process. Using primary culture of human adult liver cells, we demonstrate that HGF increases the number of cells capable of activating the insulin promoter, and augments pancreatic hormones gene expression. However, while increasing the expression of several pancreatic endocrine transcription factors such as NeuroD1 and Ngn3, it decreases the expression of Nkx6.1 and MafA which are involved in mature beta-cells differentiation and function. To potentially restore Nkx6.1 activity, we ectopically expressed Nkx6.1 in Pdx1 and HGF treated human liver cells. This molecular manipulation enhanced the pancreatic differentiation without decreasing the number of trans-differentiated cells. Nkx6.1 increased the expression of pancreatic markers and enhanced the glucose dependent c-peptide secretion. Surprisingly Nkx6.1, which is a beta-cells specific factor, promoted also the differentiation of alpha-like cells. Our data suggests a complementary promoting effect of Nkx6.1 and HGF. HGF increases the number of liver cells predisposed to activate the pancreatic lineage, while Nkx6.1 promotes the pancreatic maturation of these cells. The combined effects of pancreatic transcription factors and growth factors may increase the efficiency of generating insulin producing cells from liver, and bring us one step closer to cell replacement therapy for diabetes. PROLIFERATION OF DEDIFFERENTIATED ADULT HUMAN PANCREATIC BETA CELLS IN VITRO REVEALED BY CELL-LINEAGE TRACING

H.A. Russ1, Y. Bar1, P. Ravassard2, S. Efrat1

1Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel, 2UMR 7091, National Center for Scientific Research, Hopital Pitie Salpetriere, Pierre and Marie Curie University, Paris, France

Expansion of insulin-producing beta cells from adult human cadaver islets could alleviate donor shortage for cell-replacement therapy of diabetes. A major obstacle to development of effective expansion protocols is the rapid loss of beta-cell markers in the cultured cells. Here we report a genetic cell-lineage tracing approach for following the fate of cultured beta cells. Cells dissociated from isolated human islets were infected with 2 lentiviruses, the first expressing Cre recombinase under control of the insulin promoter, and the second a reporter cassette with the structure CMV promoter- loxP-DsRed2-loxP-eGFP. Beta cells were efficiently labeled by the dual virus system. Label-positive, insulin-negative cells derived from beta cells were shown to proliferate for a maximum of 16 population doublings, with an approximate doubling time of 7 days. Labeled cells could be isolated and expanded in the absence of other pancreas cell types, if provided with medium conditioned by non-beta pancreatic cells. Our findings provide for the first time direct evidence for dedifferentiation, survival, and replication of cultured adult human beta cells. The tracing system will allow optimization of beta-cell expansion for transplantation. These findings demonstrate the feasibility of cell-specific labeling of cultured primary human cells, using a genetic recombination approach that was previously restricted to transgenic animals. ATTENUATION OF ACUTE KIDNEY INJURY BY SV40 CAPSID PROTETINS

D. Uzi1, M. Abd El-Latif1, V. Butin-Israeli1, G. Pizov2, Y. Haviv3, A. Oppenheim1

1Department of Hematology, Hebrew University of Jerusalem, Hadassah Medical School, Jerusalem, Israel, 2Department of Pathology, Hadassah Medical Center, Jerusalem, Israel, 3Department of Nephrology, Hadassah Medical Center, Jerusalem, Israel

Acute kidney injury (AKI) is a severe disease, caused by sepsis, ischemia and nephrotoxic agents, and compromising patient prognosis, with no effective medical treatment. The mortality of AKI in critically ill patients remains high despite the increasing ability to support vital organs. The major underlying pathogenic mechanisms are apoptosis and necrosis of kidney tubular cells. We established mercury-based AKI toxicity model, which is reproducible, and used a dose of 6.5 mg/kg to induce illness. Recombinant VP1, the major SV40 capsid protein, produced in insect cells, spontaneously assembles into DNA- free virus like particles (VLPs). These VLPs present natural tropism to the kidney as shown by anti-VP1 antibody. We studied the effect of SV40 VLPs in vivo in the mercury AKI mouse model with ~12% animal survival. VLP treatment increased survival rate to >60%. Dose response was wide, ranged between a total 0.003 - 3 mg/kg, administered in 3 daily consecutive injections. VLPs significantly reduced serum creatinine and urea as well as oxidative stress. Histological and pathological examination of AKI mice cortex pointed to widespread kidney failure. In contrast, in VLP-treated mice cell injury were dramatically diminished. This study demonstrates that SV40 VLPs, devoid of nucleic acids, may potentially be used as a prophylactic agent for AKI. APOPTOTIC CELL THERAPY IN GRAFT-VERSUS-HOST DISEASE

J. Zazoun, D. Mevorach

Laboratory for Cellular and Molecular Immunology, Hadassah Medical School, Hebrew University of Jerusalem, Israel

Allogeneic hematopoietic stem-cell transplantation (HSCT) has become a mainstay for the treatment of hematopoietic malignancies, inherited hematopoietic disorders, and aplastic anemia. However, a high percentage of patients with advanced cancers who undergo allogeneic sibling HSCT die from complications related to the procedure. Many of them die or experience significant morbidity due to graft versus host disease (GvHD) or the immunosuppressive treatments associated with GvHD. In GvHD, a central and critical role of host and donor antigen-presenting cells (APCs) was established for T-cell activation and the generation of pro-inflammatory cytokines. We have showed that ingestion and even the presence of a dying cell may shutdown a potentially inflammatory APC, converting it to be in a state of tolerization (Krispin et al. Blood, 2006). In the current study, we checked whether intravenous infusion of apoptotic cells enhances bone marrow engraftment across major histocompatibility barriers and avoid GvHD. We have established a mice model of acute GvHD. After having been lethally irradiated, CB6F1 mice were divided to three groups that included mice with no additional treatment, mice treated with HSCT and splenocytes, and mice treated with HSCT, splenocytes and apoptotic cells. Results: We observed that 33% of the mice treated with apoptotic cells survived more than 70 days before dying from GVHD whereas the mice that received only bone marrow cells and splenocytes died after 40 days. Conclusion: Apoptotic cell treatment significantly ameliorates survival by preventing or reducing GvHD. IDENTIFICATION AND CHARACTERIZATION OF NOVEL ADIPONECTIN RECEPTOR 1 ISOFORMS

R. Ashwal1, R. Rosenblum1, E. Yissachar1, M. Hanochi2, L. Granot3, A. Karasik2, R. Hemi2, H. Kanety2

1Everard Goodman Life Sciences Faculty, Bar-Ilan University, Ramat Gan, Israel, 2Institute of Endocrinology, Chaim Sheba Medical Center, Tel Hashomer, Israel, 3Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Israel

Adiponectin, an adipocyte-derived abundant plasma protein, gained recognition as a potential mechanistic link between obesity, diabetes and cardiovascular disease. It has insulin-sensitizing, anti-inflammatory and anti-atherogenic properties. The biological activities of adiponectin are mediated by two distinct adiponectin receptors, AdipoR1 and AdipoR2. Alternative mRNA splicing is an important mechanism for the generation of structural and functional diversity in proteins, therefore, the aim of the current study was to identify and characterize novel alternatively spliced variants of human AdipoR1. By using bioinformatics methods we identified alternatively splice variants of the human adiponectin receptor 1 and verified their expression in various human tissues. Quantitative RT-PCR analysis revealed a distinct expression pattern of the transcripts in adult and fetal tissues. Whereas most of the transcripts encode the WT-R1 protein, one novel transcript encodes a truncated receptor isoform, Trun.-R1, with a distinct C-terminus sequence. Protein dot blot and Western blot analysis with isoform specific antibodies demonstrated that WT-R1 is abundant in the muscle, Trun.-R1 in the brain, and WT-R2 is highly expressed in the liver. In addition, AdipoR1 isoforms display a distinct molecular mass in different cell lines and tissues, suggesting that they are subjected to tissue specific post-translational modifications. We found that the Trun.-R1 migrates on SDS-PAGE at 10-kDa higher than its predicted molecular mass and it is probably due to SUMO and/or ubiquitin modifications. Immunoprecipitation experiments demonstrated the ability of Trun.-R1 to form hetero-multimers with WT-R2 in the liver. We also over expressed the WT- R1 and the Trun.-R1 in HEK293 cells and examined the effect of their over expression on the phosphorylation of several molecules involved in adiponectin signaling. While over expression of the WT-R1 enhanced the basal level as well as adiponectin-induced phosphorylation of these proteins, over expression of the truncated isoform did not alter or even reduced their phosphorylation, suggesting that the truncated isoform may have an inhibitory activity. The function of the distinct adiponectin receptors and the interplay between the different isoforms is currently under investigation. Hopefully it will enhance our understanding of the mechanisms underlying adiponectin biological activities. ADRENAL 20ALPHA HYDROXYSTEROID DEHYDROGENASE (20HSD) EXPRESSION IS RESTRICTED TO THE X-ZONE

L. Hershkovitz1, F. Beuschlein2, M. Krup1, Y. Weinstein1

1Department of Microbiology and Immunology, Ben Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Internal Medicine II, University Hospital Freiburg, Germany

Progesterone plays an essential role in the maintenance of pregnancy of most mammals. The enzyme 20alpha hydroxysteriod dehydrogenase (20HSD) is a progesterone catabolizing enzyme which is highly expressed in the ovary and the adrenal gland. Whereas the functional significance of ovarian 20HSD for the induction of parturition has been defined in details, regulation and distribution of adrenal 20HSD expression has not been determined. As we demonstrate herein expression of 20HSD within the adrenal is restricted to the X-zone, a transient zone between the adrenal cortex and the medulla of yet unknown function. We found a direct correlation between X-zone growth and regression, the presence of 20HSD expressing cells, and 20HSD enzymatic activity: High 20HSD activity and a large X- zone was present in the adrenals of young females while a regressed X-zone and lack of 20HSD positive cells were found in males older than 5 weeks. Testosterone treatment induced rapid involution of the X-zone that was associated with the disappearance of the 20HSD positive cells and X-zone regeneration. Reappearance of 20HSD expressing cells in male animals was evident after gonadectomy. Moreover, pregnancy was accompanied by loss of adrenal 20HSD activity and X-zone regression. Interestingly, this loss of adrenal 20HSD was partially restored in animals that were kept from nursing their pups. We also reveal that treatment with prostaglandin F2alpha (PGF2alpha) during pregnancy which is known to induce the expression of 20HSD in the ovary following a decline in progesterone blood level and abortion; did not affect its expression within the adrenal. Taken together, these data indicate that the expression pattern of 20HSD in the adrenal differs from that in the ovary. In addition, in contrast to the transcriptional regulation of 20HSD expression in the ovary, adrenal 20HSD expression seems to be dependent mainly on the particular growth dynamics of the X-zone. DETERMINANTS OF PANCREATIC BETA CELL PROLIFERATION AND MASS: A PARABIOSIS APPROACH

A. Khalaileh1,2, Y. Dor1

1Department of Cellular Biochemistry and Human Genetics, Hebrew University of Jerusalem, Israel, 2Department of Surgery, Hadassah University Medical Center, Jerusalem, Israel

Most tissues undergo significant cellular turnover throughout adult life, yet maintain their cell number within a narrow, predicted range. This suggests that cell number is continuously monitored and adjusted. Regeneration experiments provide further evidence that a set-point for tissue mass exists and is actively monitored, but the signals that govern tissue homeostasis and regeneration are largely unknown. One basic question is whether tissue mass is regulated by local interactions or by systemic, blood-borne factors. To examine the contribution of systemic factors to tissue homeostasis and turnover we performed parabiosis experiments, where the circulatory systems of two mice are joined. When pancreatic beta cells of one parabiont are eliminated using a beta cell specific toxin, diabetes develops in this mouse but not in its partner. Using immunohistochemical analysis we show increased proliferation rate of beta cells and increased beta cell mass in the pancreas of healthy mice connected to a diabetic partner. These results indicate that the blood of diabetic animals contains a beta cell mitogen. HORMONAL REGULATION OF LUTENIZING HORMONE(LH)-BETA AND GONADOTROPIN RELEASING HORMONE (GnRH) mRNA EXPRESSION IN CULTURED GRANULOSA CELLS

N. Litichever1, E. Gershon1, T.D. Schirman-Hildesheim1, N. Ben-Aroya1, N. Dekel2, Y. Koch1

1Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel, 2Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are synthesized and released by the anterior pituitary and function to stimulate various gonadal activities. Lately, these glycoprotein hormones were found to be produced also in the mammalian testes and fish ovary. Recently we found that the rat ovary expresses LHbeta, FSHbeta and the common alpha subunit mRNA. In the present study we examined the regulation of LHbeta and of GnRH mRNA expression in rat ovarian granulosa cells that were isolated from either: (1) estrogen treated rats, a treatment that induces proliferation of the granulosa cells without affecting the maturation of the follicles; or (2) rats that were treated with pregnant mare serum gonadotropin (PMSG) that leads to maturation of the follicles towards ovulation, including proliferation and maturation of granulosa cells. We found that granulosa cells purified from PMSG primed rats expressed lower amounts of LHbeta and GnRH mRNA as compared to cells purified from estrogen primed rats. In both cell types, GnRH agonist treatment resulted in a decrease in LHbeta expression. However only in mature granulosa cells derived from PMSG treated rats, GnRH agonist treatment produced an increase in GnRH mRNA expression. The expression of GnRH mRNA was vastly increased by LH treatment of cells from PMSG primed rats, whereas the expression of LHbeta did not change. In contrast, FSH reduced the expression of LHbeta mRNA in granulosa cells from estrogen primed rats, but had no effect on the expression of GnRH mRNA. These results indicate that the expression of LHbeta in the ovary is dependent on the maturation state of the follicle and is regulated by locally produced GnRH and by pituitary/ovarian LH. Thus, the hormonal regulation of gene expression in granulosa cell culture depends both on the expression of the relevant receptor and on the maturational state of the cells. PARATHYROID-SPECIFIC KNOCK-OUT OF THE VITAMIN D RECEPTOR DEMONSTRATES THE ROLE OF VITAMIN D IN PARATHYROID PHYSIOLOGY

T. Meir, R. Levi, T. Naveh-Many, J. Silver

Department of Nephrology, Hadassah University Medical Center, Jerusalem, Israel

One of the main regulators of serum calcium concentration is parathyroid hormone (PTH), which acts to elevate serum calcium by enhancing its release from bone, and promoting the production of the active metabolite of vitamin D (VD) in the kidney, which in turn enhances calcium absorption in the intestine. VD acts as a negative regulator of the PTH, and thus creates a negative feedback loop. VD down-regulates PTH levels through transcriptional repression, by activating the vitamin D receptor (VDR). However this feedback loop is based on the effect of administered VD. The role of VD in normal physiology of the parathyroid is not known. Total body VDR knock-out mice showed various phenotypes, including high PTH levels, hypocalcemia, bone malformations, rickets, alopecia etc. Most, but not all of the phenotypes were found to be corrected by rebalance of serum calcium concentration. To investigate the regulation of PTH expression by the VD/VDR in as close as possible to the physiological conditions, we used a specific Cre/lox system, to knock out VDR specifically in the parathyroid (VDR-PT-KO). A significant reduction of VDR expression (although not a complete absence) was observed in the parathyroid glands of VDR-PT-KO mice. These mice exhibited a generally normal phenotype, and their parathyroid glands responded normally to changes in serum calcium concentration. Noteworthy, serum PTH levels were significantly increased in these mice compared to control littermates, without significant changes in the serum calcium concentration. Systemic administration of VD reduced serum PTH levels to the same values as wt VD treated mice, with a slight elevation of serum calcium. This elevation in serum calcium may have caused the reduction in serum PTH levels. The increase in basal PTH in the VDR-PT-KO mice indicates that the VD sets the levels of PTH expression in normal physiology. ARE STEROIDS DISPENSABLE FOR LH/HCG TRIGGERED RESUMPTION OF MEIOSIS IN MAMMALS

S. Motola, M. Popliker, A. Tsafriri

Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

In fish and amphibians resumption of meiosis in the oocytes is triggered by steroids produced by follicle cells. Such role of steroids in mammals has not been confirmed until recently when a series of publications presented evidence that steroids mediate the LH action on resumption of meiosis in the mouse. In these studies it was claimed that progesterone, estrogen or testosterone can stimulate resumption of meiosis and that specific antagonists of each of these steroid receptors can block this action of LH. Here were examined the suggested role of these steroids in the mediation of LH- stimulated resumption of meiosis in the rat and the mouse follicle-enclosed oocytes (FEOs) and cumulus-enclosed oocytes (CEOs). In CEOs, we tested whether steroid antagonists block maturation and whether steroids overcome the inhibition of maturation by hypoxanthine (Hx), a mild inhibitor of phosphodiesterases. The progesterone antagonists RU 486 and Org 31710, as well as the estrogen antagonist faslodex did not prevent LH-triggered maturation of rat or mouse FEOs or the spontaneous maturation of CEOs. In accordance, the progesterone agonist, promegestone and estradiol did not stimulate the resumption of meiosis in rat and mouse FEOs, and both did not overcome the Hx inhibition of meiosis in CEOs. Flutamide, an androgen antagonist, did block meiosis in rat FEOs, but this action could not be affected by adding dihydrotestosterone (DHT), suggesting that it was not androgen receptor mediated. Flutamide did not affect spontaneous maturation of rat CEOs and DHT could not stimulate meiosis inhibited by Hx. Contrary to recent claims, we could not provide evidence that steroids mediate LH stimulation of meiosis in rodents. Thus, in stark contrast to lower vertebrates, these steroids do not seem to serve as an obligatory signal by which the somatic cells of the follicle transfer the gonadotropic stimulation of meiosis to the oocyte. FETAL ORIGINS OF ADULT METABOLIC DISEASE: THE ROLE OF PANCREATIC BETA CELLS

M. Stolovich-Rain, Y. Dor

Department of Cellular Biochemistry and Human Genetics, Hebrew University - Hadassah Medical School, Jerusalem, Israel

A large body of evidence indicates that experience during fetal life is an important determinant of adult health. Conditions such as gestational diabetes and intrauterine growth restriction predispose to a wide spectrum of diseases including diabetes, cardiovascular disease and the metabolic syndrome. It is believed that the basis for this phenomenon is developmental plasticity, where patterns of embryonic growth are shaped, in an irreversible manner, according to environmental cues in utero such as blood glucose levels. This so called fetal programming may prove maladaptive under certain postnatal conditions, for example when fetal malnutrition is followed by postnatal high energy western diet. However, the critical fetal target organs of gestational diabetes or intrauterine growth retardation are not known. We employ a transgenic mouse system that allows for the specific killing of pancreatic beta cells at a desired time in order to investigate the long term consequences of beta cells deficiency during embryogenesis. Mice whose beta cells were killed during embryonic development die from diabetes in the first week of life. Ablation of beta cells until embryonic day 15.5 allows for partial recovery of beta cell mass by birth and survival to adulthood. Although fed and fasting blood glucose levels slowly normalize in adult mice whose beta cells were killed until E15.5, these mice show a lasting defect in glucose tolerance, deteriorating with time. These mice present normal insulin tolerance, but ~ 2.5 fold reduction in glucose stimulated insulin secretion in vivo and failure to catch up beta cell mass until 7 months age. The above findings suggest a “memory” phenotype of the embryonic insult. The effect of embryonic beta cell ablation on various organ systems is under investigation. These experiments are expected to shed light on the singular contribution of embryonic beta cells to adult health. BOTYRTIS CINEREA INDUCE SENESCENCE AND IS INHIBITED BY AUTOREGULATED PRODUCTION OF IPT GENE

D. Swartzberg1,2, B. Kirshner3, D. Rav-David3, Y. Elad3, D. Granot1

1Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel, 2Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 3Institute of Plant Protection, Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel

Botrytis cinerea is a non-specific, necrotrophic pathogen that attacks many plant species, including Arabidopsis and tomato. Since senescing leaves are particularly susceptible to infection by B. cinerea we hypothesized that the fungi might induce senescence as part of its mode of action and delaying leaf senescence might reduce the severity of B. cinerea infections. To examine these hypotheses, we followed the expression of Arabidopsis SAG12, a senescence-specific gene, upon infection with B. cinerea. Expression of SAG12 is induced by B. cinerea infection, indicating that B. cinerea induces senescence. The promoter of SAG12, as well as that of a second Arabidopsis senescence-associated gene, SAG13, were previously analyzed in tomato plants and were found to be expressed in a similar manner in the two species. These promoters were previously used in tomato plants to drive the expression of isopentenyl transferase (IPT) from agrobacterium to suppress leaf senescence (Swartzberg et al., 2006). In this study we examined the expression of these promoters following infection of tomato plants with B. cinerea. Both promoters exhibit high expression levels upon B. cinerea infection of non-senescing leaves of tomato plants, supporting our conclusion that B. cinerea induces senescence as part of its mode of action. Expression of IPT, under the control of the SAG12 and SAG13 promoters in response to infection with B. cinerea resulted in suppression of B. cinerea-induced disease symptoms, substantiating our prediction that delaying leaf senescence might reduce susceptibility to B. cinerea. EATING SALAD, IS IT GOOD FOR YOUR BONES? PLANT-DERIVED MICRONUTRIENTS INHIBIT ESTROGEN SIGNALING IN BREAST CANCER BUT NOT IN BONE CELLS

A. Veprik, M. Khanin, K. Hirsch, M. Danilenko, J. Levy, Y. Sharoni

Department of Clinical Biochemistry, Faculty of Health Science, Ben-Gurion University of the Negev, Beer-Sheva, Israel

The advantages of fruit and vegetable rich diet for human health are well known. Micronutrients from such diet were found by us to inhibit sex steroid signaling, the major risk factor, in breast and endometrial cancer. In addition, we recently found that micronutrients from fruit and vegetables stimulate the antioxidant response element (ARE) transcription system and the Nrf2 transcription factor. Moreover, using siRNA for Nrf2 and chromatin immuno-precipitation (ChIP) assay we showed that Nrf2 is involved in the inhibition of estrogen activity in breast cancer cells. Although estrogen effect in breast and endometrial cancer is harmful, it is beneficial for bone formation. Thus, the aim of the present work was to determine whether micronutrients, which inhibit the harmful signals of estrogen in cancer cells, do not inhibit its positive action in bone cells. We used MC3T3-E1 mouse calvaria osteoblast like cells, which respond to estrogen and MG-63 human osteosarcoma cells, stably ,We found that carotenoid derivatives .(בtransfected with estrogen receptor alpha (ER polyphenols and isothiocyanate do not inhibit estrogen activity in bone cell lines. Moreover, some of them stimulated the estrogen induced reporter gene activity. However, these micronutrients activated the Nrf2 transcription system in bone cells similar to the activation of this system in breast cancer cells. Using real-time PCR we examined transcription of various markers of bone differentiation such as Osteocalcin, Osteoprotegerin and Alkaline Phosphatase. We found that the combination of micronutrients with estrogen up-regulated transcription of these genes, in contrast to the inhibition of estrogen-induced gene transcription in breast cancer cells. In conclusion, dietary micronutrients, which inhibit estrogenic activity in cancer cells, do not inhibit and even stimulate estrogen signaling in bone cells. The mechanism for this differential activity of plant-derived micronutrients is under study. LONG-ACTING INSULIN ANALOGUES HAVE MITOGENIC AND ANTIAPOPTOTIC ACTIVITIES

D. Weinstein1, M. Simon1, Z. Laron2, H. Werner1

1Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel, 2Endocrine and Diabetes Research Unit, Schneider Children’s Hospital, Petah Tikva, Israel

Long acting insulin analogues have been developed to improve diabetes care. These analogues include structural modifications in specific regions which do not participate in mediating insulin binding to its receptor. However, these regions appear to have an important role in determining ligand affinity towards the IGF-I receptor (IGF-IR), a transmembrane receptor with important roles in tumor biology. Our aim was to test whether long-acting insulin analogues Glargine (Lantus®, Sanofi Aventis) and Detemir (Levemir®, Novo Nordisk) exhibit IGF-I-like activities, including enhanced mitogenic and antiapoptotic effects. For this purpose, Colon (HCT116), prostate (PC3) and breast (MCF7) cancer-derived cell lines were incubated with IGF-I, regular insulin (rINS), glargine, or detemir for different time intervals and then harvested and counted with a hemocytometer. The potential antiapoptotic activities were evaluated using an Annexin V/FITC kit and the activated signaling cascades were identified by Western immunoblotting. Results obtained showed that in HCT116 and PC3 cells both analogues significantly (p<0.05) stimulated cell proliferation in a similar fashion to IGF-I. rINS, in contrast, had essentially no effect. A similar proliferative potential was exhibited by the analogues in MCF-7 cells. Apoptosis measurements in HCT116 cells demonstrated that after 12 hours of incubation, glargine and detemir exhibited an antiapoptotic effect, similar to that elicited by IGF-I, whereas the portion of apoptotic cells after rINS or vehicle (control) treatment was significantly higher. Western blot analysis revealed that glargine activates both the MAPK and PI3K pathways, the major signaling cascades of both the insulin and IGF-I receptors. Finally, the effect of glargine on AKT phosphorylation was stronger than that of IGF-I. In conclusion, glargine and detemir exhibit potent mitogenic and antiapoptotic activities. These activities significantly exceed the extent of the effects elicited by rINS and seem to resemble IGF-I actions. The clinical implications of these findings remain to be established. IDENTIFICATION AND CHARACTERIZATION OF NOVEL SPLICE VARIANTS OF THE ADIPONECTIN RECEPTOR-2 (AdipoR2)

E. Yissachar1, R. Ashwal1, U. Nir1, A. Karasik2, R. Hemi2, H. Kanety2

1The Mina and Everard Goodman Life Sciences Faculty, Bar-Ilan University, Ramat Gan, Israel, 2Institute of Endocrinology, Sheba Medical Center, Tel Hashomer, Israel

Adiponectin, an abundant adipocyte derived plasma protein, gained recognition as a potential mechanistic link between obesity and its related morbidities. It has insulin- sensitizing, anti-inflammatory, and anti-atherogenic properties. Recently two distinct adiponectin receptors were cloned (AdipoR1 and AdipoR2), which mediate the biological activities of adiponectin. Here, we used bioinformatics tools and real-time PCR analysis in order to characterize the human adiponectin receptor AdipoR2. We identified novel mRNA splice variants of this receptor, and revealed distinct distribution of the splice variants in a range of human tissues, suggesting a tissue specific regulation of their expression. In addition to the previously reported R2T1 transcript, we found another abundant splice variant, R2T2, which similar to R2T1, encodes the WT protein but contains additional 22nt in the 3`UTR of the first exon. Relatively high levels of another transcript, R2T7, were identified in RNA extracts from vein, artery and hypothalamus. The potential protein that is encoded by R2T7 contains only the first N-terminus 155 amino acids of the WT protein, and it lacks the trans-membrane region. The expression of AdipoR2 splice variants during embryonic development, and in adult tissues was also examined. In adults AdipoR2 is highly abundant in the liver and has much lower expression in muscle and brain tissues. Increased levels of total AdipoR2 and its transcripts were noticed in the developing fetus, with peak levels in adult tissues. We also analyzed AdipoR2 levels in subcutaneous and omental fat biopsies from obese women, and found that AdipoR2 expression is significantly lower in the omental fat compared with the subcutaneous fat, and is inversely correlated with the women's BMI and serum triglyceride levels. Since adiponectin has autocrine activities, possibly also in lipid metabolism, this altered expression of AdipoR2 may contribute to the differences between subcutaneous and omental fat. Hopefully, the identification and characterization of the new splice variants of adiponectin receptors will enhance our understanding of the role of adiponectin in health and disease. PHOSPHORYLATED ACIDIC PROTEINS IN ESCHERICHIA COLI

A. Akerman1, R. Rosen2, A. Sacher1, D. Biran1

1Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel, 2Agentek (1987) Ltd., Tel Aviv, Israel

Bacteria contain highly phosphorylated acidic proteins, that accumulate under stress conditions or under conditions, which overload the proteolytic system. These proteins can be seen as an acidic banc in two dimensional gels. This group of proteins includes many cellular proteins, such as chaperones, biosynthetic, and metabolic enzymes. It is possible that the highly phosphorylated proteins represent a group of proteins tagged for degradation by phosphorylation. Such a tagging process may be involved in a general bacterial degradation pathway. ROLE OF THE SCF UBIQUITIN LIGASE IN CANDIDA ALBICANS MORPHOGENESIS

A. Atir-Lande, T. Gildor, D. Kornitzer

Faculty of Medicine and the Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel

Candida albicans is a fungal pathogen that causes superficial infections in human but also life threatening systemic infection in immunocompromised patients. C. albicans cells can reversibly switch between several growth forms: the yeast spherical form, and the more elongated pseudohyphal and true hyphal forms. Morphogenesis, the ability to switch between different morphological growth forms, contributes to its virulence. Environmental conditions induce the morphogenetic switch through signal transduction pathways such as the cAMP/protein kinase A pathway or the MAPK cascade. In addition, it has been shown that cell cycle cyclins that regulate cell cycle progression also affect morphogenesis. We found that a C. albicans mutant deleted for CaCDC4, a homolog of the S. cerevisiae component of the SCFCDC4 ubiquitin ligase, is viable and displays constitutive filamentous growth. This phenotype suggests that ubiquitin-mediated protein degradation is involved in the regulation of the dimorphic switch of C. albicans, and implies that one or more substrates of the SCFCaCDC4 may be regulators of the yeast-to-mold switch. We identified potential substrates of the SCFCaCDC4 ligase based on homology to substrates of SCFCDC4 in S. cerevisiae. No obvious homolog of the cell cycle inhibitor Sic1, the critical substrate of S. cerevisiae Cdc4, could be identified in the C. albicans genomic sequence. We therefore used a functional assay to identify C. albicans Sol1 as a functional homolog of Sic1. We found that Sol1 is a substrate of SCFCaCDC4, and that its function only partially overlaps with that of Sic1. However, neither Sol1, nor CaTec1 or CaFar1, homologs of SCFScCDC4 substrates, are essential for the cacdc4- /- hyphal phenotype. COMPARTMENTALIZED ER MANNOSIDASE I IS REQUIRED FOR EXTENSIVE N-GLYCAN TRIMMING WHICH LEADS TO ER- ASSOCIATED DEGRADATION OF GLYCOPROTEINS

E. Avezov, G.Z. Lederkremer

Department of Cell Research and Immunology, Tel-Aviv University, Ramat Aviv, Israel

An elaborate quality control mechanism in the Endoplasmic Reticulum (ER) assures that only properly folded glycoproteins will leave this organelle and proceed through the secretory pathway. Recently it became clear that the timing of protein folding attempts is linked to trimming of mannose residues from the precursor Glc3Man9GlcNAc2, which is found on the majority of proteins traversing the secretory pathway. Although it was initially thought that trimming of one mannose residue occurs, recent evidence gathered in our lab and others implies further trimming of three to four a1,2-linked mannose residues. These extensively trimmed species lack a mannose residue, which acts as an acceptor for reglucosylation by the folding-sensor enzyme UDP-Glc:glycoprotein glucosyltransferase (UGGT). In the absence of this glucose residue, binding of misfolded glycoproteins to the chaperones calnexin and calreticulin is blocked, leading to termination of rescue attempts of misfolded glycoproteins by the ER folding machinery and delivering them to ER- associated degradation (ERAD). A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. This enzyme removes only one mannose residue in vitro, at physiological concentrations, however at very high concentrations it can excise up to 4 a1,2-linked mannose residues. Using siRNA knock-down of ERManI, we show that this enzyme is required for extensive trimming and for ERAD in vivo. We found that the intercellular localization of ER ManI is restricted to a pericentriolar compartment that we had termed the ER-derived quality control compartment (ERQC). Upon proteasomal inhibition, this microtubule-dependent, membrane-enclosed compartment accumulates misfolded proteins and the chaperones calnexin and calreticulin but not BiP, PDI, UGGT or ERp57. ERManI knock-down prevents accumulation of misfolded proteins in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and that passage through this compartment would allow timing of folding attempts and ERAD. TURNOVER OF StAR PROTEIN: ROLES FOR THE PROTEASOME AND MITOCHONDRIAL PROTEASES

A. Bahat1, Z. Granot1, S. Eimerl1, O. Kobiler2, B. Lu3, A.B. Oppenheim2, C.K. Suzuki3, J. Orly1

1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Department of Molecular Genetics and Biotechnology, Hebrew University - Hadassah Medical School, Jerusalem, Israel, 3Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA

Steroidogenic Acute Regulatory protein (StAR) is a mitochondrial protein essential for massive synthesis of steroid hormones in the adrenal and the gonads. Our studies suggest that following translation, StAR preprotein either associates with the outer mitochondrial membrane to mediate transfer of cholesterol substrate required for steroidgenesis, or it is degraded by the proteasome. Proteasome inhibitors can prevent the turnover of StAR preprotein and other matrix-targeted preproteins. Once imported, excessive accumulation of inactive StAR in the matrix is avoided by a rapid turnover. Unexpectedly, mitochondrial StAR turnover can be inhibited by two proteasome inhibitors, i.e., MG132 and clasto-lactacystin beta-lactone (lactacystin, IC50= 3 micro Molar), but not epoxomicin considered to be the most specific inhibitor of the proteasome. To identify the mitochondrial protease involved in StAR degradation we expressed murine StAR in genetically manipulated E.coli strains null for each of the bacterial proteases. We identified Lon as the only protease capable of degrading StAR expressed in these cells. Moreover, in Lon-null bacteria, StAR degradation was fully restored by co-expressing human Lon. The relevance of these studies to degradation of StAR in the mammalian mitochondrial was demonstrated by use of siRNA mediated Lon knockdown experiments, Lon over-expression data, and cell-free assays confirming that that inhibition of StAR or casein degradation by Lon is readily inhibited by the proteasome inhibitors. Further studies are currently carried to unveil the mechanism by which the proteasome inhibitors affect a Ser-Lys (and not catalytic threonine) protease like Lon. polyQ DISEASES IN YEAST MODEL: AGGREGATION, TOXICITY AND QUALITY CONTROL IN GROWING OR NON-DIVIDING YEAST

N. Alumot, A. Cohen, S. Bar-Nun

Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel

“Polyglutamine disorders” are neurodegenerative diseases associated with polyglutamine (polyQ) proteins. The variable polyQ length is inversely correlated with the age of the disease onset. The toxicity is accompanied by polyQ-containing aggregates found in affected individuals, and impaired protein quality control and ubiquitin-proteasome system. We adopted the yeast model to explore polyQ aggregation and its relation to quality control. Expression of human-originated polyQ proteins caused toxicity and aggregation in yeast, in a length-dependant fashion. Impairment of the AAA-ATPase Cdc48, a quality control component, caused substantial increase in polyQ aggregation and toxicity. Interestingly, aggregation was also “age” dependant, as aggregation increased in non-dividing yeast. This can be explained, at least in part, by the compromised ubiquitin-proteasome system. Hence, yeast proves a valuable model for polyQ aggregation and interrelations between aggregation and quality control can be elucidated using yeast mutants. Also, polyQ aggregation indicates quality control failure, thus providing new insights into this fundamental system. Based on the conservation of the quality control system from yeast to man, studying correlations between aging, quality control and polyQ aggregation in non-dividing yeast may lead to the identification of components whose function is compromised in aging individuals. Hopefully, finding tools to boost such components would ameliorate the severity of neurodegenerative disorders symptoms. RNF121 – ANOTHER PLAYER IN THE ERAD MACHINERY?

A. Darom, L. Broday

Department of Cellular and Developmental Biology, Sackler School of Medicine, Tel-Aviv University, Ramat Aviv, Israel

RING finger proteins can function as E3 ligases in the ubiquitin system, thus play an important role in the regulation of protein stabilities and activities. ER associated degradation (ERAD) is a mechanism in which misfolded and unassembled protein complexes that fail the ER quality control are selectively retro-translocated from the ER into the cytosol and degraded by the proteasome. In this study we characterize RNF-121, a highly conserved newly identified RING finger protein in the C. elegans model system. Using C-terminal GFP fusion approach we show that RNF-121::GFP is localized to the membrane of the muscle sarcoplasmic reticulum as well as the ER membrane of other cells and tissues. We use a deletion mutant and RNAi experiments to analyze RNF-121 role in ER stress. The rnf-121 mutant worms show increased sensitivity to tunicamycin and elevated expression of the ER stress reporter hsp-4. Moreover, PAT-3/beta-integrin::GFP accumulates into inclusions in rnf-121(RNAi) muscle cytoplam, suggesting that PAT-3 may be a substrate for RNF-121 E3 ligase activity. These results indicate a possible role for RNF-121 in ERAD, and in the regulation of integrin maturation under normal and ER stress conditions. STUDYING A POSSIBLE QUALITY CONTROL ROLE FOR RHOMBOID, AN INTRA-MEMBRANE SERINE PROTEASE

E. Erez, E. Bibi

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Improperly folded or misassembled membrane proteins are usually toxic: (1) they might alter the ion permeability properties of the membrane, leading to cell death; and/or (2) they might cause severe damages by associating with functional integral membrane proteins and interfering with essential membrane processes. Removal of these defective membrane proteins is therefore crucial for cell survival and is accomplished by membrane-related quality control activities including proteolysis. We study intra-membrane proteolysis as mediated by the E. coli intra-membrane serine protease GlpG as a model, both in vivo and by biochemical means. GlpG is a member of the rhomboid family, one of the most widespread families of membrane proteins in all living cells. The notion that GlpG may cleave defective membrane proteins was examined, utilizing a series of aberrant (truncated) integral membrane proteins in-vivo. Two of these proteins were efficiently cleaved only by wild type GlpG but not by its active site mutant (S201A), both in-vivo and in-vitro. The cleavage sites of both proteins were determined by N-terminal sequencing. These results thus extend the substrate profile of rhomboids. Previously, rhomboids were thought to be specific for type-I or type-II membrane proteins, whereas the current study revealed for the first time that they also cleave type-III membrane proteins. This extended substrate recognition profile thus makes rhomboids good candidates that may play a role in intra-membrane quality control pathways. MANNOSE TRIMMING AND ERAD SUBSTRATE LOCALIZATION POSSIBLE INVOLVEMENT OF TRIMMING IN SUBSTRATE RELEASE FROM EDEM1

B. Groisman, E.Z. Ron, G.Z. Lederkremer

Department of Cell Research and Immunology, Tel-Aviv University, Ramat Aviv, Israel

A proper balance between synthesis, maturation and degradation is crucial for cell survival. The ER-associated degradation (ERAD) pathway directs ubiquitin-mediated degradation of a variety of ER-associated misfolded proteins. This process requires substrate recognition, ubiquitination and retrograde transport across the ER membrane. The progressive trimming of sugar chains is a main regulator of the substrate’s fate in the pathway. The importance of mannose trimming for glycoprotein degradation was widely demonstrated by many research groups, nevertheless not much is known about detailed events prior and after the trimming. The effect of the trimming on substrate localization and binding to proteins involved in ERAD pathway was addressed in this study. Working with our model ERAD substrate the uncleaved precursor of asyaloglycoprotein receptor H2a we have previously demonstrated involvement of two E3 ubiquitin ligases in its degradation: HRD1, - a tranmembrane protein exposing a RING H2 finger domain to the cytosol and the cytosolic lectin Fbs2. For the sugar chain of a carrying substrate recognized by cytosolic Fbs2 the glycoprotein must undergo retrotranslocation from a luminal compartment as occurs in ERAD. Therefore, the existence of Fbs2/H2a complexes served us as marker for substrate localization in the cytosol. Analyzing the effect of mannose trimming on substrate binding to several ERAD factors, we show that inhibition of the trimming process decrease H2a binding to E3 ligases, but increases its binding to EDEM1. Immunofluorescence analysis supported this observation and calculation of overlapping regions in the cells treated with the mannosidase inhibitor kifunensine showed separation between H2a and the E3s. Increase in H2a/EDEM complexes in these conditions, and the fact that EDEM interacts with H2a in cells lacking ER ManI expression, indicate that trimming is not required for EDEM binding, rather it is most likely needed for release. Similar observations were obtained with H2a lacking all its three glycosylation sites, indicating possible involvement of mannose trimming in non-glycoprotein ERAD. THE EFFECT OF MODULATED LEVELS OF THE REGULATORY SUBUNIT OF Clp PROTEASE ON THE EXPRESSION OF FOREIGN PROTEINS IN TOBACCO CHLOROPLASTS

M. Ish-Shalom, L. Naveh, Z. Adam

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem, Rehovot, Israel

Clp is an ATP-dependent stromal protease, consisting of a proteolytic core and a molecular chaperon regulatory subunit ClpC a member of the Hsp100 family. Transgenic tobacco plants were generated, in which ClpC1 was expressed under the control of the constitutive 35S promoter. Transgenic plants were screened for higher levels of ClpC1 accumulation. Transformants were found to have higher transcript levels, and accumulated 50-100% more ClpC1 protein compared with WT plants. The phenotypic effect of altered expression of ClpC1 was examined on endogenous protein stability and several aspects of plastid function. With the exception of one transgenic line, all plants looked quite similar to WT plants, and their chlorophyll and protein levels were normal. Overexpression of ClpC1 affected neither the sensitivity to photoinhibition nor the overall protein profile of the chloroplast membrane and soluble fractions. However, levels of ClpP1 and ClpP6, components of the proteolytic core, were 50-100% higher in ClpC1 overexpressors, suggesting co-regulation of the proteolytic and regulatory subunits. The plastid represents a very attractive compartment for high-level expression of recombinant proteins. Maintaining the stability of those proteins in plastids may facilitate the use of plastid transformation as a production platform. To test the hypothesis that overexpression of ClpC1 may affect the level of foreign proteins expressed in the chloroplast, we obtained seeds of tobacco plants expressing viral proteins (such as rotavirus VP6) in their chloroplasts genome. These recombinant proteins were shown to be unstable: high level in young tobacco leaves, whereas the amount of protein declines as the leaves mature. The ClpC1 overexpressing tobacco lines were crossed with transplastomic lines and double transgenic plants were obtained. On-going experiments will indicate whether increased chaperon levels of ClpC1 could stabilize foreign proteins so that plastid transformation could be used for mass production of pharmaceutical proteins. SUMOYLATION OF CYTOPLASMIC INTERMEDIATE FILAMENT PROTEINS IN C. ELEGANS ARE REQUIRED FOR FILAMENT ASSEMBLY

L. Broday1, R. Kaminsky1, C. Denison2, S.P. Gygi2

1Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel, 2Department of Cell Biology, Harvard Medical School, Boston, MA, USA

Sumoylation is a post-translational modification essential for viability. Modifications of proteins by SUMO play a role in many processes including transcriptional regulation, cell division, chromosome integrity and DNA damage response. Here we performed a proteomic approach that identified ~250 candidate targets of sumoylation in C. elegans. The identified proteins belong to few functional groups that highlight possible novel targets for this modification. One group are cytoplasmic intermediate filaments (cIFs) proteins. cIFs play essential role in providing mechanical strength to cells. IFB-1 is a cIF protein expressed in circumferential stripes in the worm epidermal cells and provides mechanical strength to the muscle-epidermal attachment structures. We examined the expression pattern of IFB-1 in the C. elegans SUMO (smo-1 gene) deletion background and observed abnormal pattern of expression in the epidermis and formation of cytoplasmic protein inclusions. These observations suggest a novel role for sumoylation in cIFs folding and filament assembly. We are currently testing the functional relation between SUMO and IFB-1. ALL THREE J-DOMAIN PROTEINS OF THE ESCHERICHIA COLI DnaK CHAPERONE MACHINERY ARE DNA BINDING PROTEINS

C. Katz, E. Gur, A. Parket, E.Z. Ron

Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

DnaJ, DjlA and CbpA are the J-domain proteins of DnaK, the major Hsp70 of Escherichia coli. CbpA was originally discovered as a DNA binding protein. Here, we show that DNA binding is a property of DnaJ and DjlA as well. Of special interest in this respect is DjlA, as this cytoplasmic protein is membrane bound and, as shown here, its affinity for DNA is extremely high. The finding that all the three J-proteins of DnaK are DNA binding proteins sheds new light on the cellular activity of these proteins. CHARACTERIZATION OF A Cdc20 HOMOLOGUE IN LEISHMANIA DONOVANI AND ITS ROLE DURING CELL CYCLE

T. Listovsky, D. Zilberstein

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Cdc20 is a key player in cell cycle control. It activates the Anaphase Promoting Complex/Cyclosome (APC/C) during mitosis, enabling chromosome segregation. This protein is also one of the sensors of the spindle pole checkpoint that prevents cells from entering premature mitosis. Cdc20 role in mitosis has been characterized in mammalian cells and yeast, not in parasitic protozoa. We have recently cloned a CDC20 homologue (Linj 10.0150) from the intracellular parasitic protozoan Leishmania donovani, the causative agents of kala-azar in humans. L. donovani promastigotes cell culture was used to study the role of LdCdc20 during the parasite mitosis and early G1 phase. LdCdc20 activity during cell cycle was approved by performing a complementation assay in Saccharomyces cerevisiae. We showed that both wt LdCdc20 and a truncated mutant are cell cycle regulated. After releasing from synchronization at G2/M, the proteins levels decreases during the first 4 hours and then start to accumulate again. We also suggest that the decrease in proteins levels is due to degradation via the ubiquitine-proteosome pathway. When proteosome inhibitor was added the decrease was significantly abolished. This is consistent with the presence of two putative destruction box motifs in LdCdc20 which defines proteins for degradation via the ubiquitine-proteosome pathway. These results demonstrate that in parasites protein degradation via proteosome is again one of the main factors in cell cycle regulation. CONNECTING THE PROTEOME, THE DEGRADOME AND THE MHC- PEPTIDOME OF HUMAN CANCER CELLS

E. Milner1, E. Barnea1, E. Kario2, A. Navon2, A. Admon1

1Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel, 2Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins. The repertoire of these peptides (the MHC peptidome) correlates better with the cellular protein degradation scheme (the degradome) than with the cellular proteome. This research focuses on the relation between the proteome of MCF-7 cells and the processes by which the degradome is transformed into the MHC peptidome, which is an indicator of the health status of the cell. We have begun to define in detail the repertoire of proteins that make up the cellular proteome with relatively high certainty using gel slices or alternatively by multi-dimensional peptide identification technology (MudPIT). The MHC peptidome was examined by solubilizing the cells with non-ionic detergents and then purifying the MHC molecules with their bound peptide cargo. Alternatively, we isolated secreted-soluble MHC molecules recovered from the conditioned medium of stably transfected cells. The MHC-peptide complexes were purified by immunoaffinity chromatography using the anti-native MHC mAb W6/32. The recovered MHC molecules were denatured by mild acid treatment and the peptides were separated from the large subunits of the MHC molecules by ultrafiltration. The definition of the MCF-7 cells degradome is based on the in vitro proteolysis of cellular proteins by the 20S and 26S proteasome. All the peptides originating from the proteome, MHC peptidome and degradome are resolved by ion-exchange followed by reversed phase capillary chromatography, connected to the same Orbitrap mass spectrometer for tandem mass spectrometry. In total, our analyses include thousands of identified proteins and their proteasomal proteolysis products as well as thousands of MHC peptides. Thus, we have begun to establish a detailed comparison between the proteome, the degradome and the MHC peptidome of cultured human cancer MCF-7 cells. REQUIREMENT FOR THE ACETYL PHOSPHATE PATHWAY FOR ATP- DEPENDENT PROTEOLYSIS IN ESCHERICHIA COLI

I. Mizrahi, D. Biran, E.Z. Ron

Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

Protein degradation is a central component of the protein quality control system. Here we show that efficient proteolysis in Escherichia coli requires the active acetyl phosphate pathway. Deletion of this pathway, leading to depletion of acetyl phosphate, results in temperature sensitivity and reduced rate of ATP-dependent proteolysis. The effect on proteolysis is general, as can be seen from the slowing down of the degradation of unstable proteins, including puromycin-derived peptides. In addition, reduced intracellular concentrations of acetyl phosphate brings about an increase in the levels of protein aggregates, which contain a wide range of proteins, as expected if a broad spectrum of substrates are involved. Additional outcomes of acetyl phosphate deficiency are elevation in the levels of heat shock genes and increased thermotolerance. The acetyl phosphate pathway is involved in major cellular processes and is the only source of acetyl phosphate, which is a key metabolic compound. We present evidence for the general role of the acetyl phosphate pathway in protein degradation. THE SACCHAROMYCES CEREVISIAE AS A MODEL SYSTEM FOR THE IDENTIFICATION OF THE E3 UBIQUITIN LIGASE, WHICH FACILITATES THE FORMATION OF n-TERMINAL UBIQUITIN CONJUGATED CHAIN

B. Bercovich, T. Noy, A. Ciechanover

Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Many cellular proteins are targeted for degradation by the ubiquitin proteasome system (UPS) following tagging with ubiquitin via three sequential enzymatic reactions. One of the substrates of the ubiquitin-proteasome system is the myogenic transcription factor MyoD that is degraded along the myogenic differentiation program in phosphorylation dependent and independent manner. It was assumed that the first event in the degradation of MyoD is the attachment of ubiquitin to a Lys within the protein. However, subsequent mutagenesis of all Lys to Arg residues in MyoD demonstrated that the lysine-less MyoD (LL MyoD) is still a UPS substrate; and that blocking of the alpha-NH2 group in the N-terminus of LL MyoD inhibited its degradation. This result suggests that an additional ubiquitin docking site resides on the alpha-NH2 of the first amino acid. In this study we used the Saccharomyces cerevisiae as a model system for the identification of the E3 ubiquitin ligase which facilitates the formation of N-terminal ubiquitin conjugated chain. We demonstrated that both WT MyoD and LL MyoD are degraded in Saccharomyces cerevisiae in a proteasome dependent manner, with a half life of ~10 min. Furthermore, we showed that deletion of Ubc6 and Ubc7 inhibited the degradation of LL MyoD, implying that ubiquitination of LL MyoD is required for its degradation. We also demonstrated that LL MyoD was stabilized following deletion of 30 amino acids from its N-terminus, suggesting that “destruction signal” resides in this domain of MyoD. CYCLIN-DEPENDENT KINASE REGULATION BY THE C. ALBICANS CDK INHIBITOR Sol1

A. Ofir, A. Atir-Lande, D. Kornitzer

Department of Molecular Microbiology, Bruce Rappaport Faculty of Medicine, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel

Candida albicans, an important human pathogen, is able to switch between yeast and hyphal morphologies in response to environmental conditions. This ability is thought to contribute to C. albicans pathogenicity. Cell cycle-dependent cellular morphogenesis is regulated in part by cyclin-dependent kinase (CDK) activity in many eukaryotes, particularly in budding yeasts. The C. albicans genome contain a Cdk1 homologue, three homologues of the S. cerevisiae G1 cyclins, and only two B- type cyclins homologues. In addition, Sol1, a CDK inhibitor (CDKI) analogous to the main S. cerevisiae CDKI Sic1, was recently identified. The C. albicans sol1-/- mutant displayed a moderate growth defect and a long delay in re-entering the cell cycle from stationary phase. Although Sol1 was identified as a functional homologue of S. cerevisiae Sic1, the overexpression phenotypes of the two proteins differ: Sic1 causes cell cycle arrest at G1/S in both C. albicans and S. cerevisiae, whereas Sol1 does not. Conversely, Sol1 has a stronger effect on morphogenesis than Sic1, inducing elongated buds that resemble hyphal germ tubes, supporting a role for Sol1 in C. albicans morphogenesis, and suggesting that Sic1 and Sol1 have distinct cyclin-CDK specificities. The difference between Sic1 and Sol1 activity might explain the relaxed coordination between morphogenesis and S-phase initiation in C. albicans as compared to S. cerevisiae. To understand the differences in the activities of these CDKIs, we assayed the activities of different cyclin-Cdk1 combinations in vitro, in the presence of increasing concentrations of Sic1 and Sol1. Our results suggest a different inhibition pattern of Sic1 vs. Sol1 on ScClb2 and ScClb5, explaining the differential effect of these CDKIs in S. cerevisiae; however, they did not reveal any significant differences in the CDK-cyclin specificity of those two CDKI for the C. albicans mitotic cyclins, or for the G1 cyclins. THE ROLE OF Ssz1 IN THE ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION

O. Ohayon, E. Rabinovich, S. Bar-Nun

Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel

Endoplasmic reticulum (ER)-associated protein degradation, also known as ERAD, is part of the ER quality control system. It ensures the elimination of aberrant proteins from the ER by the ubiquitin-proteasome system (UPS). The Cdc48-Ufd1-Npl4 complex functions in the recognition of several polyubiquitin-tagged proteins in the ERAD process. Specifically, this complex facilitates the dislocation of ERAD substrates from the ER back to the cytosol, to be presented to the UPS. In a search for new factors contributing to ERAD, we performed a genetic screen for suppressors that, when over-expressed, restore the impaired ERAD of 6myc-Hmg2 in the cdc48- 10 conditional yeast mutant. In that screen, the cytosolic protein Ssz1, a member of the Hsp70 family, was identified. Ssz1 participates in the pleiotropic drug resistance (PDR) network as a post-translational activator of Pdr1. Pdr1 controls the expression of the 26S proteasome and as shown by us of Cdc48, by upregulating the transcription factor Rpn4. Indeed, over-expression of Pdr1 or Rpn4 restored the impaired ERAD in cdc48-10. However, Ssz1 restored ERAD inִ rpn4 mutant, indicating that Ssz1 may function via a pathway other than PDR. We hypothesize that Ssz1, bypassing Rpn4, restores ERAD independent of Cdc48. This possibility was supported by our finding that Ssz1 over-expression also restored the impaired ERAD in the npl4-1 mutant. Experiments are set to determine if Ssz1 functions via Pdr1, whether it operates in chaperone-related manner replacing Cdc48, or Ssz1 is a regulator that activates Cdc48 novel ERAD partners other than Ufd1 and Npl4 or ERAD components that substitute for Cdc48 itself. YEAST ANTIZYME MEDIATES DEGRADATION OF YEAST ORNITHINE DECARBOXYLASE BY YEAST BUT NOT BY MAMMALIAN PROTEASOME

Z. Porat1, G. Landau1, Z. Bercovich1, D. Krutauz2, M.H. Glickman2, C. Kahana1

1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 2Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Polyamines are small aliphatic charged molecules essential for various cellular functions. Polyamine levels are strictly regulated, as low levels impair cell growth and polyamine excess is toxic. The key enzyme regulating polyamine levels, ornithine decarboxylase (ODC), has a unique regulatory mechanism via a small polyamine- induced protein termed antizyme. Mammalian antizyme (mAz) accelerates ODC degradation, in a ubiquitin independent manner, and inhibits polyamine uptake by a yet-undefined mechanism. While yeast antizyme (yAz) stimulates the degradation of yeast ODC (yODC), we show here that it has only a minor effect on polyamine uptake by yeast cells. A segment of yODC that parallels the Az binding segment of mammalian ODC (mODC) is required for it’s binding to yAz. Although demonstrating almost no homology to mAz, yAz seems to preserve its mechanism of action, namely altering ODC conformation and exposing the degradation signal that in the case of yODC resides at the N-terminus. Interestingly, interaction with yAz provokes degradation of yODC by yeast but not by mammalian proteasomes. This differential recognition of yODC may be valuable for investigating proteasome function and for the identification of the proteasome subunit that recognizes ODC. THERMOREGULATION OF ESCHERICHIA COLI hchA TRANSCRIPT STABILITY

A. Rasouly, Y. Shenhar, E.Z. Ron

Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

Heat shock protein 31 (Hsp31), the product of the hchA (yedU) gene, is a conserved chaperone. hchA mutants are severely defected in their ability to recover from exposure to lethal temperatures. Recent results indicated that the level of Hsp31 is induced upon temperature upshifts even in an rpoH mutant lacking 32sigam. It was shown that the hchA gene is transcriptionally regulated by the sigamS transcriptional activator, which regulates the general stress response in Escherichia coli, and that the heat shock induction is presumably due to derepression by H-NS (Molecular Microbiology Jun;60(6):1576-89). Here we show the existence of an additional, H- NS-independent thermal induction of hchA. This mechanism involves a significant increase in the stability of hchA transcripts upon a shift to higher temperatures. This is a novel mode of thermoregulation to control the temperature-dependent expression of a chaperone. CHARACTERIZATION OF RHOMBOID PROTEASES IN ARABIDOPSIS THALIANA CHLOROPLASTS

R.R. Knopf, A. Feder, Z. Adam

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem, Rehovot, Israel

The rhomboid protein family is a recently discovered family of membrane-bound serine proteases, which is unique in that the proteolytic cleavage reaction occurs within the membrane bilayer. Rhomboids are ubiquitous within nearly all sequenced genomes, however they have been functionally linked to only a few biological processes. Rhomboids’ existence in mitochondria suggested that they might also be found in chloroplasts. Homology search revealed at least 10 putative rhomboids in Arabidopsis. We fused targeting sequences of candidate genes to GFP, and transiently expressed them in protoplasts. Rbd7 and Rbd8 were targeted to chloroplasts, Rbd1 to mitochondria, and Rbd3 and Rbd5 to the cytosol and peroxisome, respectively. Import assays of in-vitro synthesized Rbd7 and Rbd8 into isolated chloroplasts revealed that they can be imported and targeted to thylakoid membranes. Transcript analysis demonstrated that Rbd7 and Rbd8 expression level is higher in leaves, consistent with their localization to chloroplasts. While Rbd7 was up-regulated during light stress, Rbd8 was up-regulated in response to cold stress, suggesting different physiological roles for each one of them. Complementation assays in a Providencia stuartii mutant revealed their inability to substitute for endogenous rhomboid activity, suggesting that different rhomboid proteases have different specificities. Null mutant plants for these genes, as well as double mutants, displayed no visible phenotype under different growth conditions, and no differential sensitivity to photoinhibition. Additional experiments with these plants, as well as with epitope-tagged transgenic plants, are currently under way. We hope that these will enable us to identify the function of rhomboid proteases in chloroplasts. THE COMBINED ACTIVITY OF ER MANNOSIDASE I AND EDEM1 IS REQUIRED FOR ER-ASSOCIATED DEGRADATION

E. Ron, E. Avezov, G.Z. Lederkremer

Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, Israel

Secretory proteins that fail to acquire their proper folding are targeted for proteasomal degradation, a process known as ER-associated degradation (ERAD). The processing of sugar moieties on glycoproteins plays a crucial role in their ER quality control. Previous results in our lab demonstrate that ERAD involves extensive trimming of mannose residues. We have determined that two ER resident proteins, ER mannosidase I (ERManI) and EDEM1, which share a certain degree of homology and have been implicated in ERAD, have the same effect on the stability of a model substrate for ERAD, uncleaved precursor of the soluble asialoglycoprotein receptor, H2a. Overexpression of either ERManI or EDEM1 accelerates H2a degradation while knock-down of each of them is sufficient for robust stabilization of the ERAD substrate suggesting that both factors act sequentially in the same pathway and not in parallel. Since mannosidase activity in vitro was detected for ERManI but not for EDEM1, the last has been proposed to act as a lectin that recognizes the sugar form on glycoproteins trimmed by ERManI, possibly mediating their retrotranslocation into the cytosol for proteasomal degradation. This hypothesis is challenged by the fact that overexpression of either ERManI or EDEM1 can compensate for the substrate stabilizing effect caused by knock-down of EDEM1 or ERManI. This ability of EDEM1 overexpression to partially fulfill the function of ERManI suggests a mannosidase activity for EDEM1 which is also supported by other results in our lab. Furthermore, a mutant of EDEM1 which lack's its carbohydrate recognition domain preserves the ability of wt EDEM1 to interact with H2a. Therefore, rather than acting as a lectin receptor in ERAD, EDEM1 must have an active role in the sugar processing events. A MASON'S APPROACH TO THE 19S PROTEASOME REGULATOR

R. Rosenzweig-Geideman1, P.A. Osmulski2, M. Gaczynska2, M.H. Glickman1

1Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel, 2Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX, USA

The 26S proteasome is a multi-subunit complex that is responsible for degradation of cellular proteins selected by the ubiquitin system. It is comprised from two main components: the 20S core particle (CP), and the 19S regulatory particle (RP). The 19S RP binds ubiquitinated substrates, and subsequently de-ubiquitinates, unfolds and translocates them into the 20S CP for degradation. While the functions of the 19S RP are well defined, little is known about its fine structure or the relationship between its subunits and the 20S CP, which it regulates. Currently, the molecular structure of the 19S RP is defined at a 2.8 nm resolution as an asymmetric oblong particle that can be neatly dissected into two stable subcomplexes: base and lid. Here we show that the base is built from a central tubular foundation wrapped by peripheral ATPases. The foundation alone is sufficient to recruit proteins with ubiquitin-like domains, and to gate the translocation channel into the 20S CP; functions previously thought to require the complete base. By single particle analysis, we identify the primary components of the foundation as Rpn1 and Rpn2, individual HEAT-repeat containing toroids stacked as a chimney directly at the epicenter of the 20S CP surface. This subunit arrangement offers a new view of the 19S RP, bearing not only on how substrates are recruited and translocated through the gated pore into the 20S CP for processing, but also on how other nucleotide-dependent molecular machines function. ROLE OF CAND1 AND NEDDYLATION IN SCF UBIQUITIN LIGASE ACTIVITY

N. Sela, A. Lande-Atir, D. Kornitzer

Department of Molecular Microbiology, Bruce Rappaport Faculty of Medicine, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel

Polyubiquitination – conjugation of multiple ubiquitin moieties – to protein substrates tags these proteins for degradation by the 26S proteasome. Polyubiquitination is promoted by ubiquitin ligases (UBLs), which bind both the substrate and the ubiquitin-carrying ubiquitin conjugating enzymes (UBCs). SCF, a UBL complex, consist the proteins CUL1, ROC1, SKP1 and a variable F-box protein (FBP). CUL1 is an extended protein that serves as scaffold for the complex. It binds the UBC via ROC1 at its C-terminus, and the substrate via SKP1 and the FBP, the substrate receptor, at its N-terminus. Additional SCF regulators are NEDD8, ubiquitin-like protein that is conjugated to the C-terminal domain of CUL1(“neddylation”)and may promote binding of the UBC, and CAND1, a large protein that wraps around CUL1 and prevents both UBC and SKP1-FBP binding. The role of CAND1 in SCF function is unclear, beyond the fact that its binding is mutually exclusive with neddylation in vitro. To address this role, we are comparing two yeasts- S. cerevisiae, which lacks CAND1, and in which neddylation plays no essential role, and C. albicans, which possesses a CAND1 homolog (CaTip120). We find that overexpression of CaTip120 in S. cerevisiae carrying the C. albicans cullin homolog is toxic. Prevention of neddylation enhances CaTip120 toxicity further, in line with the in vitro results. In contrast, genetic analysis of CaTIP120 and neddylation mutants in C. albicans indicates that both are positive regulators of SCF activity. The double mutant shows additive defects, suggesting that these factors act independently. A CaTip120 toxicity suppression screen in S. cerevisiae uncovered potential factors that may explain the opposite effects of CaTip120 in the two yeasts. We suggest that the role of CaTip120 and neddylation is to enable exchange of FBPs on the cullin scaffold, facilitating the adaptation of the cell to variable external and internal conditions. REGULATION OF THE HEAT SHOCK RESPONSE BY TEMPERATURE- DEPENDENT CHANGES IN TRANSCRIPT STABILITY

Y. Shenhar, A. Rasouly, E.Z. Ron

Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv, Israel

The genetic response of bacteria to elevated temperature is one of the most important and best studied bacterial stress responses. Upon shift to elevated temperature, the level of a set of genes (the heat shock genes) is elevated to cope with the stress, however over expression of these genes may be harmful. Thus, the unique feature of the heat shock response is its transient nature – shortly after the induction, the rate of synthesis of the heat shock proteins decreases, even if the temperature remains high. We show that the shut down of the heat shock response is due to a decrease in transcript stability of heat shock genes. ER STRESS INDUCES ALTERNATIVE NON-PROTEASOMAL DEGRADATION OF ER PROTEINS BUT NOT OF CYTOSOLIC ONES

M. Shenkman, G.Z. Lederkremer

Department of Cell Research and Immunology, Tel-Aviv University, Ramat Aviv, Israel

Inhibition of protein folding in the endoplasmic reticulum (ER) causes ER stress, which triggers the unfolded protein response (UPR). To decrease the biosynthetic burden on the ER, the UPR inhibits in its initial stages protein synthesis. At later stages it upregulates components of ER-associated degradation (ERAD) and of the ubiquitin/ proteasome system, which targets ER as well as cytosolic proteins. Here we report that at later stages the UPR also activates an alternative non-proteasomal pathway of degradation, which is resistant to proteasome inhibitors, and is specific for ER substrates (assessed with uncleaved precursor of asialoglycoprotein receptor H2a and unassembled CD3delta) and not for cytosolic ones (p53). To mimic the initial inhibition of translation during UPR we incubated cells with cycloheximide. After this treatment, degradation of ERAD substrates was no longer affected by proteasomal inhibition, similarly to the observed outcome of UPR. The degradation also became insensitive to abrogation of ubiquitination in a cell line carrying a thermosensitive E1 ubiquitin activating enzyme mutant. Of all protease inhibitors tested only the metal chelator o-phenanthroline could block this non-proteasomal degradation. Preincubation of o-phenanthroline with Mn2+ or Co2+, but not with other cations, reversed the inhibition. Our results suggest that upon inhibition of translation an alternative non-proteasomal pathway is activated for degradation of proteins from the ER. This involves a Mn2+/ Co2+ dependent metalloprotease or other metalloprotein. The alternative pathway selectively targets ERAD substrates to reduce the ER burden, but does not affect p53, the levels of which remain dependent on proteasomal control. ODC PARALOG: FROM MODE OF DEGRADATION TO EFFECTS ON POLYAMINE HOMEOSTASIS AND PROLIFERATION

Z. Snapir, A. Keren-Paz, Z. Bercovich, C. Kahana

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthesis pathway in mammalian cells is characterized ay a short intracellular half-life. ODC degradation is stimulated by specific polyamine induced protein termed antizyme (Az), which in turn is regulated by an ODC related protein termed antizyme inhibitor. Recently another ODC paralogue termed ODCp was suggested to function as AzI on the basis of its ability to increase ODC activity in transiently transfected cells and inhibit Az stimulated ODC degradation in an in vitro degradation reaction. It was therefore termed AzI2. We show here that ODCp is indeed capable of negating Az functions as reflected by its ability to increase ODC activity and polyamine uptake in stably transfected cells and in its ability to provide growth advantage to these cells. However, ODCp is less efficient than AzI1 in stimulating ODC activity, polyamine uptake and growth rate. The superiority of AzI1 compared to ODCp in inhibiting the Az stimulated ODC degradation was also demonstrated in an in vitro degradation reaction. We show that the basis for the inferiority of ODCp as an AzI is its lower affinity towards Az (Az1 and 3). As we have previously demonstrated for AzI1 and as recently demonstrated for ODCp, we demonstrate here that ODCp is degraded in a ubiquitin dependent manner in a reaction that does not require interaction with Az nor the C-terminus that in the case of ODC mediates its recognition by the proteasome. We further show that not only that ODCp degradation does not require interaction with Az, but that Az actually stabilizes ODCp by interfering with its ubiquitination. This stabilization that results in maintaining Az in a stable complex with ODCp, is a central feature contributing to the ability of ODCp to function as AzI. WHY AND HOW p53 UNDERGOES UBIQUITIN INDEPENDENT PROTEASOMAL DEGRADATION

P. Tsvetkov, N. Reuven, Y. Shaul

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Intrinsically unstructured proteins (IU proteins or IUPs), also known as natively unfolded proteins, lack well-defined secondary and tertiary structure under physiological conditions. In recent years, growing experimental and theoretical evidence has accumulated, indicating that certain proteins either fully or partially are unstructured under physiological conditions, and that they play significant roles in diverse cellular processes. We show here that 20S proteasomes digest only IUP sequences, under conditions in which native, and even molten globule states, are resistant. Furthermore, we show that protein-protein interactions can protect IUPs against 20S proteasomal action. These results suggest that IUP sequences can be identified by their susceptibility to 20S proteasomal degradation in-vitro. We have previously shown that the 20S proteasome mediates a novel ubiquitin-independent degradation pathway of p53 in-vivo. The process takes place by default but can be blocked by a number of mechanisms. P53, a well-characterized tumor suppressor, has IU domains at the N- and C-termini. To further characterize this pathway, using p53 as a model protein, we show that the N-terminus of p53 is necessary for 20S proteasomal degradation and that p53 can be degraded only from the N-terminus. Remarkably, HDMX that binds p53 at the N-terminus can prevent p53 degradation by the 20S proteasome in-vitro. We further show that HDMX stabilizes p53 on the protein level in-vivo and this stabilization effect can be achieved in cells lacking activated ubiquitin (TS20). Thus, the p53 IU N-terminus sensitizes p53 to ubiquitin independent degradation, provided that it is unoccupied by a partner. We propose a more general principle whereby the unstructured proteins are susceptible to 20S proteasomal ubiquitin-independent degradation (degradation by default) unless are masked by an associated partner. STUDYING REGULATORY ASPECTS OF THE SRP PATHWAY IN E. COLI: QUALITY CONTROL ACTIVITY OF Ffh

I. Yosef, E. Bibi

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

In Escherichia coli, early stages of membrane protein biogenesis are mediated by the signal recognition particle (SRP) and its receptor FtsY. During this process ribosomes translating membrane proteins are targeted to the cytoplasmatic membrane and to the translocon. In this work, we checked the effect of Ffh over-expression on expression of cytosolic and membrane proteins. Our results show that the expression of the polytopic membrane protein, LacY, is dramatically decreased under Ffh over- expression conditions. In contrast, the expression of the cytosolic protein, LacZ, is only slightly affected. Moreover, the inhibitory effect of Ffh over-expression not restricted to LacY since the expression of many other membrane proteins was also inhibited under these conditions. Together, our results support the notion that Ffh plays a quality control role function by preventing premature membrane protein synthesis in the cytoplasm. The mechanism by which Ffh mediates its negative effect is currently under study. THE POLYCOMB PROTEIN Ring1B GENERATES SELF ATYPICAL MIXED UBIQUITIN CHAINS REQUIRED FOR ITS IN VITRO HISTONE H2A LIGASE ACTIVITY

D. Zaaroor, R. Ben-Saadon, A. Ciechanover

Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Polycomb complexes mediate gene silencing, in part by modifying histones. Ring1B and Bmi1 are RING finger proteins that are members of the Polycomb repressive complex 1 (PRC1). Ring1B is an E3 that mediates its own polyubiquitination and monoubiquitination of histone H2A. In contrast, Bmi1 has no self-ubiquitinating activity. We show that unlike other RING finger proteins that are believed to mediate their own ubiquitination and degradation, Ring1B and Bmi1 are degraded by an exogenous E3, independent of their RING domain. The RING domains of both proteins mediate their association and subsequent stabilization. Consistent with the nonproteolytic self-ligase activity of Ring1B, it generates atypical mixed K6-, K27-, and K48-based polyubiquitin chains, which require the presence of all these lysine residues on the same ubiquitin molecule. The modification is required for Ring1B ability to monoubiquitinate H2A in vitro, unraveling an as yet undescribed mechanism for ligase activation via noncanonical self-ubiquitination. THE LINKAGE PROFILE OF THE UBIQUITINOME

I. Ziv, O. Kleifeld, M.H. Glickman

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. The ubiquitin molecule is generally transferred to an epsilon-amino group of an internal lysine residue in the substrate, generating a covalent isopeptide bond between this group and the free carboxy terminus of ubiquitin. Ubiquitin itself contains seven lysine residues which can also serve as receptors for ubiquitin. In the classical model, a uniform polyubiquitin chain linked only through Lys48 is required for recognition and degradation by the 26S proteasome. However, it has been shown that ubiquitin chains containing all possible isopeptide linkages do exist in vivo. The participation of these "alternative" polyubiquitin chains in various cellular processes, including proteolysis, is still not fully understood but is currently the topic of intense research. In this study, we use ubiquitin mutants in which all but one of the seven lysine residues were replaced by arginine. By expressing these mutants in yeast we promote the formation of select linkages and follow the ubiquitination pattern. Our initial results support the findings that all of the seven lysine residues participate in ubiquitination of targets. Furthermore, we have findings that coincide with a new perception in which modifications by multiple monoubiquitin residues per substrate account for a large portion of the “ubiquitinome”. We are now working on a technique to affinity-isolate the ubiquitinome from each ubiquitin mutant strain and analyze both linkage profile and substrate repertoire using mass spectrometry. Applying powerful quantitative proteomic techniques such as Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and Absolute Quantification analysis (AQUA) alongside mutants in carefully chosen components of the ubiquitin system allows us to find new targets for ubiquitination, and correlate them with the ubiquitin linkage profile. THE EDITING ENZYME ADAR1 AND THE mRNA SURVEILLANCE PROTEIN hUpf 1 INTERACT IN THE NUCLEUS

L. Agranat1, O. Raitskin1, J. Sperling2, R. Sperling1

1Department of Genetics, Hebrew University of Jerusalem, Israel, 2Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel

Pre-mRNA molecules undergo several processing events: 5' capping, editing, splicing and 3' polyadenylation. RNA editing is the phenomenon in which RNA molecules undergo changes in sequence and in length (not due to splicing and polyadenylation) and therefore become different from the original DNA that coded for their transcription. One of the types of RNA editing is the deamination of adenosine (A) to inosine (I). This deamination is catalyzed by the ADAR family of proteins. RNA transcripts also undergo several surveillance mechanisms. Nonsense Mediated mRNA Decay - NMD - is one of the surveillance mechanisms: mRNA molecules that have a premature stop codon, and whose translation could have negative consequences, are degraded by the NMD pathway. Major components of NMD are the hUpf family of proteins. One of them, hUpf1, was also shown to be involved in another NMD- independent, yet stop codon dependent mRNA degradation pathway. We found by co- purification and co-immunoprecipitation with Sm proteins, that like the A-to-I editing enzyme ADAR1, the RNA surveillance protein hUpf1 is associated with supraspliceosomes – the pre-mRNA processing machine in mammalian cells. We also show, by co-immunoprecipitation and cross-linking experiments, that ADAR1 and hUpf1 are found associated together within additional nuclear complexes. As an indication for the potential physiological significance of the association between ADAR1 and hUpf1, we show that the RNAi-mediated downregulation of ADAR1 was accompanied by upregulation of a number of genes previously shown to undergo A-to-I editing and to be downregulated by hUpf1. We suggest a novel downregulatory pathway of RNA polymerase II transcripts by a combination of ADAR1 and RNA degradation, presumably with the aid of hUpf1. THE DYNAMICS OF MAMMALIAN P BODY TRANSPORT, ASSEMBLY AND DISASSEMBLY IN VIVO

A. Aizer, Y. Shav-Tal

The Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

The fate of exported mRNAs in eukaryotic cells is highly regulated. As transcripts emerge from the export machinery, they are targeted for protein translation. mRNA degradation is also an important step in mRNA turnover. mRNAs are degraded by several RNA decay mechanisms, some involving decapping processes. One of the most surprising observations about the decapping complex is its enrichment in cytoplasmic foci termed processing bodies or P-bodies (PBs). PBs are aggregates of translationally repressed mRNPs associated with the translation repression or the mRNA decay machinery. Dcp2 is the decapping enzyme and Dcp1 proteins stimulate this activity. We followed the dynamic properties of PBs in living human cells using stably integrated fluorescently-tagged Dcp proteins and time-lapse microscopy. The goal of this work was to quantitatively measure the kinetics of PB transport in cells. We asked whether PBs actively scan the cytoplasm in pursuit for RNAs or if the biophysical mechanism of interaction is diffusion-based. Single particle analysis showed that PB mobility was mostly random but confined, due to anchoring to the cytoskeleton. Still, some PBs exhibited directional motion with high velocities. We examined the fate of PBs during the disruption of the cytoskeletal network, and the relation to the transcription and translation processes. The findings relating to PB assembly and disassembly during these stages were then tested biochemically. We have identified post-translational modifications that might regulate the interactions between PB components. Our measurements and calculations indicate that PB complexes and their protein components are highly dynamic. However, an active cytoplasmic PB-scanning mechanism does not occur and the diffusive motion of RNAs targeted for degradation or translational silencing is sufficient for targeting into PBs. THE ROLE OF A-TO-I RNA EDITING IN CHRONIC MYELOID LEUKEMIA

Z.S. Barbash1,2, M. Safran2, E.Y. Levanon3, Y. Cohen2, A.J. Simon2, N. Amariglio2, I. Goldstein2, G. Rechavi

1Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, 2Cancer Research Center, Sheba Medical Center, Tel Hashomer, Israel, 3Department of Genetics, Harvard Medical School, Boston, MA, USA

Chronic Myeloid Leukemia (CML) is hematologic malignancy resulting from the fusion of the BCR and ABL genes leading to an activated tyrosine kinase. Interferon alpha (IFNalpha) was considered as first line therapy for this disease until recently, when TK inhibitors were introduced. The mechanism of action of IFNalpha in CML is not known. Adenosine to inosine (A-to-I) RNA editing is a site-specific modification within precursor mRNAs, catalyzed by members of the double stranded- RNA (dsRNA) specific ADAR. A-to-I RNA editing is a widespread modification affecting tens of thousands of sites in the human transcriptome. The majority of the editing sites are found in non coding RNA sequences. The longer form of ADAR1 (p150) is an IFNalpha inducible protein, thus, we speculate that RNA editing might play a role in the response of CML cells to IFNalpha. In order to examine our theory we evaluate ADAR1 activity and RNA editing levels in response to IFNalpha treatment, and compare the response to IFNalpha in cells that posses the ADAR1 activity and in ADAR1 knock down cells. Using FACS analysis, western blot analysis and proliferation assays, we found that IFNalpha causes changes in cell cycle and proliferation rate of K562 cells. We examined the levels of RNA editing in these cells in response to IFNalpha treatment using PCR followed by sequencing and by the Sequenom MassArray technology .The results show that A-to-I RNA editing is increased significantly. All the changes in editing found in response to IFNalpha treatment were not detected in cells where ADAR1 is knocked down. Our finding suggests that ADAR1 may play a role in the response of CML cells to IFNalpha. Identifying RNA editing as a key player in the treatment of CML may help to develop new therapies based on new editing modifying drugs THE ROLES OF NUCLEAR-ENCODED MATURASES IN PLANT ORGANELLAR GROUP II INTRON RNA SPLICING

A. Bezawork, I. Keren, M. Kolton, F. Shaya, O. Ostersetzer

Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Bet-Dagan, Israel

Group II introns are large catalytic RNAs, whose structures and splicing activity resemble those of the nuclear spliceosome. Group II introns, which are found in bacteria and yeast mitochondrial genomes, are particularly prevalent in plant organellar genomes. In bacteria and yeast, the splicing of group II introns is facilitated by ORFs encoded within the introns themselves (maturases). Plant organellar group II introns, however, are highly degenerated, lost their ability to self-splice in vitro and also lost their intron-encoded maturases. Interestingly, the nuclear genomes of Arabidopsis and rice contains several genes homologous to yeast and bacterial intron- encoded maturases, which exist “out of the context” of their cognate introns. These are all predicted to localize to the organelles, and thus, we anticipated that the nuclear maturases are likely to be involved in the splicing of at least several of the ~ 40 group II introns found in the mtDNA and cpDNA. Accordingly, the aims of this research are to (1) establish the cellular location of each of the four Arabidopsis nuclear maturase; (2) indentify their RNA ligands in vivo; (3) analyse lose-of-function mutants in each nuc-Mat gene. GFP-localization analyses demonstrated that three nuc-Mat’s are targeted to the mitochondria, while the forth paralog GFP signal was localized to the plastids. A mutation in one of these genes (nuc-Mat-1; At5g46920) resulted with low respiration activity, late flowering and reduced fertility phenotypes. RNase protection assays revealed that nuc-Mat-1 facilitates the splicing of several mitochondrial group II introns (cox2, nad1 inton2, nad5 intron4, and nad7 intron2). Immunoblot analysis demonstrated a lower accumulation of COX2 and complex-I subunits in the mutant. POST TRANSCRIPTIONAL ADDITION OF HETEROPOLYMERIC TAILS TO RNA IN CYTOPLASM OF HUMAN CELLS

E. Bronshtein, G. Schuster

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

The addition of poly(A)-tails to RNA is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3' ends of most nuclear-encoded mRNAs. In prokaryotes, chloroplasts and plant mitochondria, polyadenylation stimulates RNA degradation. Here we show that in human cells mRNA undergoes transient and internal polyadenylation in addition to the stable poly(A)-tail at the 3’ end. Interestingly, a fraction of the post-transcriptionally added tails are composed of poly(A)-rich sequences, containing the other nucleotides in addition to adenosine. These tails were found in vaccinia virus transcripts which are restricted to the cytoplasm and do not enter the nucleus. This is the first description of heteropolymeric tails decorating mRNAs in human cells. Our results suggest that a polyadenylation stimulated degradation pathway is functioning in the cytoplasm of human cells. DYNAMIC REFOLDING OF INTERFERON-GAMMA mRNA ITS DUAL FUNCTION AS TRANSLATION TEMPLATE AND ACTIVATOR OF PKR

S. Cohen-Chalamish, A. Hasson, D. Weinberg, L.S. Namer, Y. Banai, F. Osman, R. Kaempfer

Department of Molecular Virology, Faculty of Medicine, Hebrew University of Jerusalem, Israel

We showed previously that the human interferon (IFN)-gamma gene uses a remarkable mechanism to regulate its expression: the mRNA activates the RNA- dependent protein kinase PKR through a pseudoknot located within the 5’-UTR and thereby attenuates its own translation by as much as 30-fold. Except for the involvement of the pseudoknot, the nature of this RNA activator of PKR remained unknown. We now report that the RNA activator comprises not only the entire 5’- UTR but enters deeply into the IFN-gamma open reading frame. This implies that during protein synthesis, when the mRNA structure is disrupted by ribosomes, it must undergo dynamic refolding to enable its dual function as translation template and as activator of PKR. Through gain-of-function mutations, we provide direct evidence that the PKR activator structure is indeed dynamic and can adopt alternate RNA conformations in two regions essential for PKR activation, the pseudoknot loop and a helix that contains the AUG initiation codon. This, we propose, facilitates efficient refolding of the mRNA. The PKR activator structure in IFN-gamma mRNA is intricate and includes as essential features a tetrahelical junction characterized by orientation-sensitive base pairs and a non-canonical kink-turn motif that enables formation of the pseudoknot stem by base pairing of remote RNA sequences. Pairwise stacking of these four helices will generate two dsRNA domains of sufficient length to permit trans-autophosphorylation of the PKR dimer needed for its activation. These properties render the IFN-gamma RNA activator of PKR an exceptional structure that has evolved to permit expression of this key cytokine to the right level, allowing for protective immunity yet avoiding excess associated with diseases. This is the first example of a natural sensor and activator of PKR in mRNA for which structure- function relationships have been resolved. ISOLATION OF SUPRASPLICEOSOMES ASSEMBLED ON A SPECIFIC TRANSCRIPT IN VIVO

A. Dahan1, Y. Brody3, M. Angenitski1, Y. Shav-Tal3, Y. Sperling2, R. Sperling1

1Department of Genetics, Hebrew University of Jerusalem, Israel, 2Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel, 3The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

From the time of its formation until it leaves the nucleus, each RNA polymerase II transcript is packaged in an RNP complex - the supraspliceosome - in which splicing occurs in vivo. The supraspliceosome contains components necessary for splicing, capping, editing and 3' end processing. It is composed of four substructures (native spliceosomes), which are active in splicing. Isolation of splicing complexes assembled on specific transcripts has been previously accomplished in vitro, but not in vivo. As the supraspliceosome constitutes a general processing machine for all pre-mRNAs, the transcript heterogeneity of the supraspliceosome population within cell nuclei make structural analyses quite a challenge. To obtain a homogeneous population of supraspliceosomes we used an affinity purification procedure. Supraspliceosomes assembled on a single type of transcript that contains the MS2 aptamer sequence, were purified using the recombinant Maltose Binding Protein (MBP)-MS2 protein. The MBP-MS2 protein binds to the MS2 aptamer-tagged transcript through its C- terminus and interacts with the amylose beads through its N-terminus. The RNA- tagged supraspliceosomes were affinity isolated and eluted with maltose. For purification of in vivo assembled specific supraspliceosomes we used a human cell line stably transfected with a multi-intronic construct derived from beta-globin, in which 18 repeats of the MS2 aptamer were inserted at the 3’end. Electron microscopy visualization revealed that supraspliceosomes were affinity purified. RT-PCR analysis confirmed the specificity of the affinity purification, as the purified supraspliceosomes were found assembled on the beta-globin transcript, but not on endogenous beta-actin. These results open the way for the first time for the structural analyses of supraspliceosomes isolated from living cells and assembled on a specific transcript. INVOLVEMENT OF THE TOM COMPLEX IN mRNA LOCALIZATION TO THE MITOCHONDRIA'S MEMBRANE

E. Eliyahu, D. Melamed, L. Pnueli, Y. Arava

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

The vast majority of mitochondrial proteins is encoded in the nucleus, and need to be transported into the mitochondria. This transport is done through the mitochondrial multi-protein TOM complex (Translocase of the Outer Membrane). It was suggested that in order to facilitate transport, mRNAs encoding for mitochondrial proteins are preferentially enriched in the mitochondria's vicinity. For few tested mRNAs, this enrichment was shown to involve elements within their 3' UTR. Recently, the RNA binding protein Puf3, which interacts mainly with mRNAs encoding for mitochondrial proteins, was found to be localized to the mitochondria membrane. We therefore hypothesized that Puf3 interacts with the TOM complex, and this interaction assists in mRNAs localization to the vicinity of the mitochondria's membrane. To test this, we examined the interactions of Puf3 with several proteins of the TOM complex in the yeast S. cerevisiae. Our initial results reveal a genetic interaction between Puf3 and Tom20 proteins, as a strain deleted for both genes can not grow on a non fermentable medium. In a strain deleted of the TOM20 gene, fractionation assays revealed that the localization of Puf3 to the mitochondria is hindered. Moreover, a genome-wide analysis of mRNAs associated with mitochondria revealed a significant decrease of mRNAs containing Puf3 binding sites in the TOM20delta strain. Taken together, this data suggests an interaction between Puf3 and Tom20, which assist in localization of a subset of mRNAs. The effect of this interaction on protein transport into the mitochondria is yet to be determined. TWO POLYPYRIMIDINE-LIKE BINDING PROTEIN (PTB) IN TRYPANOSOMA BRUCEI: THEIR ROLE IN MESSENGER RNA STABILITY AND SPLICING

S.K. Gupta, M.Z. Stern, M. Salmon-Divon, O. Barda, T. Hacham, S. Levi, S. Michaeli

The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel

In trypanosome, all mRNA are processed by trans-splicing. In this process, a small exon (SL) is donated to all mRNA from a small RNA, the SL RNA. It is currently unknown if splicing is constitutive or is a regulated process. We were therefore interested to identity in trypanosomes factors that are known to function in splicing regulation in metazoa such as hnRNP, SR and the polypyrimidine tract biding proteins (PTB). PTB is an abundant splicing factors that functions in alternative splicing, but also in 3' end formation, mRNA localization and mRNA stability. Here we report the multiple role of two proteins that resemble the mammalian PTB protein. Down regulation of PTB 1 and PTB 2 by RNAi in the T. brucei demonstrate the essentiality of these factors for the parasite. PTB affects the steady state level of SL RNA and its nascent transcription. Microarray analysis performed on PTBI and PTBII silenced cells indicate that these factors affect the transcriptome differentially, thus regulating different subset of mRNAs. PTB1 and PTB2 substrates were identified and verified by immunoprecipitation with cognate antibodies. Both factors function in mRNA stability and can either stabilize or destabilize mRNAs. Bioinformatic analysis on PTB substrates suggest that PTB binds to sites located in both 3’ UTR but also in splicing regulatory sequences upstream to 3’ splice site. PTB was shown to bind pre- mRNA lacking polypyrimidine tract that carry only PTB binding sites and thus function as splicing activator. However, PTB binding sites were also found imbedded within polypyrimidine tracts. In mammals PTB acts as splicing repressor but in trypanosomes this factor acts both as activator or repressor. This study suggests that trans-splicing is a regulated process and that different mRNA may utilize different proteins for recognizing the polypyrimidine tract. A GENOMIC-TAGGING STRATEGY TO VISUALIZE ENDOGENOUS mRNA IN LIVING YEAST

L. Haim, G. Zipor, J.E. Gerst

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

mRNA localization may play a role in a wide variety of cellular processes, thus, there is a need for specialized tools to monitor mRNA trafficking in individual cells. The localization of endogenous mRNA has been examined using fluorescence in situ hybridization (FISH) with labeled RNA probes, but necessitates the use of fixed material. Plasmid-based expression systems are used to exogenously express mRNAs bearing binding sites for RNA binding proteins (RBPs; i.e. MS2-CP) and, when co- expressed with an RBP fused with green fluorescent protein (i.e. MS2-CP-GFP), allows for mRNA localization in living cells. However, plasmid expression results in mRNA levels higher than endogenous, which could result in mRNA mislocalization. Endogenous mRNAs in living cells have been tracked using fluorescent hybridization probes, but these techniques are not easy to employ and the signals are transient. Thus, a simple technique allowing for the sustained visualization of endogenous mRNA in vivo is required. To facilitate our studies on mRNA localization in yeast, we developed a genomic-tagging strategy (m-TAG) which utilizes a PCR-generated DNA fragment to insert binding sites for MS2-CP between the coding region and 3'- UTR of any gene of interest by homologous recombination. Upon co-expression of MS2-CP fused with GFP(X3), we are able to demonstrate the localization of endogenous mRNAs in live yeast for the first time. This technique is currently being used to generate a general mRNA localization map in yeast. IDENTIFICATION AND CHARACTERIZATION OF SMALL NUCLEOLAR RNAS AND PROTEINS INVOLVED IN TRYPANOSOME SPECIFIC rRNA PROCESSING

A. Hury, Y. Ziporen, S. Barth, R. Katz, S. Michaeli

The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel rRNA maturation to produce the large (LSU) and small (SSU) rRNA is a highly regulated complex process that requires the concerted action of many proteins and small nucleolar RNAs (snoRNAs). Trypanosomes, unlike most eukaryotes, possess unique additional cleavage events of LSU to produce two large rRNA subunits and four small rRNA (srRNA). It is currently unknown if these trypanosome-specific cleavages are mediated by snoRNAs, enzymes or both. So far, only two snoRNAs involved in rRNA processing were described in trypanosomes such as U3 and snR30 homologues but many of snoRNAs that exist in other eukaryotes are still missing. RNAi silencing of NOP1 (that bind the C/D snoRNA RNA) showed rRNA defects in releasing the srRNA fragment. In this study, we characterize the role of two C/D snoRNAs (92 and 76 nt long) in trypanosome-specific rRNA processing. Bioinformatics analysis demonstates that these RNAs can potentially bind to mature rRNA and internal transcribed spacer (ITS). These snoRNAs were silenced by RNAi, and rRNA defects were observed by RT-PCR and RNase protection assays. In addition, the interaction of these RNAs with their target was detected using in-vivo psoralen-mediated UV cross linking. SnoRNA 92 is involved in the trypanosome specific cleavage near the small rRNA6 while snoRNA76 is involved in processing of the small subunit near ITS1. In addition to snoRNAs, we demonstrate that RNaseIII enzymes are also involved in rRNA processing. Two proteins containing RNAseIII motif were silenced by RNAi and were shown to play a role rRNA processing. Together these data suggest that trypanosome utilize a combination of unique C/D snoRNA, conserved snoRNAs and enzymes for rRNA processing. THE RNA ACTIVATOR OF PKR ESSENTIAL FOR SPLICING OF HUMAN BETA-GLOBIN mRNA

L. Ilan, F. Osman, S. Cohen-Chalamish, G. Haviv, M. Eitan, Y. Banai, R. Kaempfer

Faculty of Medicine, Hebrew University of Jerusalem, Israel

We have found that splicing of human beta-globin mRNA intron 1 depends totally upon the activation of PKR, the RNA-activated protein kinase that downregulates translation by phosphorylating initiation factor eIF2alpha. This property is imparted by a cis-acting beta-globin RNA element that is a potent activator of PKR. The ability to activate PKR requires the presence of intron 1 and is abrogated upon intron excision, yet we show that the PKR activator element is exonic and maps to the 5’- terminal portion of the pre-mRNA, comprising all of the 5’-UTR and the first 74 nt of the beta-globin open reading frame. Truncation of the stem-loop in the 5’-UTR abolished the ability of beta-globin pre-mRNA template to activate PKR, as did shortening of the globin-encoding segment. We analyzed the structure of the PKR activator by 'in-line' probing for spontaneous cleavage of the RNA. Excision of intron 1 induces a conformational change in the exonic RNA structure that shuts off the ability to activate the PKR kinase. Directed mutagenesis and compensatory changes demonstrate an essential role for short helical regions within the RNA structure that activates PKR. By itself, each helix would be too short to permit PKR activation but sufficient double-stranded RNA length can be achieved through helical stacking. Mutation of the RNA element that diminished the ability to activate PKR also reduced splicing of beta-globin pre-mRNA template in HeLa cell nuclear extract wheras compensatory mutation restored both activation of PKR and mRNA splicing. As a result, mature beta-globin mRNA is a highly effective template for translation, as demanded in erythropoiesis. As sensor and activator of PKR, this novel intrinsic RNA element renders beta-globin gene expression responsive to stress signaling. A POTENTIAL ROLE FOR INITIATOR-tRNA IN PRE-mRNA SPLICING REGULATION

E. Kamhi1, Y. Hacham1, R. Sperling2, J. Sperling1

1Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel, 2Department of Genetics, Hebrew University of Jerusalem, Israel

Consensus 5‘ splice site (5'SS) sequences are highly abundant within introns, yet they are not selected for splicing under normal conditions (latent 5‘SSs). A genomic survey revealed that more then 95% of the latent 5’SSs are preceded by an in frame stop codon (IFSC). This finding invoked an RNA surveillance mechanism proposed to prevent the use of latent 5‘SSs that otherwise would generate transcripts with premature termination codons. The occurrence of such a stop codon-mediated suppression of splicing (SOS) mechanism was previously shown in two model gene systems by eliciting latent splicing upon the mutating all IFSCs (1). Furthermore, SOS was shown to be distinct from NMD and independent of protein translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism. The detection of IFSCs by the SOS surveillance mechanism requires a register to establish the reading frame. We have previously shown that AUG sequences are required to sustain the SOS mechanism, because mutations in such sequences elicited latent splicing in constructs with intact IFSCs (2). An appealing trans acting element for this register is the initiator tRNA (ini-tRNA). Here we show that the initiator tRNA (ini-tRNA) may act, in a manner that is independent on its role in protein translation, as an SOS trans-factor that marks the AUG to establish a reading frame. Consistent with this finding, we show that ini-tRNA, but neither the elongator methionyl-tRNA nor lysyl-tRNA, fractionated with specific nuclear ribonucleoprotein complexes that had been shown to harbor splicing activity (3), suggesting a possible involvement of ini-tRNA in splicing regulation

1. Li, B., et al., (2002), Proc. Natl. Acad. Sci. USA 99, 5277 2. Kamhi, E., et al., (2006), Nucl. Acids Res. 34, 3421 3. Azubel, M., (2006), J. Mol. Biol. 356, 955 CRM: A NOVEL RNA BINDING DOMAIN WITH SIMILAR ARCHITECTURE AND BINDING PROPERTIES TO THE RRM MOTIF

I. Keren1, L. Klipcan2, A. Bezawork1, O. Ostersetzer1

1Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel, 2Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

CRM (chloroplast RNA splicing and ribosome maturation) domain is a recently- recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. While in bacteria CRM domains exists as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins containing between one to four repeats. Several members of this family with multiple CRM domains were shown to be required for the splicing of specific plastidic group-II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated that CRS1 binds with high affinity and specificity to the single group-II intron whose splicing it facilitates, the atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its catalytically active form. To understand how multiple CRM domains cooperate to achieve high affinity, sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; while CRM3 binds tightly to the RNA, CRM1 associates specifically with a unique region found within atpF intron domains I. CRM2, which demonstrated only low binding affinity may also functions in protein-protein interactions. We show further that CRM domains share structural similarities and RNA-binding characteristics with the well known RNA recognition motif, the RRM domain. FUNCTIONAL ANALYSIS OF A NOVEL PLAYER IN THE ATM- MEDIATED DNA DAMAGE RESPONSE WITH PROPERTIES OF AN RNA- PROCESSING ENZYME

R. Khosravi, Y. Shiloh

Department of Human Molecular Genetics and Biochemistry, Tel Aviv University, Ramat Aviv, Israel

The ATM protein kinase is a master regulator of the cellular responses to DNA double strand breaks (DSBs). ATM mobilizes an extensive network of pathways by phosphorylating key players in its various branches. The ATM gene is mutated in patients with the genomic instability syndrome ataxia-telangiectasia (A-T), characterized by neuromotor dysfunction, immunodeficiency, cancer predisposition and radiation sensitivity. We identified the protein encoded by cDNA clone KIAA0082 as a novel ATM target. We have localized the KIAA0082 protein to the nucleus and identified a functional nuclear localization signal in its amino-terminus. Using proteomic analysis we have found that it interacts with the RNA helicase, DHX15, the human ortholog of the yeast splicing factor Prp43, and with the Rpb1 and Rpb2 subunits of RNA polymerase II; we mapped the interaction sites with these partners. Importantly, we observed that following DNA damage the KIAA0082 protein dissociates from RNA polymerase II and is phosphorylated by ATM in vitro and in vivo on at least two sites. We hypothesize that the KIAA0082 protein serves as a link between the ATM-mediated DNA damage response and RNA processing. We are generating knockout mice for the corresponding gene and are knocking- in the WW domain that binds RNA polymerase II. These mice should provide a powerful experimental system to study the physiological roles of the KIAA0082 protein and the functional significance of the WW domain following DNA damage. UNRAVELING THE IDENTITY OF THE RNA POLYADENYLATION ENZYMES IN THE CHLOROPLAST OF DIFFERENT PLANTS

S. Larom, G. Schuster

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

RNA Polyadenylation is a posttranscriptional process characterizing eukaryotic mRNA as well as other types of transcripts. The addition of poly(A)-tail to mRNA in eukaryotes is important for its stability and translation initiation. However, RNA molecules in prokaryotes and organelles are transiently polyadenylated as part of the polyadenylation-dependent RNA degradation pathway in which polyadenylation tags the RNA for rapid degradation. Polyadenylation has been analyzed in chloroplasts and mitochondria of several plants. Previous Studies using spinach chloroplasts revealed heteropolymeric tails containing all four nucleotides, indicating that polynucleotide- phosphorylase (PNPase) is the major polyadenylating enzyme. However, homopolymeric poly(A) tails were found in the Arabidopsis chloroplast, suggesting that poly(A)-polymerase (PAP) is responsible for the polyadenylation activity. In order to reveal the identity of the polyadenylation enzyme in chloroplasts of different organism, we analyzed the tail's compound in various organisms including plants such as wheat, barley, tomato, pea, maize, tobacco, spinach and Arabidopsis as well as green algae and the moss Physcomitrella. The results disclosed transcripts with both homo- and hetero-polymeric at different ratios in the different organisms. Therefore, both PAP and PNPase are present in the chloroplast of higher plants but the division of work in polyadenylating the RNA significantly differs. The evolutionary consequent of this discovery is discussed. LINGUISTIC CHARACTERIZATION OF NON-CODING RNA

I. Lebenthal, R. Unger

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

In recent years, the recognition of the importance of non-coding RNA (ncRNAs) has increased. It is assumed that the genome of all organisms and in particular of multicellular organisms contains many ncRNA molecules that have not yet been identified, both as new members of known ncRNA families and as members of yet unknown families. Bioinformatics methods have proven successful in the identification of novel protein-coding genes, however, ncRNAs often lack the characteristic features used by gene-finding algorithms for protein-coding genes. Thus, computational detection of ncRNAs is far more difficult than detection of protein-coding genes. In this study we analyzed sequences of known ncRNAs of different classes from Drosophila melanogaster in order to identify linguistic features that distinguish the different classes of ncRNA. We analyzed the distribution of different "words" (sequences of 1-7 bases) in some classes of ncRNA (tRNA, miRNA, snoRNA and snRNA) as compared to other genomic regions (CDS, UTR, intronic and intergenic regions). Although we did not find typical "words" for all ncRNA, their distribution along the genome is statistically distinct. ncRNAs are often characterized more by their secondary structure than by their primary sequence. Thus, ncRNAs sequences should be enriched in their ability to form palindromes. Therefore, we compared the potential to form palindromes in ncRNAs with that of other regions. tRNA and miRNA were found to have higher potential to create palindromes as compared to intergenic regions, while snRNA and snoRNA showed a similar trend (to a lesser extent) only when G-U pairing were allowed. These linguistic features found in the known ncRNAs may enhance our ability to predict novel ncRNAs through the development of new bioinformatics tools. NMD MECHANISM AS A GENETIC MODIFIER OF THE RESPONSE TO READTHROUGH TREATMENT

L. Linde1, S. Boelz2,3, M. Nissim-Rafinia1, Y.S. Oren1, M. Wilschanski4, Y. Yaakov4, G. Neu-Yilik2,3, A.E. Kulozik2,3, E. Kerem4, B. Kerem1

1Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Israel, 2Molecular Medicine Partnership Unit, University of Heidelberg and European Molecular Biology Laboratory, Germany, 3Department for Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Germany, 4CF Center, Hadassah University Hospital, Mount Scopus, Jerusalem, Israel

Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients all carrying the W1282X nonsense mutation. We have demonstrated that there are patients in whom the level of CFTR nonsense transcripts is markedly reduced while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) mechanism might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines, even derived from the same tissue. Variability in NMD efficiency was found also for the beta-globin transcripts carrying the beta-thalassemia-causing PTC NS39. Furthermore, this variability was found for endogenous physiologic NMD substrates which included an alternatively spliced PTC-bearing transcript, RPL3; transcripts with introns in their 3 ΠUTR, SC35 1.6 kb and SC35 1.7 kb (two splicing isoforms); a transcript with a uORF, ASNS; and another bona fide NMD substrate with an unknown NMD-inducing feature, CARS. Importantly, our results demonstrate existence of cells in which NMD of all transcripts was efficient, while others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and led to enhanced CFTR chloride-channel activity in response to gentamicin. Altogether, our results provide new insights into the molecular basis of the response to treatments that promote readthrough of PTCs, by showing a potential role for the NMD mechanism in regulating this response. The results provide evidence for variable NMD efficiency as a general phenomenon and an inherent character of a given cell. WHO DEGRADES THE MITOCHONDRIAL RNA?

V. Liveanu, G. Schuster

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

The control of RNA stability is an important step in regulation of gene expression in both cytoplasm and mitochondria. However, while our understanding of RNA degradation in the cytoplasm is quite advanced, knowledge of these processes in the mitochondria is still limited. As mitochondria have a bacterial origin they would be expected to have similarities in their mechanisms of RNA degradation, but this seems not to be the case. Enzymes that are involved in RNA decay are only partially conserved between the mitochondria of different organisms. Examining the degradation mechanism of bacteria, yeast plant and animal mitochondria, and its regulation reveals significant differences acquired during the evolution. In order to characterize the proteins involved in RNA degradation in animal mitochondria, we have fractionated ribonuclease activities by classical biochemical techniques. Fractionation of bovine liver mitochondrial extract on gel filtration column Superdex 200, anion exchange and heparin columns have revealed that mitochondrial extract contains at least three RNA degradation activities. Since there is yet no known homolog of the ribonucleases located in the mitochondria, the identification of the proteins responsible for the ribonuclease activity is of great importance STRUCTURE OF THE RNA ACTIVATOR OF PKR THAT REGULATES SPLICING OF HUMAN TUMOR NECROSIS FACTOR mRNA

L.S. Namer, F. Osman, S. Cohen-Chalamish, R. Jung, A. Biran, Y. Banai, R. Kaempfer

Faculty of Medicine, Hebrew University of Jerusalem, Israel

A cis-acting 3’-UTR element in human tumor necrosis factor (TNF)-alpha mRNA renders splicing not only fully dependent on the activation of the RNA-dependent protein kinase PKR but also highly efficient. Thus, TNF-alpha mRNA is spliced exceptionally well once PKR is activated, fitting with the indispensable role of TNF in protective immunity. This 104-nt RNA element, 2-APRE, is located upstream of the AU-rich element that imparts mRNA instability. When the 2-APRE is present, mRNA splicing becomes sensitive to inhibition by 2-aminopurine or dominant- negative mutant PKR, yet even more efficient when PKR is overexpressed. Deletion of the 2-APRE element from the TNF-alpha 3’-UTR led to loss of the PKR- dependence for splicing. 2-APRE RNA activated PKR more strongly than did dsRNA and induced eIF2alpha phosphorylation yet did not impair translation of the spliced mRNA. Using directed mutagenesis combined with ‘in-line’ structure probing for susceptibility to spontaneous cleavage coupled with V1 nuclease and Pb2+ sensitivity mapping, we show that the RNA element harbors a tetrahelical junction generated by three hairpins and pairing of the 5’- and 3’-terminal sequences, each helix being critical for the activation of PKR. Mutations in the 2-APRE that abrogated its ability to activate PKR abolished its activity as enhancer of TNF-alpha mRNA splicing. Whereas certain mutations within short helical regions, including reversal of base pair orientation, had a negative effect, other mutations strongly stimulated PKR activation. These results show that the 2-APRE is a suboptimal activator of PKR, yet sufficiently potent to fulfill the need for activated PKR in TNF-alpha mRNA splicing. Most likely, limited activation of PKR is important for 2-APRE function to avoid excessive expression of TNF-alpha that can cause inflammation and autoimmune disease. As RNA sensor and activator of PKR, the 2-APRE is an intrinsic RNA element that renders splicing of TNF-alpha mRNA highly responsive to stress. SUPPRESSION OF SPLICING IN THE NEMATODE CAENORHABDITIS ELEGANS

Y. Nevo1, G. Saper1, J. Sperling2, R. Sperling1

1Department of Genetics, Hebrew University of Jerusalem, Israel, 2Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel

Choosing the correct splice site is an important role of the splicing machinery. We have previously shown that only ~20% of the sequences that resemble the 5' splice site (5'ss) consensus in humans are selected for splicing under normal growth conditions, whereas the rest remain latent. The utilization of over 94% of these sites would have introduced an in frame stop codon, resulting in aberrant and possibly harmful mRNAs. We have termed the regulatory mechanism that prevents splicing at latent sites nonsense-mediated suppression of splicing (SOS). This mechanism has been found to be independent of translation and distinct from NMD. It has also been found to be heat shock sensitive, as latent sites can be activated under heat shock conditions. These experiments portray SOS as a novel mechanism that prevents the formation of nonsense transcripts. Here we show that the regulation of splice site selection in the nematode C. elegans deals with similar decisions and is probably done through a similar mechanism. Bioinformatic analysis of the complete C. elegans genome shows that latent 5'ss are found in ~25% of the worm's introns. There is a significant correlation between the utilization of these sites and the disruption of the open reading frame (ORF). Looking at the splicing pattern of a small sample of these sites shows no activation of the latent sites under normal growth conditions in a wild type (N2) strain, as well as in several NMD-deficient (smg) strains. Furthermore, these sites can be activated after heat shock treatment. These findings show the necessity of discriminating between authentic and latent sites in the worm and suggest the existence of an SOS-like mechanism. Finally, the use of mini-gene constructs inserted into the worm's genome provides a direct evidence for the correlation between maintaining the ORF and the choice of the 5'ss. INITIAL CHARACTERIZATION OF THE A-TO-I RNA EDITING PHENOMENON DURING hESCs DIFFERENTIATION

S. Osenberg

Cancer Research Center, Sheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel

Adenosine to inosine (A-to-I) RNA editing is a site-specific modification within precursor mRNAs, catalyzed by members of the ADAR proteins (Adenosine Deaminase Acting on RNA) family. RNA editing occurs at relatively very high level in humans RNA which include thousands of different sites. Some of the known sites are found in protein coding regions but most of them are in non coding regions, such as 3’UTRs, 5’UTRs and introns, especially in Alu elements. Although ADAR1 and ADAR2 enzymes are expressed ubiquitously, the highest levels of editing can be found in the brain. By using animal models it was shown that RNA editing is crucial for normal development and vitality. It was also shown that editing levels are increased during embryogenesis and after birth. Here we show an initial characterization of the RNA editing phenomenon in normal pluripotent stem cells of hESCs (Human Embryonic Stem Cells) and during spontaneous and neuronal differentiation. We found that RNA editing indeed occurs in hESCs and its differentiated derivatives. The level of editing and the ADARs genes expression are not constant throughout differentiation and they depend on the differentiation stage and type. To our surprise we could not detect higher level of editing in the neuronal culture in comparison to the spontaneous differentiation and undifferentiated cells. While we detected no general pattern in the editing levels of the sites in coding regions as a result of differentiation we saw that there is a global decrease in the editing levels of non coding Alu sites, particularly in the neuronal differentiation. By using the RNAi method we show that the high editing levels in the Alu elements in undifferentiated hESCs are ADAR1 depend and we believe that this phenomenon might be important for early differentiation decisions of human embryonic stem cells. ALTERED ADENOSINE TO INOSINE RNA EDITING IN HUMAN CANCER

N. Paz1, E.Y. Levanon2,3, N. Amariglio1, A.B. Heimberger4, Z. Ram5, S. Constantini6, Z.S. Barbash1, K. Adamsky1, M. Safran1, A. Hirschberg1, M. Krupsky7, I. Ben-Dov8

1Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Ramat Aviv, Israel, 2Compugen Ltd., Tel Aviv, Israel, 3Department of Genetics, Harvard Medical School, Boston, MA, USA, 4Department of Neurosurgery, Brain Tumor Center, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA, 5Department of Neurosurgery, Sourasky Medical Center, Tel Aviv, Israel, 6Department of Pediatric Neurosurgery, Dana Children's Hospital, Sourasky Medical Center, Tel Aviv, Israel, 7Department of Internal Medicine, Chaim Sheba Medical Center, Tel-Hashomer, Israel, 8Pulmonary Institute, Chaim Sheba Medical Center Tel-Hashomer, Israel, 9Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, Detroit, MI, USA, 10School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Ramat Aviv, Israel

Adenosine-to-inosine (A-to-I) RNA editing was recently shown to be abundant in the human transcriptome, affecting thousands of genes. Employing a bioinformatic approach, we identified significant global hypoediting of Alu repetitive elements in brain, prostate, lung, kidney and testis tumors. Experimental validation confirmed this finding, showing significantly reduced editing in Alu sequences within THRAP1 transcripts in brain tissues. Looking at editing of specific recoding and non-coding sites, including in cancer-related genes, a more complex picture emerged, with a gene-specific editing pattern in tumors vs. normal tissues. Additionally, we found reduced RNA levels of all three editing mediating enzymes, ADAR, ADARB1 and ADARB2, in brain tumors. The reduction of ADARB2 correlated with the grade of malignancy, with glioblastoma multiforme, the most aggressive of brain tumors, displaying a 99% decrease in ADARB2 RNA levels. Consistently, overexpression of ADAR and ADARB1 in the U87 glioblastoma multiforme cell line resulted in decreased proliferation rate, suggesting that reduced A-to-I editing in brain tumors is involved in the pathogenesis of cancer. Altered epigenetic control was recently shown to play a central role in oncogenesis. We suggest that A-to-I RNA editing may serve as an additional epigenetic mechanism relevant to cancer development and progression. THE INTERPLAY BETWEEN RNA POLYADENYLATION AND DEGRADATION IN THE THREE DOMAINS OF LIFE

V. Portnoy, G. Schuster

Department of Biology, Technion-Israel institute of Technology, Haifa, Israel

Polyadenylation is a phenomenon common to almost all organisms. In eukaryotes, a stable poly(A)-tail is added to the 3’-end of most mRNAs and it is important for nuclear export, mRNA stability and translation initiation. In prokaryotes and organelles, RNA molecules are polyadenylated as a part of polyadenylation- stimulated RNA-degradation mechanism. Interestingly, mammalian mitochondria include stable poly (A) tails at the 3’- end side by side with truncated polyadenylated RNA fragments, similarly to prokaryotes and organelles. Our work discovered halophilic and some methanogenic archaea, as well as Mycoplasma as organisms in which there is no polyadenylation. In the absence of polyadenylation RNA degradation performed in these organisms by the conserved 3’-5’ hydrolytic exoribonuclease RNase R. Interestingly, all other archaea groups metabolize their RNA by polyadenylation-stimulated degradation mechanism in which the multiprotein complex, the archaeal exosome both polyadenylates and degrades the RNA. Together, our results described the first organisms that metabolize RNA without polyadenylation and the archaeal exosome as a polyadenylating enzyme. The evolutionary consequence of these results will be discussed. ALTERNATIVE SPLICING WITHIN THE SUPRASPLICEOSOME

O. Raitskin1, N. Sznajder1, J. Sperling2, R. Sperling1

1Department of Genetics, Hebrew University of Jerusalem, Israel, 2Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel

Eukaryotic pre-mRNAs, transcribed by RNA polymerase II undergo several nuclear posttranscriptional events, including 5’-end capping, splicing, editing and 3’ end processing. The entire population of nuclear pre-mRNAs is individually packaged into supraspliceosomes until exported to the cytoplasm. Supraspliceosomes are functional in splicing and represent the in vivo splicing machine. It was shown previously, that the 21 MDa supraspliceosome contains essential splicing components, such as phosphorylated SR proteins and U snRNPs. Moreover, recent data shows that supraspliceosomes harbor also components of capping, editing, 3' end processing as well as nuclear export and RNA surveillance functions. Therefore, the supraspliceosome has features of the general RNA processing machine in eukaryotic nuclei. Alternative splicing modulates the expression of at least 88% of human intron containing genes. Therefore, the ability to perform alternative splicing is an essential requirement from a functional splicing complex in vivo. Here we show that several alternative splicing regulators cosediment with supraspliceosomes in glycerol gradients. Localization of some of these factors in the nucleoplasmic preparations and their levels in the supraspliceosomal fractions were found to be phosphorylation- dependent. Moreover, semiquantitative RT-PCR analysis of the distribution of a number of alternatively spliced endogenous transcripts such as ADAR1, hnRNP A/B and SMN2 revealed that both exon included and exon skipped mRNAs of the above genes are predominantly found in supraspliceosomes. The predominant presence of alternatively spliced transcripts as well as regulators of alternative splicing in the supraspliceosomal fractions, together with the finding that pre-mRNAs are assembled in supraspliceosomes, strongly indicate that the supraspliceosome is the machine in which alternative splicing occurs in mammalian cell nuclei. In addition, alterations in the levels and/or phosphorylation state of regulators of alternative splicing within supraspliceosomes, is likely to represent a way for regulation of alternative splicing in mammalian cell nuclei. FIRST CRYSTAL STRUCTURE OF THE CLASS II MONOMERIC HUMAN MITOCHONDRIAL PheRS AT 2.2A resolution.

L. Klipcan1, N. Kessler1, I. Levin1, I. Finarov1, N. Moor2, M. Safro1

1Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel, 2Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia

Phenylalanyl-tRNA synthetase is a member of class II aminoacyl-tRNA synthetases. All class II aaRSs, whose structures have been solved to date, are known to be active as functional dimers or tetramers. However, the multimeric organization is not a prerequisite for the phenylalanylation activity, as monomeric mitochondrial PheRS is also fully active. Human mitPheRS is a 415 amino acid single-chain enzyme and in fact is a chimera of the bacterial PheRs’s alpha-subunit and "RNP-domain" (B8 domain) of its beta-subunit. Both the analog of bacterial alpha-subunit and B8-domain at the C-terminus form the minimal functionally active structural set. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffract to 2.2 A resolution and has been determined by using combination of molecular replacement and MIR methods. Crystal structure of human mitPheRS displays unique position of the anticodon- binding domain different from that we observed in Thermus thermophilus enzyme. Moreover, the determination of the 3D-structure of the mitochondrial aaRSs enzyme is important in view of recent findings showing that at least half of human mitochondrial diseases originate from mutations in the mitochondrial tRNA and associated with a variety of human diseases. Understanding of the precise molecular mechanisms of these mutations is limited and it is not clear which exact effect the specific mitochondrial tRNA mutations on recognition and correct aminoacylation have. On the basis of the knowledge derived from 3D-structure of mitPheRS it is possible to modify enzyme in such a way to rescue the function of mutant tRNAs associated with MERFF (myoclonic epilepsy with ragged red fibers), MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) syndromes. Other specific features of human mitPheRS relative to its three- dimensional structure will be discussed. EUKARYOTIC RIBONUCLEASE E

A. Schein, G. Schuster

Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel

In E. coli, Ribonuclease E (RNase E) is the endoribonuclease that plays a key role in RNA processing and degradation. In bacteria, its cleavage activity is believed to be an essential step in the RNA degradation process and therefore, the control of RNase E activity is important for determining the half-life of each individual mRNA. Homologs of RNase E were found in different Archaea, Algae and higher plants. This work presents the first characterization of eukaryotic RNase E, encoded by Arabidopsis thaliana nuclear genome. The protein was isolated, based on its homology to bacterial RNase E and expressed in E. coli. Recombinant, highly purified protein was studied for endonucleolytic activity on different RNA substrates. It was found to be a chloroplast-residing, non-specific AU-rich-sequences-targeting endonuclease, very similar to its bacterial equivalent. Exploring the mode of action of the chloroplast RNase E will elucidate the molecular mechanism of the early steps in RNA degradation in this organelle. SLD – A QUALITY CONTROL MECHANISM TO DISCARD DEFECTIVE SPLICED LEADER RNA IN TRYPANOSOMA BRUCEI

H. Shaked, G. Arvatz, L. Rozenboim, S. Michaeli

The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel

Trypanosama brucei is a protozoan parasite that diverged very early in eukaryotic evolution. Its genome is transcribed polycistronically and its mRNAs undergo trans- splicing which involves the addition of a small exon, the spliced leader, to all mRNAs from a small RNA, the SL RNA. SL RNA, like snRNAs binds the core Sm proteins. Knockdown of Sm proteins resulted, unexpectedly, in accumulation of SL RNA, first in the nucleus and then in the cytoplasm in particle that we termed SL RNP-C (Mandelboim et al., JBC 2003, Biton et al., MBP 2006). SL RNP-C was purified to homogeneity by combination of conventional and affinity chromatography and proteins were analyzed by mass-spectrometry (MS). MS analysis revealed three novel trypanosome specific proteins, Tbp58, Tbp22 and Tbp72 that were chosen for further analysis. To verify that these proteins are genuine SL RNP-C proteins their genes were down-regulated by RNAi. All the tested proteins were shown to be essential for SL RNP-C formation. Tbp58 is an essential gene and its protein is localized in the cytoplasm and carries three distinct domains: SAM, Zinc finger and KH. The protein is related to eIF4A helicase. GFP-fusions and or antibodies raised to SL RNP-C proteins suggest that upon Sm silencing, the proteins accumulate in cytopalsmic speckles. We termed the process of SL RNA removal from the nucleus to the cytoplasm, SLD for SL RNA discard. We further wanted to explore if SLD is related to known quality control mechanism in eukaryotes, such as mRNA nonsense- mediated decay (NMD). The NMD factor Magho was tagged with YFP and we examined its localization upon Sm depletion. Magho migrates and accumulates in the cytoplasm upon Sm depletion, suggesting that SLD is a distinct mechanism but may share protein factors with the NMD machinery. CLASSIFYING RNA-BINDING PROTEINS BASED ON ELECTROSTATIC PROPERTIES

S. Shazman, Y. Mandel-Gutfreund

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Protein structure can provide new insight into the biological function of a protein and can help to design better experiments to learn its biological roles. Moreover, deciphering the interactions of a protein with other molecules can contribute to the understanding of the protein’s function within cellular processes. In this study, we apply a machine learning approach for predicting and classifying RNA-binding proteins from their three dimensional structures. The method is based on characterizing unique properties of electrostatic patches on the protein surface. Using an ensemble of general protein features and specific properties extracted from the electrostatic patches, we have successfully trained a support vector machine (SVM) to discriminate RNA-binding proteins from other positively-charged proteins that do not bind nucleic acids. Furthermore, by applying a multi-class SVM we show that we are able to automatically classify the RNA-binding proteins based on their RNA target, for example, whether it binds a ribosomal RNA (rRNA), a transfer RNA (tRNA) or messenger RNA (mRNA). Finally, our method does not rely on sequence or structural homology and could be applied to predict novel RNA binding proteins with unique folds and/or binding motifs. MASS-SPECTROMETRY BASED HIGH THROUGHPUT QUANTITATIVE ANALYSIS OF A-TO-I RNA EDITING

M. Safran1, O. Sherf1, N. Paz1, Z.S. Barbash1, S. Ausenberg1, D. Dominissini1, G. Hout-Siloni1, Y. Cohen1, N. Amariglio1, G. Rechavi1,2

1Cancer Research Center, Hemato-Oncology Laboratory, Chaim Sheba Medical Center, Tel Hashomer, Israel, 2Tel Aviv University, Ramat Aviv, Israel

A-to-I RNA editing is a global RNA modification that alters transcript sequence and function. RNA editing is catalyzed by the ADAR group of enzymes that are capable of converting adenosine to inosine at a specific location in a double strand RNA structure. It has been shown by bioinformatic analyses that RNA editing is a widespread phenomenon, affecting tens of thousands of sites and more than 1,600 different genes. Until recently the most accurate method to measure editing levels in specific sites was DNA sequencing. The homogeneous MassEXTEND (hME) assay is a simple and robust method for the analysis of single nucleotide polymorphisms (SNP). The hME assay is based on the annealing of an oligonucleotide primer adjacent to the SNP of interest. The addition of a DNA polymerase along with a mixture of terminator nucleotides allows extension of the hME primer through the polymorphic site and generates allele-specific extension products, each having a unique molecular mass. The resultant masses of the extension products are then analyzed by MALDI-TOF MS and a genotype is assigned in real time. Since RNA editing is a change of nucleotides on the RNA molecule we have applied the hME technology to detect and quantify the levels of the editing in a site specific manner. In order to compare the accuracy of the hME assay to the direct sequencing method we have designed primers for editing sites both in ALU sequences and coding regions. Based on our results we can conclude that the hME assay is at least as accurate as direct sequencing and that the error of the assay is less than 5% (exactly like the sequencing method). The use of this technique allows us to examine several dozen editing sites in one assay with only a very little amount of purified RNA. A NEW STRATEGY FOR ISOLATING mRNA BINDING PROTEINS

B. Slobodin, J.E. Gerst

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Intracellular trafficking and localization of mRNA is a fundamental feature of living organisms. This process is pivotal for the establishment and maintenance of cell polarity, which is necessary for a variety of processes, like cell division, organelle biogenesis, secretion, cell-cell communication, motility and others. mRNA localization is usually achieved via the interaction of RNA-binding proteins (RBPs) with cis motifs present in the message to form a ribonucleoprotein (RNP) complex that undergoes trafficking to discrete sites within the cell. Although many mRNAs have been shown to be actively transported and localized in a variety of different organisms, the identity of the participating RBPs is more obscure. In addition, there is no means of isolating specific mRNA molecules without dissociating RBPs important for mRNA localization and/or stability. We are currently developing a method that allows for the specific pull-down of mRNAs of interest together with their interacting proteins. This technique requires tagging of the mRNA with a MS2 bacteriophage- derived RNA sequence and utilizes a RBP that recognizes the tagged message and is fused to streptavidin-binding protein. The technique should preserve the integrity of the mRNPs, therefore, allowing for protein identification via mass spectrometry. This method should enable researchers to identify new proteins responsible for mRNA transport and localization. STABLE PNPASE RNAI SILENCING; ITS EFFECT ON THE PROCESSING AND ADENYLATION OF HUMAN MITOCHONDRIAL RNA

S. Slomovic, G. Schuster

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

Polynucleotide-phosphorylase (PNPase) is a diverse enzyme, involved in RNA polyadenylation, degradation, and processing in prokaryotes and organelles. However, in human mitochondria, PNPase is located in the inter-membrane space (IMS), where no mitochondrial RNA (mt-RNA) is known to be present. In order to determine the nature and degree of its involvement in mt-RNA metabolism, we stably silenced PNPase by establishing HeLa cell lines expressing PNPase-shRNA. Processing and polyadenylation of mt-mRNAs were significantly affected; however, to different degrees in different genes. For instance, the stable poly(A)-tails at the 3' ends of COX1 transcripts were abolished, while COX3 poly(A)-tails remained unaffected and ND5 and ND3 poly(A) extensions increased in length. Despite the lack of polyadenylation at the 3’ end, COX1 mRNA and protein accumulated to normal levels, as was the case for all 13 mt-encoded proteins. Also, cell lines with silenced PNPase showed a decrease in mitochondrial membrane potential and likewise seek a decrease in total cellular ATP . Interestingly, ATP depletion also altered poly(A)-tail length, demonstrating that adenylation of mt-RNA can be manipulated by indirect, environmental means and not solely by direct enzymatic activity. When both PNPase and the mitochondrial poly(A)-polymerase (mtPAP) were concurrently silenced, the mature 3' end of ND3 mRNA lacked poly(A)-tails but retained oligo(A) extensions. Furthermore, in mtPAP-silenced cells, truncated adenylated COX1 molecules, considered to be degradation intermediates, were present, but harbored significantly shorter tails. Together, these results suggest that an additional mitochondrial polymerase, yet to be identified, is responsible for the oligoadenylation of mt-RNA and that PNPase, although located in the IMS, is involved, most likely by indirect means, in the processing and polyadenylation of mt-RNA. SL RNA BIOGENESIS AND THE UNIQUE SETS OF HEPTAMERIC Sm/Lsm CORE PROTEINS COMPLEXES IN TRYPANOSOMA BRUCEI

I.D. Tkacz, A. Hury, S. Cohen, M. Salmon-Divon, S. Michaeli

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

In trypanosomes all mRNAs are processed by trans-splicing. In trans-splicing a common exon SL is donated to the mRNA from a small RNA, the SL RNA. We are studying the proteins factors that participate in trans-splicing. Sm and Sm like (Lsm) proteins are central to RNA metabolism, and are involved in splicing and rRNA processing. There are two main Lsm heptameric ring complexes Lsm1-Lsm7 that bind U6 snRNA and Lsm2-Lsm8 that are involved in mRNA degradation. The Sm proteins bind to snRNAs involved in splicing (U1, U2, u5 and U5). The goal of our study was to identify and characterize the Sm/Lsm complexes in T.brucei and find which core proteins bind the SL RNA. A search in the T. brucei genome database revealed several proteins carrying Sm motifs. RNAi, TAP-tag purification and fluorescent- tagging methodologies were used to study the function of these proteins. We identified the canonical Sm proteins, as well as the Lsm complex that binds to U6 snRNA. Surprisingly, we identified additional three proteins containing Sm motifs that bind specifically to U2 and U4 snRNAs that we termed specific spliceosomal Sm proteins (SSm). SL RNA was found to bind the Sm canonical proteins. Next, we studied the nuclear localization of these proteins. Using in-situ hybridization and GFP-fused Sm proteins, we found that SL RNA assembly with Sm proteins takes place in a special nuclear domain. In this sub-domain we also find the SL RNA transcription factor tSNAP42 and SLA1, the small RNA which guides the pseudouridylation on SL RNA, suggesting that SL RNA transcription, modification and assembly take place in this special sub-domain. We termed this nuclear domain, the SL RNP factory. We suggest, that SL RNP is assembled in this domain and then moves to nuclear sites where trans-splicing takes place. THE 3' UTR MEDIATES THE CELLULAR LOCALIZATION OF AN mRNA ENCODING A SHORT PLASMA MEMBRANE PROTEIN

A. Loya1, L. Pnueli1, Y. Yosefzon1, Y. Wexler2, M. Ziv-Ukelson3, Y. Arava1

1Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel, 2Department of Computer Science, Technion-Israel Institute of Technology, Haifa, Israel, 3Department of Computer Science, Tel Aviv University, Ramat Aviv, Israel

Co-translational synthesis of proteins into the ER is preceded by targeting of the translating mRNA once a signal peptide emerges from the ribosome exit tunnel. Because of the size of this tunnel and the minimal length of the signal peptide, this mechanism was shown to be effective for polypeptides that are at least ~50 amino acids long; how mRNAs encoding for shorter membrane proteins are targeted to the ER membrane is unknown. Herein we have tested the possible involvement of the 3' UTR in localization of the mRNA that encodes for the short S. cerevisiae Pmp1 protein. We found by Ribosome Density Mapping (RDM), sedimentation analysis, differential centrifugation and Fluorescent in Situ Hybridization that the 3' UTR is essential for the association of the entire transcript with membranes compartments. Fusion of the 3' UTR to heterologous ORFs conferred on them sedimentation pattern resembling that of the PMP1 transcript. Mutation analysis of regions within the 3' UTR revealed that a repeating UG-rich sequence is important for the membrane association. Taken together, our results reveal an essential role for elements within the 3' UTR in the localization of an mRNA that is likely to be ignored by the standard signal-dependant mechanism. INHIBITION OF pre-mRNA SPLICING BY PEPTIDE ANALOGS OF SPLICING FACTORS

M. Zhou1, S. Marom2, C. Gilon2, J. Sperling3, R. Sperling1

1Department of Genetics, Hebrew University of Jerusalem, Israel, 2Department of Organic Chemistry, Hebrew University of Jerusalem, Israel, 3Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel

Splicing of pre-mRNA occurs in an RNA-protein macromolecular complex- the spliceosome - which is composed of five spliceosomal snRNPs (U1, U2, U4/U6, U5 snRNPs) and a large number of non-snRNP proteins. Spliceosome assembly in vitro occurs in a stepwise manner. First, U1 snRNP interacts with the 5’splice site, followed by association of U2 snRNP with the branch site forming splicing complex A. The U4/U6.U5 tri-snRNP is then added to form complex B. A subsequent structural rearrangement of the spliceosome with the release of U1 and U4 snRNPs leads to the activation of the spliceosome (complex C). To trap spliceosomes in specific functional states we have developed target-designed peptides and peptide derivatives that mimic essential interactions within the spliceosome, and thus specifically inhibit protein-protein and protein-RNA interactions. Our lead peptide is a derivative of a sequence from U5 snRNP 15kD protein. U5 snPNP is a major component of the spliceosome, which mediates important steps during assembly, maturation and dissociation of the spliceosome. The 15 kD protein is one of the specific proteins of U5 snRNP. It is highly conserved evolutionarily and is essential for splicing in vivo. Several derivatives of a basic peptide sequence derived from U5 snRNP 15kD protein were synthesized, and their inhibitory effect on splicing in vitro was tested on two pre-mRNA transcripts using HeLa cell nuclear extract. The lead peptide had an IC50=30, 60 µM in an in vitro splicing reaction of beta-globin or Adeno pre-mRNA, respectively. Yet, assembly of splicing complexes A and B can still be observed in the presence of the inhibitory peptide at concentrations below their IC50 value. Furthermore, when HeLa cells, grown in tissue culture, were treated with the lead peptide or derivatives, inhibition of pre-mRNA splicing expressed from both endogenous SMN2 gene, and from transfected Adeno minigene was observed. LOCALIZATION OF mRNAs ENCODING PEROXISOMAL PROTEINS IN YEAST

G. Zipor, L. Haim, J.E. Gerst

Department of Molecular Biology, Weizmann Institute of Science, Rehovot, Israel mRNA localization is a mechanism that is utilized post-transcriptionally for targeting protein synthesis to specific subcellular sites. It is a widespread phenomenon that occurs in unicellular organisms, animal and plant tissues, and in developing embryos from a variety of animal phyla. Studies in diverse systems such as oocytes, embryos, and somatic cells have demonstrated the existence of several potential mechanisms by which mRNAs undergo localization. These include local protection from degradation, diffusion and anchoring, local synthesis, and active transport by molecular motors. In Saccharomyces cerevisiae asymmetric sorting of mRNAs to the bud has been reported for several mRNAs. The best studied example is ASH1, whose mRNA localizes specifically to the bud. Local translation of Ash1 protein regulates HO gene expression, which is involved in mating- type switching. The mechanism in which ASH1 mRNA moves to the bud tip involves sequences in its ORF and 3’-UTR regions, and several trans-acting factors. In mitochondria, several mRNAs encoded from nuclear genes were shown to be localized in its vicinity. Other studies have shown the importance of the 3’-UTR for the correct localization of these mRNAs. The importance of translation in the region where the protein is ultimately needed, as demonstrated for many organelles, has brought us to ask whether mRNA localization plays a direct role in the localization and import of peroxisomal proteins into peroxisomes. In this study we demonstrate for the first time that mRNAs encoding peroxisomal proteins may localize to the vicinity of peroxisomes. Therefore, mRNA localization may have a role in peroxisome biogenesis and function. TRANSLATIONAL CONTROL OF PROTEIN KINASE C'ETA' BY TWO UPSTREAM OPEN READING FRAMES (uORFs)

H. Amit1, A. Maissel1, M. Shapira2, E. Livneh1

1Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Protein kinase C (PKC) represents a family of serin/threonine kinases, playing a central role in the regulation of cell growth, differentiation and transformation. Post- translational control of the PKC isoforms and their activation has been extensively studied, however, not much is known on their translational regulation. Here we report that expression of one of the PKC isoforms, PKC'eta', is regulated at the translational level both under normal growth conditions and during stress imposed by amino acids starvation, the latter causing a marked increase in its protein levels. The 5'- untranslated region (5'UTR) of PKC'eta' is unusually long and GC rich, characteristic of many oncogenes and growth regulatory genes. We have identified two conserved upstream open reading frames (uORFs) in its 5'UTR and show their effect in suppressing the expression of PKC'eta' in MCF-7 growing cells. Point-mutations of each or both initiation codons (uAUGs) of these two uORFs relieved this repression. However, while the two uORFs function to maintain low basal levels of PKC'eta' in growing cells, the presence of the uORFs contributes to its enhanced expression upon amino acids starvation. Mutating both uAUGs completely abolished this stress- induced elevation. Our work further suggests that translational regulation could provide another level for controlling the expression of PKC family members, being more common than currently recognized. DECIPHERING THE EFFECT OF CODON USAGE ON TRANSLATION EFFICIENCY AND PROTEIN LEVELS IN S.CEREVISIAE

Z. Bloom, O. Mizrachi-Man, O. Dahan, Y. Pilpel

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Codon usage bias, the nonrandom use of synonymous codons, is common to both unicellular and multicellular organisms. This is most extreme in highly expressed genes in which the codon usage is biased toward "optimal" codons that correspond to the most abundant tRNA isoacceptors in cells. The translation efficiency of a gene is commonly defined as the gene's codon adaptation to the tRNA cellular pool, and serves as a proxy for the speed of translation. This study aims to examine the effects of modifications in codon usage on the translation efficiency of genes and their corresponding protein expression levels. This is done by introducing synonymous mutations that result in a worse/better fit to the cell's tRNA pool. To evaluate translation efficiency in S.cerevisiae we chose the tRNA adaptation index (tAI). To calculate the tAI of a transcript we take the geometric average, over all its codons, of the relative concentrations of the corresponding tRNAs. These concentrations are approximated by the number of gene copies found in the genome for each tRNA. We chose a number of genes with a moderate tAI value, that would allow both lowering and raising of translation efficiency. A total of 54 mutations were designed for each gene (27 to lower/raise the tAI), such that the change in the gene's tAI corresponds to a change of at least four-fold in predicted protein levels. To allow a gradual alteration of the protein levels, the mutations are incorporated in three mutagenesis rounds in each direction, using PCR primers containing the relevant substitutions. Each mutated gene is fused to N-terminal and C-terminal tags, enabling the measurements of protein expression levels, using sandwich Elisa assays. In conclusion, the proposed methodology may serve as a mean to study the effect of codon usage on translation efficiency and protein levels in cells. AMP KINASE AND THE TUBEROSCLEROSIS COMPLEX MEDIATE ANOXIA-DEPNDENT TRANSLATIONAL REPRESSION OF TOP mRNAs

O. Cheshin, O. Meyuhas

Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel

TOP mRNAs, which encode many components of the translational apparatus, are translationally regulated by mitogenic and nutritional signals through their 5' Terminal OligoPyrimidine motif (5'TOP). We now show that TOP mRNAs are translationally repressed when cells are subjected to anoxia, as exemplified by their shift from polysomes into messenger ribonucleoprotein. Oxygen deficiency induces the accumulation of the transcription factor HIF1alpha, and thereby transcriptional activation of its target genes. In addition, anoxia results in energy starvation that leads to activation of the AMP-dependent kinase (AMPK) and its effector, tuberosclerosis complex (TSC1/2) that consists of a TSC1 and TSC2 dimer. Using genetic and biochemical approaches we have examined the role of these two pathways in the apparent translational repression. Our results imply that it is the AMPK/TSC pathway, rather than the HIF1alpha pathway, that mediates the anoxia-dependent translational repression of TOP mRNAs. Thus, down-regulation of AMPK by a specific inhibitor, compound C, relieves the selective translational repression, typically observed under anoxia. Likewise, knockout of TSC1 or TSC2 rescues TOP mRNAs from anoxia- or chemically-induced activation of AMPK. In contrast, TOP mRNAs remain sensitive to anoxia in cells lacking HIF1alpha, indicating that the respective pathway is disposable for this mode of regulation. In summary, these results demonstrate that the signal of oxygen deficiency is transduced into translational repression of TOP mRNAs through diminished cellular energy charge, and consequently to activation of the repressive AMPK/TSC1/2 pathway. A NOVEL INTERACTION BETWEEN 3' UTR FRAGMENTS AND THEIR ORF AS SUGGESTED BY THEIR SIMILAR SEDIMENTATION IN SUCROSE GRADIENT

N. Eldad, L. Pnueli, Y. Arava

Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel

The 3' UTR of eukaryotic mRNAs has important roles in mRNA stability, localization and translation. To determine if large protein complexes, ribosomal subunits or even ribosomes are bound to the 3' UTR of yeast mRNAs, we performed RDM analysis in which several polysomes-associated mRNAs were cleaved at the stop codon. The cleavage products were separated on a sucrose gradient and their sedimentation position was determinate by northern analysis. We found that the 3' UTR fragments of all tested mRNAs have a bi-modal sedimentation pattern, with a group that sediments as free of ribosomal subunits and a group that sediments in heavy fractions. The first group probably represents mRNAs on which ribosomes fully dissociated at the stop codon. The second group of 3' UTRs, which sediments in the heavy fractions, varied significantly between the tested mRNAs. Intriguingly, the sedimentation position of these 3' UTRs was exactly the same as their respective ORF-containing fragment. We hypothesized that the similar sedimentation of the 3' UTR fragment and the rest of its mRNA is due to association between them which maintains them together even after the cleavage. A possible mean for the association between the 3' UTR and rest of the mRNA is the known interaction between eIF4F and Pab1. To test this, we disrupted this interaction by several treatments, including removal of the polyA tail from the mRNA, titration of Pab1 or utilization of yeast strains deleted of various proteins. None of these treatments affected the sedimentation of the 3' UTR, suggesting that the similar sedimentation is through interactions that are in addition to the known eIF4F and Pab1. Although the basis of this interaction is yet to be determined, it is interesting that it differs between mRNAs. This variation may suggest specific fine tuning of translation mechanism for each mRNA. A SEARCH FOR FACTOR THAT MEDIATE THE IRES ACTIVITY OF CrTMV

S. Hen, O. Elroy-Stein

Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Initiation of protein synthesis of most eukaryotic mRNA is believed to occur via cap- dependent ribosome scanning mechanism in which the eIF4F (composed of eIF4E, eIF4G and eIF4A) initially binds the m7GpppN cap at the 5'-end of the mRNA via its cap binding subunit, eIF4E. However, some mRNA contain Internal Ribosome Entry Site (IRESes), which direct the 40S ribosome to internal site(s) within the mRNA 5'- untranslated region (5'UTR), and thereby promote translation independently of the 5'- cap, with the aid of cellular IRES-trans-acting-factors (ITAFs). IRESes have been found in many animal viruses and cellular gene, and in two plant viruses, Tobacco Etch Virus (TEV) and Crucifer Tobamovirus (CrTMV). As opposed to most known IRESes, crTMV's IRES has a remarkable cross- kingdom conservation of activity: it is active in plant (wheat germ) and animal (rabbit reticulocyte and Krebs-2) cell-free translation systems, as well as in plant (tobacco and Arabidopsis), animal (HeLa cells) and fungal cells in vivo. This indicates that the ITAF(s) mediating the internal translation initiation by the CrTMVs IRES are also broadly conserved. We demonstrated that CrTMVs IRES is fully active in HEK293 cells and our goal is to identify and characterize the ITAF(s) from animal cells. In order to isolate the IRES- binding proteins, we employ the RNA Affinity in Tandem (RAT) method specifically designed for affinity purification of in-vivo assembled RNPs (Hogg and Collin 2007). We further plan to identify the pulled-down proteins by mass spectroscopy and analysis of their ITAF activity. AMINO ACIDS CONTROL mTOR SIGNALING PATHWAY IN MAMMARY EPITHELIAL CELLS

R. Ladovsky Prizant, I. Barash

Institute of Animal Science, ARO, The Volcani Center, Bet-Dagan, Israel

In addition to their role as protein building blocks, amino acids also function as signaling molecules that control the limiting step in protein production - translation initiation, through the mTOR signaling pathway. Recent studies with muscle, liver and heart tissues established a positive role for the branched-chain amino acids, especially leucine, on translation initiation. The present study focuses on the role of essential amino acids in regulating translation in differentiated mammary epithelial cells cultured under lactogenic conditions. Addition of Leucine to amino acids- deprived cells augmented the phosphorylation of S6K1 and 4E-BP1 – signaling molecules that control translation initiation. Consequently, syntheses of total- and specific milk-proteins were enhanced. Surprisingly, three amino acids exerted a negative, dose-dependent, effect on this signaling pathway. Lysine, histidin and threonin at concentration 5 times higher then in medium, inhibited phosphorylation of S6K1, 4E-BP1, as well as total and milk protein synthesis to levels lower than detected in the amino acids- deprived cells. Their repressive effects were additive and a combined addition of the three as a mix completely abolished phosphorylation of S6K1, 4E-BP1 and mTOR in the murine and bovine epithelial cells. Total protein and milk protein synthesis were significantly lower than in the amino acids deprived cells. In contrast to the negative effect of the three amino acids mix on members of the mTOR pathway, it enhanced IRS-1 phosphorylation. This surprising finding contradicts the expected effect of active S6K1 on IRS-1 phosphorylation, and suggests the involvement of additional regulators. Taken together, a mix of lysine, histidin and threonin blocks mTOR signaling, similar to rapamycin- a specific mTOR inhibitor. It may, therefore, serve as a potential natural inhibitor specific to this pathway which is involved in cancer, diabetes and obesity. THE ROLE OF LIVIN IN ONCOGENESIS REVEALS A NOVEL MODE OF GENE REGULATION

I. Lazar

Department of Hematology, Hadassah - Hebrew University Medical Center, Ein Kerem Campus, Jerusalem, Israel

Acquired resistance to apoptosis is a hallmark of all types of cancer. The Inhibitor of Apoptosis Protein (IAP) family members are able to inhibit apoptosis induced by a variety of stimuli mainly by binding to and inhibiting caspases. We and others previously identified the IAP Livin. We further showed that Livin functions as a regulator of apoptosis that can undergo cleavage by effector caspases to produce a truncated form with paradoxical pro apoptotic activity. In this study, we describe the role of Single Nucleotide Polymorphism (SNP) in the regulation of Livin in normal and melanoma cells and demonstrate how the 528 (C/T) SNP regulates Livin at the protein level. We show that Livin is expressed in a non-random monoallelic manner as only the 528T allele exists in normal blood cells and in melanoma tumor samples obtained earlier in the clinical course of the disease. The Livin protein is not detectable in these samples. In samples obtained at progression/recurrence of the disease, both 528T and 528C mRNA alleles of Livin are expressed by a mechanism of DNA overrepresentation, resulting in expression of the Livin protein. It emerges that variable expression of the Livin protein may play an important role in the progression of melanoma and correlates with the survival of affected patients. We suggest that high level of the protein can serve as an independent adverse prognostic marker. Importantly however, low-intermediate level of Livin, rather than the absence of the protein, appears to be associated with favorable prognosis. CROSS KINGDOM CONSERVATION OF IRES FUNCTION: INVOLVEMENT OF POLY(A)-BINDING PROTEIN?

L. Marom, O. Elroy-Stein

1Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

Efficient translation initiation of most eukaryotic mRNAs is 5'cap-dependent. The 5'cap is known to function synergistically with the 3'poly(A)-tail to enhance translation efficiency due to mRNA circulization which facilitates ribosomes recycling. Circulization is achieved by poly(A) binding protein (PABP) which directly binds to the 3'poly(A)-tail and indirectly binds to the 5'cap through its interaction with eIF4G. An alternative 5'cap-independent translation initiation is mediated by ribosomes recruitment to an internal ribosome entry site (IRES). The current research addresses the possibility that PABP has a role in IRES-mediated translation. We studied the Crucifer-infecting tobamovirus (CrTMV) IRES, a unique IRES that has a cross-kingdom activity due to its purine-rich element. In addition we studied and a similar sequence of the Turnip vein-clearing virus (TVCV). Using mammalian krebs-2 cell-free translation system we showed the following: (i) although both viral sequences exhibited IRES activity, the CrTMV IRES was more active than TVCV IRES; (ii) the 3'poly(A) tail had a role in IRES-mediated translation. PABP depletion from the translation extract enabled us to demonstrate that PABP has a positive effect on both IRES activities in a 3'-poly(A) tail-dependent manner. We propose a model in which PABP binds simultaneously to the 3'poly(A)-tail and to the purine-rich element within the IRES, to form a circular translated mRNA. THE CYTOSKELETAL NETWORK CONTROLS THE TRANSLATION OF THE c-JUN PROTEIN IN A UTR-DEPENDENT MANNER

M. Prizant1, A. Arditi-Duvdevany1, I. Ben-Dror1, P. Polak1, Y. Shav-Tal2, L. Vardimon1

1Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel, 2The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

The cytoskeleton is a dynamic network that undergoes restructuring during various cellular events, influencing cell proliferation, differentiation, and apoptosis. In previous studies we have demonstrated that accumulation of c-Jun, a member of the AP1 family of transcription factors that play a key role in normal and aberrant cell growth, dramatically increases upon depolymerization of the cytoskeleton, and that, unexpectedly, this increase is controlled translationally. Depolymerization of the actin or microtubule network induces an increase in c-Jun accumulation with no corresponding increase in c-Jun mRNA or in the halflife of the c-Jun protein, but rather in the translatability of its transcript. This increase is mediated by the untranslated regions (UTRs) of c-Jun mRNA, and is not dependent on activated MAPK pathway. Here we show that the first 277 bases of the c-Jun 5'UTR are sufficient to mediate the cytoskeletal dependent control of c-Jun translation. This sequence inhibits c-Jun translation in quiescent cells, but facilitates translation in cells treated by cytoskeleton disrupting agents. Using in situ hybridization analysis we demonstrate that depolymerization of the cytoskeleton does not alter the cellular localization of the c-Jun transcript, which is cytoplasmic in both treated and untreated cells. Moreover, the c-Jun 5'UTR does not contain a cryptic promoter or alternative splicing sites that are activated upon depolymerization of the cytoskeleton, but rather binding sites to specific cytoplasmic protein(s). UV cross-linking of protein extracts to radioactively labeled fragments of c-Jun UTRs identified a cytoplasmic protein band that interact specifically with the first 277 bases at the c-Jun 5'UTR. Based on these findings we suggest that the cytoskeletal network influences RNA binding protein(s), which - via interaction with specific regulatory sequences in the c-Jun 5’UTR - either repress or activate translation. This novel mechanism of c-Jun regulation might be relevant to physiological condition in which c-Jun plays a pivotal role. POST-TRANSCRIPTIONAL REGULATION OF THE ARABIDOPSIS METAL TRANSPORTER AtMHX

H. Saul, O. David-Assael, V. Saul-Tcherkas, E. Brook, I. Berezin, O. Shaul

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

AtMHX is transporter localized at the vacuolar membrane, which exchanges protons with magnesium, zinc, and ions. In vitro analyses showed that the 5' untranslated region (5'UTR) of AtMHX can repress the translation of downstream coding sequences. The major cause of the repression was efficient initiation of translation at an upstream open-reading-frame (uORF) codon included in the 5'UTR. This is indicated by the fact that a point mutation that eliminated the uORF caused derepression. Although the sequence context of the upstream AUG (uAUG) codon was highly unfavorable, it was recognized by over 90% of the scanning ribosomes. The efficient recognition of the uAUG codon was surprising, due to the weak sequence context of this codon. While the importance of the sequence-context for recognition of initiator-AUG codons is well established, there are only a few examples of the control of gene expression by the structural context of AUG codons. We investigated the possibility that the secondary structure of the 5'UTR of AtMHX facilitates the efficient recognition of its weak-context uAUG codon. Mutations designed to disrupt the pairing of a stem and loop structure, in which the uAUG codon is localized, reduced recognition of this codon, while complementary mutation restored a great extent of the recognition. The secondary structure did not have an independent effect on downstream translation, but its impact was related to the presence of the uAUG codon. The structural modification was able to affect the efficiency of downstream translation only when the sequence context of the uAUG codon was weak. These data indicated that AtMHX translation is governed by the impact of the secondary structure of its 5'UTR on recognition of the weak-context uAUG codon. TRANSLATIONAL REGULATION DURING MITOSIS OCCURS AFTER THE INITIATION STEP

G. Sivan1, N. Kedersha2, O. Elroy-Stein1

1Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel, 2Medical School, Brigham and Women's Hospital, Boston, MA, USA

Protein synthesis is down regulated during mitosis. Previous studies by several groups demonstrated that the translational inhibition is at the initiation level. By choosing to synchronize the cells in the absence of drugs that disassemble microtubules, we surprisingly found that while translation of new proteins was indeed reduced in mitotic cells, their polysomes remain intact. Moreover, we demonstrated that the mitotic heavy polysomes are less active in translation and less sensitive to puromycin. The data suggests that during mitosis, the ribosomes stall on the mRNA, implying that the translational arrest is at the elongation level. To support this notion, we showed that Stress Granules (SG) which are formed by polysomal breakdown upon arrest at the initiation level, are not detected during mitosis. This phenomenon further proves that translation is arrested at the elongation level during cellular division. ARABIDOPSIS eIF3e IS REGULATED BY THE COP9 SIGNALOSOME AND IMPACTS DEVELOPMENT AND PROTEIN TRANSLATION

A. Yahalom1, T.H. Kim2, A.G. von Arnim2, D.A. Chamovitz1

1Department of Plant Sciences, Tel Aviv University, Ramat Aviv, Israel, 2Department of Biochemistry Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA

The roles of individual eIF3 subunits are largely unclear, though some are essential, while others are thought to have regulatory roles. The “e” subunit, also known as Int- 6, is a candidate for a regulatory subunit as it is not essential for translation initiation in yeasts. eIF3e associates with the COP9 signalosome and localizes in certain tissues to the nucleus. To further elucidate the roles of eIF3e, we have taken a genetic approach using Arabidopsis as a model system. Over expression of eIF3e results in defects similar to mutations in the COP9 signalosome. eIF3e protein, but not transcript, over-accumulates in csn mutants, and eIF3e is degraded in a proteasome- dependent fashion. In vivo assays suggest that excess eIF3e inhibits translation. We conclude that CSN maintains a precise regulation of eIF3e levels which is necessary for normal development in Arabidopsis. THE CAP BINDING COMPLEX IN LEISHMANIA

Y. Yoffe, A. Zinoviev, M. Shapira

Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Trypanosomatid parasites are known for their unique molecular features that include polycistronic transcription and trans-splicing. The Spliced Leader RNA donates a complex cap-4 structure to the 5 ends of all mRNAs. It is assumed to function during maturation of the SL RNA as well as in spliceosome assembly; however its role during translation is yet unclear. In view of this structural deviation, we examined the cap binding translation initiation complex in Leishmania. In eukaryotes, the cap binding translation initiation complex, eIF4F, consists of eIF4E- cap binding protein, eIF4A- RNA helicase and eIF4G- scaffold protein that interacts with both eIF4F factors, as well as with other translation factors. Four homolgues of translation initiation factor eIF4E was found in the Leishmania genome (LeishIF4E1-4). They vary in their cap binding affinities and sub cellular distribution over sucrose gradient. Although none of them can complete the function of mutant yeast eIF4E, LeishIF4E-1 and -4 are reasonable candidates for serving as translation factors (Yoffe et al, 2006). Bioinformatics search for eIF4G homologues revealed several candidates with low phylogenetic conservation. The conserved peptide in eIF4Gs which promote the interaction with eIF4E was not found in any of the putative LeishIF4G. However, LeishIF4G-3 is a good candidate for functioning as the translation factor since it was eluted from a m7GTP-Sepharose affinity column, along with LeishIF4E-1. LeishIF4G3 is cytoplasmatic protein that co-migrates with LeishIF4E-1 and -4 over sucrose gradient. Furthermore, pull-down assays showed that LeishIF4G-3 interacts with LeishIF4E-1 and -4, but not with LeishIF4E-2 and-3. Characterization of the binding between LeishIF4E and LeishIF4G, including detailed mapping of amino acid that involves in the interaction with LeishIF4Es are currently under investigation. In view of the low phylogentic conservation between the parasite and the mammalian eIF4E and eIF4G isoforms, the interaction between them could serve as an ideal drug target. FIBROBLAST RECRUITMENT INTO OVARIAN TUMORS: TWO PHOTON INTRAVITAL MICROSCOPY

Y. Addadi, D. Granot, V. Kalchenko, N. Nevo, M. Nemman

1Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel, 2Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel

The tumor microenvironment is frequently predominated by fibroblasts and myofibroblasts, which are found in close proximity with the tumor vasculature. Although not fully characterized, these stroma cells are known to have a critical role in tumor angiogenesis. Their origin is still controversial, however it has been suggested that fibroblasts at the tumor-host interface differentiate into myofibroblasts and then into pericyte-like cells, which are proposed to guide endothelial sprouts. This hypothesis is supported by the massive fibroblastic population in epithelial tumor stroma, and by the association between functional tumor vasculature and infiltrating stromal myofibroblasts. Recent work in our lab demonstrated the recruitment of fibroblasts into ovarian tumors by MRI and near infrared fluorescence. In order to better understand the process of angiogenesis, we have designed an experimental set- up that would enable non invasive, continuous in vivo follow-up of tumor progression, for high resolution study of the interactions between tumor cells, fibroblasts and blood vessels. A skin flap window chamber system (implanted in nude mice) combined with two photon microscopy (Zeiss LSM 510 META NLO, Germany equipped with Mai Tai One Box Ti:Sapphire Tunable Laser from Spectraphysics, USA, for two photon excitation), enabled collection of high optical resolution information from samples >300 micron thick. MLS-YFP fluorescent human ovarian tumors were generated in window chambers, followed by an intra-peritoneal injection of fluorescently labeled fibroblasts (CV-1 line and human primary WI-38 fibroblasts). In order to unravel the interactions with newly formed blood vessels, dextran-FITC was intravenously injected. Using this triply labeled system we were able to monitor the recruitment of fibroblasts into the tumor mass as well as to follow their specific spatial 3D organization. Understanding the process of fibroblasts recruitment advances our knowledge regarding tumor progression and angiogenesis and could potentially be developed as a method for targeted therapy. INHIBITION OF CORNEAL NEOVASCULARIZATION FOLLOWING CHEMICAL INJURY

L. Cohen, M. Cohen, A. Amir, S. Dachir, E. Fishbine, R. Sahar, H. Gutman, V. Givant-Horwitz, J. Turetz, T. Kadar

Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, Israel

Neovascularization (NV) of the cornea is a major public health problem. It is estimated that each year, 4% of the US population develop corneal NV following infectious, inflammatory, toxic or nutritional insults. The NV is associated with scar formation and loss of transparency, leading to visual impairments and blindness. The aim of the present study was to elucidate the pathological mechanism of NV in an experimental model of chemical burn, and to test the efficacy of anti-inflammatory drugs in ameliorating this pathology. Chemical burn of cornea was performed in NZW rabbits, using sulfur mustard (SM) vapor. Corneal NV was evaluated by a clinical ocular examination, combined with morphometric analysis. Corneal thickness, as an indicative parameter for edema, was measured in vivo by pachymetry. Topical application of steroids (0.1% Dexamethasone sodium sulfate and 0.5% neomycin sulfate, Dexamycin®) was administered using different therapeutic regimes either for prevention or intervention. At the end of experiments eyes were taken for biochemical and histological evaluations. VEGF immunohistochemistry was performed using anti-VEGF mAb. Corneal NV following SM exposure developed, as early as two weeks after exposure, associated with chronic inflammation, prolonged impairment of corneal innervation, epithelial stem cell deficiency and increased levels of VEGF. Steroid treatment delayed and attenuated the appearance of NV, when given during the first week. When treatment was applied symptomatically to corneas displaying NV, a significant regression in the angiogenic process was observed. Yet, the effect was temporal and angiogenesis reappeared when therapy was terminated. A combination of steroid with metalloproteinase inhibitors and/or anti-VEGF factors is currently under investigation. MODELING NEURONAL LESIONS OF THE PRETERM NEONATE THROUGH CONDITIONAL MANIPULATIONS OF VEGF

T. Dor1, T. Licht2, E. Keshet2

1Neuropediatric Unit, Hadassah Medical School, Jerusalem, Israel, 2Department of Molecular Biology, Hebrew University Medical School, Jerusalem, Israel

Prematurity is a major cause of cerebral palsy (CP). CP is thought to result in many cases from periventricular leukomalacia (PVL), a condition characterized by bilateral necrosis of neurons and oligodendrocytes around the ventricles of the infant brain. One hypothesis ascribes PVL to ischemic insults that selectively damage periventricular blood vessels. Inspired by the Retinopathy of Prematurity (ROP) paradigm, where vascular injuries are known to result from oxygen-induced downregulation of VEGF which is essential for the survival of immature vessels, we examined whether a similar mechanism may also apply for PVL pathogenesis. A key to this hypothesis is the proposition that periventricular vessels are selectively vulnerable and hence remain VEGF-dependent whereas nearby blood vessels remain intact upon VEGF withdrawal. To test this, we devised a transgenic system for conditional (and reversible) knock-down of VEGF in the brain based on induction of a VEGF-trapping soluble receptor. VEGF blockade was exercised during consecutive time windows of brain development and resultant vascular and neuronal phenotypes were analyzed. Corresponding to the vascular deficits, the affected brain regions (i.e. areas where neuronal cell death occurs) were progressively more restricted as the onset of VEGF blockade was further delayed. Strikingly, when started at a relatively late stage of brain development (days E12.5-E13.5) neuronal death was only detected in the periventricular area while even later onset (>E14.5) did not cause a detectable brain damage. These results highlight the specific vulnerability of the periventricular zone to transient VEGF insufficiency, and assess the time table for VEGF-dependence or maturation of its blood vessel. Further studies thrive to develop this experimental system as a mouse PVL model. CARDIOVASCULAR DISEASE IS ONLY WEAKLY ASSOCIATED WITH OXIDATIVE STRESS ACCORDING TO SELECTED INDICES

Y. Dotan, D. Lichtenberg, I. Pinchuk

Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel

Background: Oxidative stress has long been associated with cardiovascular disease. Yet, most of the clinical trials designed to reduce oxidative stress by means of antioxidant supplementation have failed. This study was conducted in hope of assessing the association between various indices of oxidative stress (e.g. the plasma concentrations of Malondialdehyde (MDA), Isoprostanes, vitamin E and 8-OH-dG) and the prevalence of CVD. Methods and Results: We performed a meta-analysis of case-control studies that assessed oxidative stress in individuals with and without prevalent CVD, using different indices of oxidative stress. Studies were included only when commonly used methods were employed to assess the oxidative stress in at least 20 patients and 20 matched controls. Furthermore, included studies were either case- control or nested case-control trials. The standard mean difference was used to estimate the observed differences between groups. We analyzed 20 studies, comparing 1068 cases to 2128 controls, in which 15 common indices were used to assess oxidative stress in individuals with prevalent cardiovascular disease in comparison to matched controls. Counter intuitively, the prevalence of cardiovascular disease was associated only with MDA concentrations (SMD = 1.60,95%CI 0.75 to 2.45). Further analysis shows that even this observation may result from publication and other biases. Conclusions: Unexpectedly, the commonly accepted paradigm associating cardiovascular disease to oxidative stress appears to be overestimated. ENGINEERING VASCULARIZED PANCREATIC ISLETS IN-VITRO

K. Francis1,2, N. Weinberg3, Y. Dor3, S. Levenberg1

1Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel, 2Biotechnology Interdisciplinary Unit, Technion-Israel Institute of Technology, Haifa, Israel, 3Department of Cellular Biochemistry and Human Genetics, Faculty of Medicine, Hebrew University of Jerusalem, Israel

Insulin-dependent diabetes mellitus (IDDM) is a chronic life threatening disease, resulting from the destruction of the insulin producing beta cells in the pancreatic islets of langerhans. Islet transplantation is considered a potentially curative treatment for type 1 diabetes. However, thus far transplantation has only enjoyed moderate success. Besides the shortage of available pancreatic tissue, tissue grafts that have been used rely on vascularization from the host to provide permanent engraftment. It is estimated that during the first few days after grafting more then 50% of the transplanted islets die as a result of insufficient perfusion. We have hypothesized that greater success in islet transplantation may be achieved through the use of primarily vascularized islets, reducing the period required for vascularization after transplantation. In this study, biodegradable polymeric scaffolds have been utilized to establish a three-dimensional co-culture system of endothelial cells and adult pancreatic islets. This model strives to mimic the natural anatomical context of pancreas vascularization and analyze the effect of this vascular bed on the growth, survival, and function of the tissue and beta cells in particular. Our results revealed distinct differences in pancreatic islet survival dependent upon their co-culture with endothelial cells. Significant differences have also been detected between pancreatic islets co-cultured with endothelial cells on 2D plates or 3D polymer scaffolds. We anticipate that this unique cell-culture model will give rise to a plethora of engineered constructs, which will serve as model systems for studying the effect of a pre- fabricated vascular network on in vivo vascularization, survival, and function of transplanted pancreatic explants. In the long term, this could provide an important tool for therapeutic transplantation of islets in type 1 diabetes. THERAPEUTIC ARTERIOGENESIS FOR HIND LIMB ISCHEMIA USING VEGF ACTIVATED BONE MARROW DERIVED MONOCYTES

M. Grunewald1,2, J. Vilar2, A. Itin1, A. Recalde2, M. Faroja3, J-S. Silvestre1

1Department of Molecular Biology, Hadassah Medical School, Ein Kerem, Jerusalem, Israel, 2INSERM, Hopital Lariboisiere, Paris, France, 3Department of Surgery, Hadassah Hospital, Ein Kerem, Jerusalem, Israel

Arteriogenesis describes the remodeling of small interconnecting arterial anastomoses with almost no net blood flow to large functional arteries. This collateralization process represents the major compensatory mechanism after arterial occlusions, since capillary sprouting can only partly enhance tissue perfusion. Diabetic patients commonly suffer from cardiovascular complications, including vascular diseases. In particular, collateralization is severely impaired leading to severe tissue ischemia, poor outcome following heart infarct and often non traumatic limb amputation. Monocytes have been shown to play a pivotal role during arteriogenesis. They enter the vessel wall and provide a number of cytokines important for remodeling and endothelial and smooth muscle cells proliferation. However, their function in diabetic settings is not completely understood. Recently, we have developed a transgenic mouse model of VEGF driven neovascularization in the liver. Using this system, we are able to isolate a population of VEGFactivated monocytes with proangiogenic activities. We propose in this study to harness these cells in a model of hind limb ischemia both in healthy and diabetic mice. Monocytes isolated from transgenic livers significantly improve arterialization and tissue perfusion in healthy mice as compared to bone marrow or wild type monocytes. Interestingly, these cells also improve the perfusion of the diabetic leg to a level comparable to the perfusion of normoglycemic tissue. These results suggest that diabetic impaired collateralization could be in part due to a defective activity of the monocytes. To further confirm these findings, we performed injection of monocytes isolated from diabetic transgenic mice. As expected these cells were not able to sustain arteriogenesis in healthy mice. Diabetic monocytes impaired activity could be attributed to defective recruitment from the bone marrow, to poor invasion of the vessel walls or to decreased proangiogenic activities. We are currently exploring those different possibilities. VASCULARIZATION OF ENGINEERED CARDIAC MUSCLE TISSUE FROM HUMAN EMBRYONIC STEM CELLS

A. Lesman1, M. Habib2, O. Caspi2, Y. Basevitch3, A. Gepstein2, G. Arbel2, I. Huber2, L. Gepstein2,4, S. Levenberg1

1Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel, 2Sohnis Family Research Laboratory and the Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel, 3Biotechnology Interdisciplinary Unit, Technion-Israel Institute of Technology, Haifa, Israel, 4Department of Cardiology, Rambam Medical Center, Haifa, Israel

Introduction: Tissue-engineering of the heart muscle represents a novel experimental therapeutic paradigm aiming to improve the failing heart function. However, this strategy has been hampered by the lack of sources for human cardiomyocytes and by the significant cell death following cell transplantation into the ischemic myocardium. It was shown that tissue survival may be enhanced in-vivo by the degree of graft vascularization which can be induced by co-culture with endothelial cells. Additionally, endothelial-cardiomyocyte interactions may also play a key role in promoting cell survival and proliferation. Human embryonic stem cells derived cardiomyocytes (hES-CM) are a promising cell source in future cell therapy strategies. Therefore, to promote in-vitro tissue vascularization we constructed multi- cellular scaffold in which hESC-CMs were combined with human umbilical vein endothelial cells (EC) and embryonic fibroblast (EmF). Materials and Methods: Various methods were used in order to assess the functionality of this engineered cardiac tissue including immunostaining, ultrastructural analysis, RT and real time PCR, pharmacological, and confocal laser calcium imaging studies. For assessing the functionality of blood vessels post-transplantation intraventricular fluorescent microspheres and Lectin-HPA were used. Results and Discussion: This study demonstrates, that such multi-cellular tissue engineering strategy enables the formation of highly vascularized human engineered cardiac tissue containing a condense blood vessels network. In-vitro assessment of the engineered cardiac tissue revealed the differentiation of EmF into mural cells. Mural cells enhanced stabilization, organization, proliferation and reduction in ECs mortality. Moreover, the presence of endothelial capillaries augmented cardiomyocyte proliferation. Implantation of the engineered cardiac tissue resulted in long-term stable graft. Maturation of cardiomyocytes within the scaffold was confirmed by immunostaining; revealing the presence of elongated and well–aligned striated cardiomyocytes. The formation of human and rat-derived vasculature within the scaffold was confirmed. Perfusion assessment suggests the functionality of these vessels and integration with host tissue vasculature. THE NOVEL NON TOXIC TELLURIUM COMPOUND SAS BLOCKS ANGIOGENESIS BY INHIBITION OF CELL MIGRATION AND ADHESION: ROLE OF INTERFERENCE WITH ALPHA V BETA 3 ACTIVITY

D. Makarovsky1, V. Indenbaum1, O. Sagi-Nigel1, A. Albeck2, S. Yosef2, M. Albeck2, M. Nomizu4, H. Kleinman3, B. Sredni1

1The Mina and Everard Goodman Faculty of Life Sciences, Safdie Institute for AIDS and Immunology Research, Bar-Ilan University, Ramat-Gan, Israel, 2Faculty of Chemistry, Bar-Ilan University, Ramat-Gan, Israel, 3National Institute of Dental and Cranio-Facial Research, National Institutes of Health, Bethesda, MD, USA, 4Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan

Angiogenesis plays a pivotal role in various diseases, including tumor growth and metastasis. A novel small molecule of Te(IV), octa-O-bis-(R,R)-Tartarate Ditellurane (SAS), is shown in the present study to have a direct anti-angiogenic activity in vivo and in vitro. The activity and stability of the SAS was higher than that of the well known Te(IV) compound AS101. This was expressed in increased inhibition of endothelial cell migration induced by angiogenic stimuli in various models, including Boyden chamber, “scratch” wound closure, and single cell analysis by time lapse photography. The Te(IV) chemistry of SAS allowed it to interact with specific thiols and selectively interfere with the biological activity of cysteine-proteases like papain. This activity was associated with the ability of SAS to diminish endothelial cell adhesion to the ECM proteins, fibronectin and the bioactive laminin-derived peptide, via blocking of alpha V beta 3 integrin binding. Furthermore, signaling events that follow integrin binding were substantially inhibited. More importantly, interference with alpha V beta 3 binding by SAS was associated with both its direct anti angiogenic property on endothelial cells and its direct effect on tumor cells. The integrated results emphasize the potential use of the tellurium non toxic molecule, SAS, as a new class of anti-angiogenic, anti-cancer compound. HORMONAL REGULATION OF VEGF-C IN OVARIAN CANCER

S. Sapoznik1, B. Cohen1, Y. Tzuman1, S. Ben-Dor2, M. Neeman1

1Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel, 2Department of Biological Services, Weizmann Institute of Science, Rehovot, Israel

Lymphangiogenesis typically occurs during embryonic development as well as during tumor formation. VEGFC was reported to play a central role in tumor lymphangiogenesis, inducing hyperplasia of lymphatic vessels and increasing metastasis to draining lymph nodes. Indeed, in many tumor types, including ovarian carcinoma, lymphatic capillaries could be detected, and the expression level of VEGFC correlated significantly with metastasis and poor survival. The role of gonadotropins in ovarian cancer progression, demonstrated previously in induction of tumor cell adhesion and expression of VEGFA, is consistent with the rise in incidence and severity of the disease in post-menopausal women. In the current study, we examined the influence of in vitro stimulation of ovarian cancer cell lines by gonadotropins on the expression of VEGFC and its novel transcriptional activator LEDGF. MLS and ES2 cell lines were exposed to LH and FSH for varying periods of time. Following stimulation, total RNA was extracted and the mRNA levels of VEGFC as well as its transcriptional activator LEDGF were examined by RT-PCR. We found that hormonal stimulation induces a significant increase in the mRNA levels of VEGFC and LEDGF. This transcriptional response may lead to enhanced tumor progression and metastasis through the creation of new lymphatic vessels. LEDGF was reported to have a natural antisense transcript. The mRNA levels of this antisense were examined in the same experimental setup. Interestingly, similar hormonal regulation was observed for the sense and the antisense forms of LEDGF. This finding suggests that the LEDGF antisense is functional and may influence the translation of the gene. Thus, gonadotropins may promote progression and metastasis of ovarian carcinoma by induction of VEGFC and this effect can be mediated through activation of LEDGF induced transcription. SEMAPHORIN-3C INHIBITS ANGIOGENESIS AND HINDERS TUMOR DEVELOPMENT

T. Lange, R. Zidon, O. Kessler, G. Neufeld

Cancer and Vascular Biology Research Center, Rappaport Research Institute in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Semaphorin-3F (sema3F) had been previously found to inhibit tumor angiogenesis. To determine if semaphorin-3C (sema3C) inhibits angiogenesis and tumor progression we expressed the sema3C cDNA in HEK293 cells. Sema3C was efficiently expressed but failed to be secreted from several types of cancer cells. Domain swapping experiments revealed that the signal sequence of sema3C is active suggesting that sema3C contains retention signals that inhibit secretion. Sema3C expression did not affect the proliferation or adhesion of HEK293 cells. Nevertheless, sema3C expressing HEK293 cells formed tumors at a significantly lower incidence and the tumors were much smaller than tumors developing from empty vector transfected cells. Furthermore, sema3C expressing cells implanted in bFGF containing matrigel inhibited bFGF induced angiogenesis. To force sema3C secretion from cultured cells we expressed in HEK293 cells a fusion protein in which alkaline- phosphatase (AP) is fused in frame to the N-terminal of sema3C. Cells expressing this fusion protein repelled endothelial cells, inhibited their proliferation and induced apoptosis of endothelial cells. Interestingly, AP-sema3C induced the collapse of the cytoskeleton of cells co-expressing np2 and plexin-A4 but not of cells co-expressing np2 and plexin-A1, indicating that the identity of plexins in association with neuropilins in target cells determines biological responses to sema3C. LYMPHATIC EXPANSION INDUCED BY ENDOTHELIAL AKT1 PRECEDES VASCULAR EXPANSION

K. Ziv1, V. Kalchenko2, K. Walsh3, L.E. Benjamin4, M. Neeman1

1Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel, 2Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel, 3Division of Cardiovascular Research, Boston University, Boston, MA, USA, 4Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

Akt signaling in endothelial cells regulates multiple critical steps in angiogenesis. We previously reported that overexpression of endothelial Akt1 resulted in a systemic increase in blood volume, elevated permeability and edema (1). The aim of this study was to investigate the effect of endothelial Akt1 signaling on dermal lymphatics. For this purpose we used transgenic mice in which constitutively active Akt1 (myrAkt1) is expressed by endothelial cells in a tetracycline regulated manner. tTA:VE-cadherin x TET:myrAkt1 double transgenic mice were generated by crossing TET:myrAkt1 mice with tTA:VE-cadherin mice. MyrAkt1 expression was suppressed by addition of tetracycline in the drinking water, and was induced for 3 days prior to the intravital microscopy experiments by the withdrawal of tetracycline. Visualization of the blood vessels and lymphatic of the mouse ear was done by combining dynamic light scattering imaging (DLSI) for detection of functional blood vessels and fluorescence lymphangiography (using dextran-FITC MW 500kDa, intradermal injection) for detection of the dermal microlymphatics. Activation of myrAkt1 expression in endothelial cells resulted in elevation of lymphatic vessels density, which preceded the expansion of blood vessels. These results suggest that the lymphatics respond rapidly to activation of endothelial Akt1, either directly or possibly indirectly as response to the elevation in interstitial pressure. This work was supported by the Minerva Foundation (to MN) References 1. Phung et al. Cancer Cell 10, 159–170, 2006