Session E. Structure, Properties and Functions of Proteins and Peptides

Lectures both tissues [3]; as well as the results of DNA and protein imaging. L5.1 References: 1. Binnig G et al. (1986) Ch Phys Rev Lett 56: 930–933. 2. Matyka K et al. (2007) J Mol Recognit 20: 524–530. Selected applications of AFM in biology: 3. Jastrzebska M et al. (2008) J Mat Sci: Mat Med 19: 249–256. from single molecules to tissues Antoni Ciszewski Institute of Experimental Physics, University of Wroclaw, Wrocław, Poland e-mail: Antoni Ciszewski

The areas of application of the atomic force microscopy (AFM) and related scanning probe microscopy (SPM) techniques are constantly growing. The atomic force microscope [1] and the devices operating on the same physical base have proven to be powerful tools for bio- logical studies. The variety of biological objects imaged using AFM includes single molecules, cells, tissues and biomaterials. The main advantage of AFM in biology is its ability to operate in liquids, that is under most physi- ological conditions. A lateral resolution of better than one nanometer can be achieved and a variety of samples with sizes ranging from a few nanometers up to several mi- crometers can be imaged. Applications include not only imaging but also measuring the magnitude of forces. The high sensitivity of AFM makes it possible to measure in- teractions between two opposing surfaces down to the single molecule level, allowing to explore the smallest molecules encompassing proteins, lipids, DNA, RNA and other nucleic acids, as well as much larger objects like hu- man’s platelets, viruses and living cells. The exploration includes specific and non-specific molecular interactions, which the biological processes are governed by. These are protein-protein, -substrate, antigen-antibody, re- ceptor-ligand interactions, drug-target associations, and many others. The AFM tool allows the visualization and real-time imaging of nucleation and crystallization of macromolecular crystals or processes involved in the cell living cycle. Herein, the physical base of AFM and main variants of this technique most frequently used in biology will be briefly discussed. These will be illustrated by selected re- sults of AFM studies, which concern both the molecular interaction as well as the imaging. Highlighted will be the methods, which allowed the critical forces that unfold sin- gle proteins bonds to be measured. Some results of AFM studies carried out at the Institute of Experimental Phys- ics of the University of Wroclaw will be also presented. These will include the results of imaging and studies of mechanical properties of collagen constituent in animal pericardium tissues [2]; the results of surface topography examination of normal human aortic valve and GA-stabi- lized porcine pericardium tissue, made in order to gain a comparative insight into the supramolecular structure of Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 81

L5.2 L5.3

Proteases of Staphylococcus aureus – from Puzzling hormone-binding proteins structure and function to practical application from plants and insects Adam Dubin, Grzegorz Dubin Mariusz Jaskolski Faculty of Biochemistry, Biophysics and Biotechnology, Department of Crystallography, Faculty of Chemistry, A. Jagiellonian University, Kraków, Poland Mickiewicz University and Center for Biocrystallographic e-mail: Adam Dubin Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland Proteases are of significant importance for the virulence e-mail: Mariusz Jaskolski of S. aureus – a human pathogen causing a wide range of diseases [1, 2]. Proteases of three different catalytic Transport of hydrophobic molecules in the aqueous media classes are found among the secreted proteins. They are requires specialized protein carriers. It is believed that the encoded by genes organized in four operons: ssp (en- binding of the cargo by its carrier protein should be tight coding the SspA serine and SspB cysteine protease and and specific. Our crystallographic studies of proteins that SspC cysteine protease inhibitor); scp (endoding the ScpA bind hydrophobic hormone molecules in different organ- cysteine protease and ScpB cysteine protease inhibitor); isms show a puzzling picture, suggesting that things are spl (the claster of six serine protease-like genes which more complicated than in a simple “ideal 1:1 fit” scenario. encode for putative proteases) and aur (encoding metal- trans-Zeatin (tZ) is a plant hormone from the cytokinin loprotease and protein of unknown function) [1]. We iso- group. We have crystallized mung bean Cytokinin-Specif- lated and characterized several native S. aureus secreted ic Binding Protein (CSBP) in complex with tZ and solved proteases. Using heterologous expression system we ob- the structure at atomic resolution. The CSBP fold consists tained recombinant proteins SspC and ScpB that function of a curved β-sheet, crossed by a long α-helix, with a large as endogenous and very specific inhibitors of cysteine hydrophobic tZ-binding cavity. The binding mode of the protease SspB and ScpA (staphopains), respectively 4 CSBP molecules in the asymmetric unit is very unusual. [3, 4]. To date, four members of homologous inhibitors In each case, one (Inner) tZ molecule is bound deep in constituting a novel family (staphostatins) have been de- the pocket in the same fashion. In three pockets, there is scribed [5]. Representatives were structurally character- another (Outer) ligand. In two cases it has the same orien- ized in detail by NMR and X-ray crystallography [6, 7]. tation but in one case it is flipped. Backsoaking removes Although staphopains belong to clan CA of papain-like the sole Inner ligand before the Outer zeatins move out. proteases, staphostatins inhibit exclusively the staphylo- The structure of the CSBP-tZ complex makes us wonder coccal and not other clan members tested. Un- which of the two tZ molecules is the ligand specifically like the previously known cysteine protease inhibitors, recognized by CSBP? Juvenile hormone (JH) is a highly the staphostatin polypeptide chain spans the active site of hydrophobic molecule found only in insects, where it the protease in a manner similar to the substrate. The in- regulates the complicated pattern of development. JH hibitor escapes cleavage due to the distinct deposition of is transported in the aqueous hemolymph by Juvenile the P1 glycine residue thus resembling “standard mecha- Hormone Binding Protein (JHBP) in 1:1 stiochiometry. nism” serine protease inhibitors. The crystal structure of JHBP from wax moth shows an From the six serine protease-like (spl) genes encomposed unusual fold consisting of a long α-helix wrapped almost in one operon — three ( SplA, B and C) have been already completely in a helically twisted β-sheet. The molecule efficiently expressed and characterized [8, 9].- Theen has two hydrophobic cavities, one at each pole. The ques- zymes show restricted substrate specificity similar to that tions are, which of the cavities is used for JH binding, and of the V8 protease and epidermolytic toxins. The mech- what is the role of the second pocket? The first question anism of unusual activation of SplB protease as well as can be answered with certainty because biochemical ex- practical application in recombinant protein technology periments clearly map the interacting JHBP fragments to will be discussed. the cavity near the N- and C-termini. The second question References: is more difficult because so far nobody has suspected that 1. Dubin G (2002) Biol Chem 383: 1075–1086. JHBP had two binding sites. It is of note that JHBP-like 2. Dubin G (2003) Acta Biochim Polon 50: 715–724. fold is found in some mammalian lipid-binding proteins, 3. Rzychon M et al. (2003) Mol Microbiol 49: 1051–1066. where it occurs in tandem. We may speculate that JHBP 4. Rzychon M et al. (2003) Protein Sci 12: 2252–2256. represents an ancestral fold capable of fulfilling the same 5. Dubin G et al. (2007) Biol Chem 388: 227–235. function as its evolutionarily more advanced relations. 6. Dubin G et al. (2003) Biochemistry 42: 13449–1346. 7. Dubin G et al. (2004) J Biomolecular NMR 28: 295–296. Both, CSBP and JHBP have hydrophobic ligand-binding 8. Popowicz G et al. (2006) J Mol Biol 358: 270–279. cavities formed between a curved β-sheet and an α-helix. 9. Dubin G et al. (2008) J Mol Biol 379: 343-356. This may be an ancient structural motif used by nature to bind hydrophobic molecules in aqueous media. 82 The Congress of Biochemistry and Cell Biology — Abstracts 2008

L5.4 L5.5

Macromolecules involved in juvenile hormone Mechanisms of actin filament binding protein traffic and its gene expression regulation by tropomyosins Marian Kochman Joanna Moraczewska Department of Biochemistry. Faculty of Chemistry, Wrocław Department of Experimental Biology, Kazimierz Wielki University of Technology, Wrocław, Poland University in Bydgoszcz, Bydgoszcz, Poland e-mail: Marian Kochman e-mail: Joanna Moraczewska

Juvenile hormone (JH), together with ecdysone, controls Actin filaments are a universal system present in muscle insect growth, development, and reproduction. In the in- and non-muscle cells implicated in contraction, cell mor- sect hemolymph over 99.8% of JH is bound to juvenile phology and various forms of cellular motility. Muscle hormone binding protein (JHBP). Previously, we demon- thin filaments and majority of non-muscle microfilaments strated that JHBP from the hemolymph of G. mellonella is a are complexed with a regulatory protein – tropomyosin single-chain, basic glycoprotein containing two disulfide (TM). Tropomyosin molecules form a coiled-coil dimer, bonds. The interaction between JH and JHBP results in a which binds as continuous strains on both sides of the profound conformational transition of the JH carrier, as filament via head-to-tail interactions. In skeletal muscle judged from changes in electrophoretic mobility, UV dif- each TM molecule is complexed with troponin, a protein ference spectra, sedimentation coefficients, and resistance which binds Ca2+ and activates muscle contraction. In to proteolysis. Recently, the 3D structure of this protein mammalian cells about forty TM isoforms are created by was resolved in cooperation with a team lead by Dr. Jas- alternative splicing from four genes and selection of alter- kolski. Sequencing the jhbp gene we found four introns. native transcription promotors. The first, third and fourth introns are in phase 1, whereas TM controls muscle contraction and motility of non-mus- the second is in phase 2. Six putative regulatory elements cle cells through regulation of the thin filament interac- [Hunchback, Heat shock factor binding element, Ultrabit- tions with myosin motors and actin-binding proteins. horax, Broad-Complex Z3, Elf-1 and Chorion factor 1/ul- Regulatory properties of TM isoforms are quantitatively traspiracle (CF1/Usp)] were found in the distal promoter and, in some cases, also qualitatively different, however of the jhbp gene. To date, there hasn’t been an experiment the structural basis for these differences is poorly under- done on the jhbp promoter function in JHBP expression. stood. The regulation of actin-myosin interactions is de- In this report we show proteins which form specific com- scribed by a classic steric blocking mechanism, however plexes with JHBP core promoter elements (TATA box, Inr many lines of evidence indicate that allosteric conforma- and DPE). Deletion of the jhbp promoter in the distal and tional changes in actin are also important in this process. core regulatory elements suggests that the jhbp promoter Actin site-directed mutagenesis is an approach used is is TATA- and Inr-driven. CF1/Usp elements exhibit inhib- several laboratories to understand specificity of interac- itory effects on jhbp gene expression. Footprinting analy- tions between TM and actin. The results obtained with ses indicated that the interaction between Usp and DNA actin carrying mutations within subdomains 2, 3 and 4 is dependent on recognition of the consensus sequence clearly demonstrate that TM affects actin functions not (GGGTCA) and on ionic interactions of several phosphate only by direct interactions with charged surface residues groups outside of this element. [1], but also through long-distance transitions [2, 3]. Lim- To shed some light on JHBP traffic in insect tissues, the ited proteolysis of actin within two regions involved in interaction of this carrier with other proteins was investi- forming intermonomer contact site, C-terminus in sub- gated. Our studies revealed the presence of JHBP in tra- domain 1 and D-loop in subdomain 2, show that the cheal epithelium and fat body cells in both membrane and integrity of the actin-actin interface is important for TM cytoplasmic sections. The interaction between JHBP and binding and regulation of actin-myosin interactions [4, 5]. membrane proteins occurs with saturation kinetics and Our recent work has also revealed that through allosteric is specific and reversible. The membrane ATP synthase interactions actin C-terminus is involved in TM isoforms was identified as a JHBP membrane binding protein. The recognition (unpublished work). interaction of JHBP with hemolymph apolipophorin, References: arylphorin and hexamerin was detected. These results 1. Cammarato A et al. (2005) J Mol Biol 347: 889–894. provide the first evidence of the physical interaction of 2. Korman VL, Tobacman LS (1999) J Biol Chem 274: 22191– JHBP with membrane and hemolymph proteins involved 22196. in JHBP molecule traffic. 3. Gerson JH, Bobkova E, Homsher E, Reisler E (1999) J Biol Chem 274: 17545–1755. Acknowledgement: 4. Moraczewska J et al. (2004) J Biol Chem 279: 31197–31204. This report was supported by a grant from Ministry of Education 5. Śliwińska M et al. (2008) Biophys J 94: 1341–1347. and Scientific Research 2 P04A 044 30. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 83

L5.6 L5.7

Proteins with FBAR/BAR domains and the Combinatorial chemistry as a tool of synthesis mechanism of generation of membrane and selection of serine proteinase substrates invagination during endocytosis Adam Lesner Elżbieta Wyroba Faculty of Chemistry, Gdańsk University, Gdańsk, Poland Department of Cell Biology, Nencki Institute of Experimental e-mail: Adam Lesner Biology PAS, Warszawa, Poland e-mail: Elżbieta Wyroba Combinatorial chemistry emerge in the 1988 when the hungarian scientist Arpad Furka described his methods called “More peptide by less labor”. Soon in the 90. of the last century the combinatorial chemistry enter into the blooming phase. Several methods of synthesis and further determination of active sequence (compounds) have been described. In this work the application of com- binatorial chemistry methods for synthesis and selection of chromogenic substrates of physiologically important members of serine protease will be discussed. Our previ- ous studies focused on the model enzymes like bovine β-trypsin and α-chymotrypsin and procedure of the solid phase synthesis of chromophore carrying peptides was established. The same approach we applied for selection of chromogenic substrates that were selectively and ef- ficiently hydrolyzed by human neutrophil serine protei- nases (NSP). This group of proteolytic enzymes consist of cathepsin G, neutrophil elastase and proteinase 3 is secreted by the activated neutrophiles and takes part in early immune response. Their uncontrolled action yields several pathology like tissue damage or severe inflamma- tion. Therefore monitoring of the activity of these pro- teases is crucial for the proper therapeutical treatment. The simplest method for determination of protease activ- ity is the use of synthetic substrates. They are also use- ful for characterization of the enzyme specificity. In this work we report selection of chromogenic and fluorogenic substrates of human serine neutrophil and mastocytes proteases applying combinatorial chemistry approach. Peptide libraries were synthesized by the portioning- mixing method. Deconvolution of synthesized libraries was performed using iterative approach in solution. 5- Amino-2-nitro benzoic acid attached to the C-termini of synthesized peptides served as a chromophore released upon the interaction of peptide with enzyme. Additional introduction of 7-methoxy-4-coumaryl acetic acid or 2- amino benzoic acid on α-amino groups of the chromog- enic substrates converted them into FRET displaying compounds. The most active substrates were subjected to further modification applying non-proteinogenic amino acids. As a result, one of the most selective substrates of these proteases, published to date were obtained. Acknowledgements: This work was supported by University of Gdańsk under grant BW-8000-5-0451-8. 84 The Congress of Biochemistry and Cell Biology — Abstracts 2008

Oral Presentations O5.2

O5.1 Conservative regions of vertebrate fructose 1,6-bisphosphatases and their Applications of atomic force microscopy in possible role in calcium binding protein investigations — some examples Marek Zarzycki Iwona Mróz Department of Molecular Physiology of Animals, Institute of Institute of Experimental Physics, University of Wroclaw, Zoology, University of Wroclaw, Wrocław, Poland Wrocław, Poland e-mail: Marek Zarzycki e-mail: Iwona Mróz Fructose-1,6-bisphosphatase (FBPase) catalyses the hy- Atomic force microscopy (AFM) is useful for direct visu- drolysis of fructose-1,6-bisphosphate in the presence of alization of single protein molecules and protein assem- divalent cations such as magnesium, manganese or zinc. blies. We discuss the experimental conditions necessary Two isoenzymes: muscle and liver specific have been dis- for obtaining protein images of the best quality, e.g. of the covered in mammalian tissues. The former participates highest spatial resolution. Typical and modified modes in glycogen synthesis from lactate (glyconeogenesis), the of imaging as well as AFM design, especially useful for latter is a regulatory enzyme involved in the gluconeo- protein observations (cryo-AFM), are presented. genesis. The kinetic properties of both isozymes are virtu- The process of sample preparation and protein imaging ally identical. The fundamental difference between them in liquids is demonstrated for enzymes: aldolase and concerns their sensitivity toward AMP inhibition: the FBPase, involved into the glyconeogenesis process. The muscle enzyme is ca. 100-fold stronger inhibited by AMP. proteins are immobilized on freshly cleaved mica and im- A proposed mechanism for the allosteric regulation of aged under two different buffered solutions. Small struc- catalysis involves three conformational states of the loop tural details (of about 5 nm) are clearly visible. 52–72 called: engaged, disengaged and disordered (Choe We also present a short overview of the AFM-based force et al., 1998, Biochemistry 37: 11441–11450). Recently it has spectroscopy technique that allows us to manipulate sin- been reported that the muscle FBPase, unlike the liver gle molecules. The technique is used, among others, to isozyme, is strongly inhibited by calcium ions. The de- investigate protein-protein (e.g. ligand-receptor) interac- termined I0.5 was ca 0.6 μM (Gizak et al., 2004, FEBS Lett tions. The measured forces are of piconewtons. In par- 576: 445–448). Calcium influences additionally the subcel- ticular, the “forced unfolding” experiments can be per- lular localization of FBPase and so regulates the glycone- formed to investigate the unfolding pathways of proteins ogenesis (Mamczur et al., 2005, FEBS Lett 579: 1607–1612]. or single domains. Moreover, these experiments demon- Employing site-directed mutagenesis I proved that Glu69 strate the relationship between the protein structure and within the loop 52–72 acts crucial role in calcium inhibi- its mechanical properties. tion (Zarzycki et al., 2007, FEBS Lett 581: 1347–1350). In the muscle enzyme the presence of two acidic residues (Asp68 and Glu69) interacting with calcium might stabilize the loop 52–72 in the disengaged conformation. The existence of two FBPase genes has been explained by the duplica- tion which took place 300 million years ago (Tillmann et al., 2002, Gene 291: 57–66). However it would not clear up the existence of two muscle and liver specific enzymes among fish. Comparring over 60 primary structures of known FBPases I found that the amino acid residue in the position 69 is significant. Among Vertebrates (includ- ing fish) in all known muscle FBPases it is occupied by acidic residue unlike the liver isoenzymes which contain exclusively glutamine in mammals or hydrophobic resi- dues in other organisms. Hypothetically, the single point mutation (Gln69Glu) was the crucial step in FBPase evo- lution enabling the regulation of glyconeogenesis in mus- cle cells by calcium ions.The crystalographic analysis and the site-directed mutagenesis are necessary to determine the muscle FBPase binding site of calcium. The study on the determination which additional a.a. residues are in- volved in calcium binding are in progress. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 85

O5.3 Posters

Tripeptidyl peptidase II participates in P5.1 degradation of defective ribosomal products Analysis of the Duffy glycoprotein N- Łukasz P. Biały, Izabela Młynarczuk-Biały glycans: use of lectins and glycosidases Depatrment of Histology and Embryology Center of Magdalena Grodecka, Marcin Biostructure Research, Medical Warsaw University, Czerwiński, Kazimiera Waśniowska Warszawa, Poland e-mail: Łukasz Biały L. Hirszfeld Institute of Immunology and Experimental Therapy PAS, Wrocław, Poland Defective ribosomal products (DRiPs) represent “short- e-mail: Magdalena Grodecka lived” cellular proteins and polypeptides that never at- tain native structure due to errors in translation or post- The Duffy Antigen/Receptor for Chemokines (DARC) is transnational processes. DRiPs may constitute upwards a seven-transmembrane glycoprotein carrying the Duffy of 30% of newly synthesized proteins and the majority of blood group antigen (Fy), acting also as a promiscuous them are degraded by proteasomes in ubiquitin- depend- chemokine receptor. The protein has three potential N- ent manner. However, some DRiPs representing severe glycosylation sites (Asn-X-Ser sequence) at amino acid missfolded proteins can be degraded without ubiquiti- residues Asn16, Asn27 and Asn33. Recently, using DARC nylation. Moreover, DRiPs are the major source of anti- mutants with deleted glycosylation sites, we have proven genic peptides presented by MHC class I molecules. that in the recombinant Duffy glycoprotein all three sites Tipeptidyl peptidase II (TPPII) is a N-terminal protease are occupied by N-linked oligosaccharide chains. Here that supplements action of the proteasome by degrading the mutated forms were used to elucidate the profile of proteasome-deliberated polypeptides. TPPII digests pref- DARC N-glycans and to investigate the possible impact of erentially peptides that contain more than 16 aminoacids their presence on chemokine binding. Four recombinant into tripeptides. It was proposed that TPPII can substitute Duffy forms (a wild-type form and three forms with only the action of proteasomes by cleaving proteins by its “en- one glycosylation site) were expressed in human eryth- doproteolitic” activity. Various studies have shown the roid K562 cells. Duffy glycoproteins were extracted from principal role of TPPII in the generation of some antigenic the cell lysates in the presence of 1% n-dodecyl-β-D-mal- peptides presented by MHC class I molecules. Thus, the toside (DDM) and purified by immobilized metal affinity aim oh this study was to quantify the involvement of TPPII chromatography using nickel and cobalt ions. Purified in DRiPs degradation. proteins were analyzed by lectin affinity chromatography The DRiP degradation rate was assessed in human leuke- and lectin blotting performed before and after desialyla- mia U937 cells by short radioactive 35S-Met pulse fol- tion. The glycoproteins were also treated with glycosidas- lowed by cycloheximide chase and TCA protein precipi- es and identified in Western blotting using anti-Fy mono- tation. Two inhibitors (AAF-cmk and butabindide) were clonal antibody. All four recombinant DARC forms were used to inhibit TPPII. The incorporated radioactivity was completely digested by N-glycosidase F, and in some de- measured by the gamma/beta-ray counter or SDS/PAGE gree by endoglycosidase H. In all cases strong binding and autoradiography. As proteasome inhibitor MG-132 with Datura stramonium agglutinin (DSA), Lens culinaris was used. agglutinin (LCA), wheat germ agglutinin (WGA) and The obtained data showed that both the proteasome and Sambucus nigra agglutinin (SNA) was observed, while TPPII are involved in DRiP degradation. Under action of Canavalla ensiformis agglutinin (ConA) shown only weak proteasome and TPPII inhibitors the degradation ratio binding. Our results indicate that DARC oligosaccharide of newly synthesized proteins was significantly slower. chains are most probably hybrid and complex type rich in Proteasome inhibition stabilized ca. 20–25% of newly N-acetyllactosamine (Galβ1-4GlcNAc) units presumably synthesized proteins after 30 min of cycloheximide pulse. terminated with sialic acid residues; presence of high- The stabilizing effect of both TPPII inhibitors was minor mannose type glycans is less probable. reaching ca. 8% of newly synthesized proteins. Tacking Acknowledgements: into account these data (and the 30%-DRiP rate observed This work was supported by grant No 2 P05A 018 30 from the by other investigators) it could be concluded that TPPII Ministry of Science and Higher Education of Poland. seems to be involved in degradation of ca. 25% of total DRiP-pool. In this study TPPII inhibitors failed to poten- tate stabilizing effect of proteasome inhibitors on DRiPs suggesting that TPPII acts downstream to proteasome in the same degradation pathway rather than substitutes the action of proteasomes in DRiP degradation. 86 The Congress of Biochemistry and Cell Biology — Abstracts 2008

P5.2 P5.3

Matrix metalloproteinase-7 and -26 of Pathological point mutations G8313A, T8316C the umbilical cord arteries, vein and and G8328A induce alternate structures Wharton’s jelly in preeclampsia of human mitochondrial tRNALys Zofia Galewska, Lech Romanowicz, Krzysztof Olszak1, Anna Przykorska2, Edward Bańkowski Marie Sissler3, Catherine Florentz3 Department of Medical Biochemistry, Medical University of 1Department of Plant Biochemistry, 2Department of Protein Białystok, Białystok, Poland Biosynthesis, Institute of Biochemistry and Biophysics PAS, e-mail: Zofia Galewska Warszawa, Poland; 3Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France Our previous paper demonstrated that preeclampsia – as- e-mail: Krzysztof Olszak sociated accumulation of collagen and proteoglycans in the umbilical cord tissues is a result of increased biosyn- A variety of point mutations have been found in the 22 thesis and decreased degradation of these components. genes for human mitochondrial (mt) tRNAs, as linked to Metalloproteinases are enzymes engaged in degradation severe neurodegenerative disorders [1, 2]. The gene of mt of collagen and protein cores of proteoglycans, including tRNALys is a hot spot with 14 mutations described. Three those which bind peptide growth factors. We used West- of these are located in a same structural domain of the ern Immunoblot method, immunoenzymatic assay (ELI- tRNA, the anticodon branch, and are correlated with neu- SA) and zymographic technique for detection of metal- rogastrointestinal encephalopathy (G8313A), myopathy, loproteinases. We found that the umbilical cord arteries, encephalopathy, lactic acidosis, and stroke (T8316C) and vein and Wharton’s jelly of control and preeclamptic new- encephalophaty (G8328A). While mutations G8313A and borns contained MMP-7 and MMP-26. The umbilical cord G8328A do strongly decrease the aminoacylation efficien- tissues of control and preeclamptic subjects contains both cy by the cognate lysyl-tRNA synthetase (5000 to 7000- enzymes in a free form and in form of complexes with fold), mutation T8316C has no effect on this function [3]. other extracellular matrix components. Furthermore, we Here, the possible effects of mutations on tRNA structure observed a distinct increase in the amount of MMP-7 in have been investigated. In vitro transcribed were preeclamptic umbilical cord vessel walls. No significant 5’-end labeled with [32P]-ATP and submitted to enzymat- differences between control and preeclamptic Wharton’s ic probes specific to single-stranded (nucleases RnI, ChSI, jelly were found. MMP-7 and MMP-26 could activate S1) or double stranded ( V1) domains. Acces- MMP-9 by their cleavage sites in pro-MMP-9. Our results sible were highlighted by autoradiography of suggest that the high activity of MMP-9 participates in a a denaturing polyacrylamide gel on which the fragments proteolytic release of peptide growth factors from their have been separated. All tested tRNALys (WT and mu- complexes with other extracellular matrix components, tants), besides folding into the classical tRNA cloverleaf which facilitate their interaction with membrane recep- can also form 2 other variants of secondary structures tors and stimulate cell division and extracellular matrix (also predicted by mfold program). Notably, WT tRNA biosynthesis in these cells. It may be one of the mecha- is mostly a 40% and 44% mixture of form I (cloverleaf) nism of extracellular matrix remodelling in the umbilical and form II (differing in the anticodon loop); 16% fold cord of preeclamptic newborns. also into form III (completely different from the classical form). Mutant G8316C mostly folds into form II (45%) but also form III (29%) and form I (26%). Mutant G8328A folds mostly into form II (56%) mixed with some form I molecules. Mutant G8313A forms a mixture of 3 types of molecules with a majority of form III (65%). These data confirm the high structural flexibility of -hu man mt tRNA transcripts and the existence of alternative conformations [4]. They also strongly suggest that the negative impact of mutations G8313A and G8328A on aminoacylation is indirect and due to an initial impact on tRNA folding and trapping into unfavorable structures not recognized by the synthetase. This is in-line with a general trend of structural incidence of pathology-related mt tRNA mutations [1, 2]. The molecular impact of mu- tation T8316C departs from this trend and needs further exploration. References: 1. Florentz C et al. (2003) CMLS 60: 1356–1375. 2.Wittenhagen S, Kelley O (2003)TIBS 28: 605–611. 3. Sissler M et al. (2004) RNA 10: 841–853. 4. Helm M et al. (1998) NAR 26: 1636–1643. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 87

P5.4 P5.5

Study of interaction of dendrimer Chitobiosidase activity in entomopathogenic – protein by fluorescence, capillary zone fungus Conidiobolus coronatus electrophoresis and Zeta potential methods — an assay optimization Szymon Sękowski1, Andrzej Kaźmierczak2, Emilia Włóka, Mieczysława I. Boguś Magdalena Przybyszewska3, Marian Institute of Parasitology PAS, Poland Zaborski3, Teresa Gabryelak1 e-mail: Emilia Włóka 1Department of General Biophysics, 2Department of Cytophysiology, University of Lodz, Lodz Poland, 3Institute Populations of insect pests can be limited by entomopath- of Polymer and Dye Technology, Technical University of Lodz, ogenic fungi due to their ability to digest insect cuticle. Lodz Poland Entomopathogenic fungus, Conidiobolus coronatus (Ento- e-mail: Szymon Sękowski mophthorales), disrupts insect cuticle by means of fungal hyphae mechanical pressure combined with the produc- Dendrimers are relatively a new class of highly branched, tion of cuticle degrading enzymes: proteases, endo- and three dimensional polymers. They possess many interest- exochitinases as well as lipases. Virulence efficiency of ing biological and biomedical properties. There have been most entomopathogenic fungi is correlated with the ac- attempts to use these polymers as drug or DNA carriers, tivity of proteases and chitinases. imaging factors and also as chelators for heavy metal ions. Very little information about chitinases including those The interaction between dendrimers and human serum from C. coronatus is available. Chitobiosidase (1,4-b-chi- albumin is very interesting. Changes in the native struc- tobiosidase) is exochitinase that hydrolyses chitin (major ture of protein give information about a potential toxicity component of insect cuticle) to chitobiose from non-re- of dendrimers. Provided that the effects of polymers on ducing ends of homopolymer. The aim of this study was the protein conformation is not significant, dendrimers to optimize chitobiosidase assay conditions in order to could be used as potential chelators for removing heavy determine role of this enzyme in insect pathogenesis re- metal ions from human body. In our research we aimed sulted from C. coronatus assault. at investigation of the interaction between G3.5 PAMAM Activity of C. coronatus chitobiosidase was measured dendrimers and human serum albumin (HSA). The fluo- fluorimetrically in mycelial homogenate towards 4- rescence, anisotropy, capillary zone electrophoresis and Methylumbelliferyl β-D-N-N’-diacetylchitobioside (Ex= Zeta potential measurements were applied in the experi- 360 nm, Em= 450 nm). Tested reaction volumes: 0.5 ml, 1 ments. We used G3.5 PAMAM dendrimer solution in PBS ml and 2 ml. Dose of mycelial homogenate ranged from (5–100 μM) and human serum albumin (5 μM) for fluo- 10 ml up to 50 ml. Activities of chitobiosidase were meas- rescence and Zeta potential study. For capillary zone elec- ured at 20oC (optimal temperature for C. coronatus culti- trophoresis the same dendrimer and protein solutions in vation) and 30oC (temperature suitable for growing in- borate buffer were used. The effect of this dendrimer on sect host, Galleria mellonella). Optimal pH was determined human serum albumin was determined on Perkin-Elmer after application of several 50 mM Tris/HCl buffer pH LS-50B spectrofluorimeter (UK), BioFocus Capillary Elec- levels ranging form 4 up to 11. Effect of substrate concen- trophoresis 3000 System (BioRad Laboratories, Munich, tration on enzyme activity was measured using different Germany) and Malvern Instruments Zetasizer 2000 (UK). substrate concentrations: 0.00125–0.04 mM. The results show that G3.5 PAMAM dendrimer interacts During the study it was observed that chitobiosidase with human serum albumin. A decrease in fluorescence demonstrates the highest activity in 1 ml of reaction vol- and an increase in fluorescence anisotropy give informa- ume. Optimal dose of mycelial homogenate was 30 ml tion about changes in the native structure of albumin. (0.0105 mg of fungal protein/ml). The optimal reaction About 13% decrease in fluorescence compared to control time was 25 minutes. Chitobiosidase showed higher ac- was observed at the highest concentration of dendrimer. tivity at 30oC than in 20oC (316.6 dF/min/mg and 98.55 The capillary zone electrophoresis showed that dendrim- dF/min/mg, respectively). Optimal pH was 7.0 and sub- er could form complex with HSA in molar ratio 17:1. The strate concentration 0.02 mM. Zeta-potential studies confirmed G3.5 binding with albu- This work shows considerable activity of chitobiosidase, min (dendrimer:protein molar ratio 12:1). On the basis which suggesting that this enzyme may play an essential of these preliminary studies we can conclude that G3.5 role in mycosis. PAMAM dendrimer possesses an ability to interact with Acknowledgements: human serum albumin. This effect probably results from This work was supported by the Ministry of Science and Higher electrostatic bonds between dendrimer and protein mol- Education, grant No. N303 027 31/0837. ecules. 88 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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The effect of phospholipids on enolase Analogues of bradykinin B2 receptor antagonist activity and conformation Dariusz Sobolewski1, Witold Neugebauer2, Maria Stasiuk1, Karolina Leś2, Arkadiusz Kozubek1 Jérôme Côté2, Simon Bélanger2, Fernand Gobeil Jr2, Bernard Lammek1, Adam Prahl1 1Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland; 1Faculty of Chemistry, University of Gdansk, Gdańsk, 2Department of Pharmaceutics, The School of Pharmacy, Poland; 2Department of Pharmacology, Faculty of Medicine, University of London, London, United Kingdom Sherbrooke University, Sherbrooke, Quebec, Canada e-mail: Maria Stasiuk e-mail: Dariusz Sobolewski

Enolase (EC 4.2.11) catalyzes the interconversion of 2- The most potent peptidic human bradykinin (BK) B2 re- phosphoglyceric acid (PGA) and phosphoenolpyruvate ceptor antagonist is HOE-140 (Icatibant). In this study we (PEP). Enolase from rabbit muscles is a dimer of 46-48 present the synthesis of nine new analogues of HOE-140 kD subunits, which each monomer contains a complete substituted at position 7, 8 or 9 with chosen d-amino acid active site with quite uniform kinetic properties. The pri- residues and/or nonproteinogenic amino acid residues, mary sequence of this enzyme contains three Trp moieties e.g. N-cyclohexylglycine (Nchg), 1-aminocyclohexane-1- (303, 306 and 367). carboxylic acid (Acc), octahydroindole-2-carboxylic acid The effect of free phospholipids form on enolase activity (Oic), piperidine-3-carboxylic acid (Nip) and 4-phenyl- and changes of protein conformation was studied. piperidine-4-carboxylic acid (Ppc). In the next nine pep- At the micromolar and milimolar range of concentration tides we combined the above mentioned modifications all studied phopholipids inhibit enolase from rabbit mus- with the placement of 1-adamantane acetic acid (Aaa) cles, except DPPC, which increases enzyme activity above at position 0. All new analogues were tested on human 15%. The order of inhibitory potency of tested lipids was umbilical vein for their antagonistic potency. Only three as follows: phosphatidylserine > phosphatidylcholine compounds containing Nchg8, Nchg9 or Ppc9 exhibited from egg yolk > sphingomyelin. noticeable antagonistic activities on human bradykinin Interactions between enolase and phospholipids were receptors thus still less potent then original HOE-140 measured by monitoring the changes in the emission sequence. Each of them with Aaa0 showed lower activ- spectra of tryptophan residues of enolase in the presence ity then parent analogues. Peptides: d-Arg-Arg-Pro- of phospholipids. It was shown that some of used lipids Hyp-Gly-Thi-Ser-d-Phe-Nip-Arg and Aaa-d-Arg-Arg- caused increase of fluorescence of tryptophan residues Pro-Hyp-Gly-Thi-Ser-d-Phe-Nip-Arg, although strongly of enolase (it were phosphatidylcholine from egg yolk, potent antagonists against BK-induced blood pressure sphingomyelin, dimiristoylphophatidylcholine, phos- lowering responses in rats, did not show noticeable an- phatidylserine and phosphatidylinositol). Only one of tagonistic activity on BK-induced contraction of the iso- tested lipids, octadecylamine, caused significant decrease lated human umbilical vein, a well-established B2R bio- of fluorescence intensity of the tryptophan residues of the assay system, suggesting a species dependent activity of enzyme. Phosphatidic acid exhibit biphasic effect on fluo- the compounds. rescence intensity of enolase, it decreases the fluorescence at lower concentration (smaller than 0.4 mmol/litr) and increases it at the concentration above 0.4 mmole/litr. The most active of all tested lipids was EPC and PS, which increase tryptophan residues fluorescence of 110 and 60% respectively. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 89

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Methyltransferase EcoVIII and its isospecific The influence of hypo- and hyperthyroidism on homologes can modify DNA at secondary sites nuclear, cytosolic and mitochondrial fractions of sialoglycoproteins in rabbit hepatocytes Ewa Wons-Karczyńska, Tadeusz Kaczorowski Ewa Nowosadzka, Stanisława Szymonik-Lesiuk, Department of Microbiology, University of Gdansk, Gdańsk, Jacek Kurzepa, Marta Stryjecka-Zimmer Poland e-mail: Ewa Wons-Karczyńska

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Anti-aggregation (chaperon) effects of MscS as a peptidoglycan synthesis modulator homocysteinylated and other modified β-caseins Wojciech Grajkowski1, Marcin Balcerzak2, Ehud Jarosław Zimny1, 2, Reza Yousefi1, Jean-Marc Y. Isacoff2, Andrzej Kubalski1, Piotr Koprowski1 Chobert1, Andrzej Guranowski2, Thomas Haertle1 1Department of Cell Biology, Nencki Institute of Experimental 1Institut National de la Recherche Agronomique, Biology PAS,Warszawa, Poland; 2Department of Molecular Biopolymères Interactions Assemblages, équipe Fonctions Cell Biology, University of California, Berkeley, USA et Interactions des Protéines Laitières, Nantes, France; e-mail: Marcin Balcerzak 2Department of Biochemistry and Biotechnology, University of Life Sciences, Poznań, Poland MscS is an E. coli mechanosensitive chanel with homologs e-mail: Jarosław Zimny found in other bacteria, archea and plants. Mechanosen- sitive channels in bacteria jettison osmolytes and E. coli Caseins are milk proteins that belong to the family of in- cells lacking them lyse under osmotic downshocks. MscS trinsically unstructured proteins. They are known for lin- is a homoheptamer and its crystal structure revealed ear and highly flexible structure, as well as for transient or presence of large cytoplasmic cage (Bass et al., 2002). We permanent binding to several different proteic partners. found previously that cytoplasmic domains of MscS are Caseins have been recently proposed to act as “molecular mobile upon gating (Koprowski & Kubalski, 2003). It sug- chaperones”, protecting in concentration dependent man- gests that they may undergo state-dependent interaction ner, a variety of proteins against thermal, chemical or UV with binging partners. Each subunit of the cytoplasmic light induced aggregation. β Casein (βCN) is a member domain of MscS (residues 171-266, Alpha/Beta Domain of this group, possessing a short N-terminal hydrophilic Of MscS, ABDOM) was found to bind FtsZ — homologue polar domain that carries essentially all the protein net of eukaryotic tubulins. FtsZ is a main component of cell charges and a large C-terminal hydrophobic domain that division apparatus which forms Z-rings and recruits plays a crucial function in the protein aggregation and other proteins involved in the synthesis of cell wall. FtsZ micellisation process. Thus the secondary structure of inhibition leads to cell filamentation (Bi & Lutkenhaus, this protein has a distinctly and highly amphiphilic char- 1993). We found that overexpression of ABDOM as well acter. Hence βCN is a potent emulsifier suitable for use in as truncated MscS channel (MscSΔ266-286) results in cell a variety of food products. elongation. ABDOM binding to FtsZ does not affect its as- In this study, different modified β caseins were used as sembly/disassembly properties in vitro. Additionally, pat- protective agents against thermal denaturation of horse tern of murein synthesis observed during overexpression alcohol dehydrogenase and chemical denaturation of of MscSΔ266-286 indicated formation of preseptal rings insulin; monitored by dithiotreitol dependent denatura- what taken together with simultaneous cell elongation tion of insulin and random aggregation of its subunits. suggest that Z-rings are not functional in cell division. The following βCNs were tested: recombinant wild type We propose that FtsZ interacts with MscS, via the chan- βCN (WT), recombinant βCN with insertion of cysteine nel’s cytoplasmic domain in certain conformation of C6 (WT C6) and cysteine C210 (WT C210), recombinant channel which in our model was mimicked by deletion bCNs in dimeric forms, homocysteinylated (βCN-Hcy) of 266-286 residues from MscS (MscSΔ266-286) or its cy- and homoserinylated (βCN-Hse). Their anti-aggregation toplasmic domain (ABDOM). We hypothesize that MscS activity was compared with the activity of native bovine non-channel function lies in transient arrest of cell divi- βCN. Among these species only homocysteinylated βCN sion and modulation of murein synthesis in conditions of exhibited higher anti-aggregation activity than native cell envelope stress. βCN; i.e. in the present of βCN-Hcy the tested proteins References: aggregated 2-fold more slowly than in the present of na- Bass RB et al. (2002) Science 298: 1582–1587. tive βCN. Wild type, WT C6 and WT C210 proteins were Bi E, Lutkenhaus J (1993) J Baceriol 17: 1118–1125. less efficient anti-aggregation agents than native βCN, Koprowski P, Kubalski A (2003) J Biol Chem 278: 11237–11245. whereas dimerised C6 βCN and dimerised C210 βCN Acknowledgments: showed activity that was comparable to the activity of na- This work was supported by Polish Scientific Network Mobilitas. tive βCN. βCN-Hse exhibited considerably lower activity pl and Polish Ministry of Science and Education (N301 3380 33). than the native βCN. Enhanced anti-aggregation activity P.K. is participant of the Human Frontier Science Program. of βCN-Hcy can be explained by increased amphiphilic- ity of βCN after its homocysteinylation. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 91

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203KKKGK207 sequence of muscle FBPase has Galleria mellonella larvae immunological critical role in nuclear targeting of the enzyme response to Bacillus thuringiensis kurstaki upon mild heat-shock conditions Agnieszka Gizak, Darek Rakus Patryk Kowalski, Teresa Jakubowicz, Iwona Wojda Department of Animal Molecular Physiology, Zoological Institute, Wroclaw University, Wrocław, Poland Department of Invertebrate Immunology, Institute of Biology, e-mail: Darek Rakus Maria Curie-Sklodowska University, Lublin, Poland e-mail: patryk kowalski Fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) ca- talyses the irreversible reaction of hydrolysis of fructose Bacillus thuringiensis kurstaki is an entomopathogenic bac- 1,6-bisphosphate to fructose 6-phosphate and inorganic terium, that kills studied model organism, Galleria mel- phosphate. Genetic and kinetics studies demonstrated lonella, using parasporal bodies-included toxins. Number that at least two distinct forms of FBPase are expressed in of humoral and cellular factors are produced by invaded vertebrates: the liver – which is the regulatory enzyme of host within several hours after infection. We exposed gluconeogenesis and the muscle – which participates in insects to elevated temperature, that was supposed to glycogen synthesis from carbohydrate precursors, but its stimulate their immunological system to more effective physiological role is not fully understood. Our previous combat against B. thuringiensis infection. Mechanism of studies revealed that in several cell lines (cardiomyocytes, such a stimulation may be based on newly expressed heat smooth muscle cells, myogenic progenitor cells) FBPase is shock proteins, or their proteolytically derived peptides present inside cells’ nuclei and that the nuclear localiza- activity. Increasing number of research focuses on heat tion of muscle FBPase seems to correlate with capability shock proteins (Hsp) role as applicable immune system of cells to divide. boosters. Experimental methods of cancer and other civi- Recently, using homology-based molecular modelling lization-related diseases treatment utilize heat shock pro- and computational methods we hypothesized that the teins. motif responsible for nuclear targeting of FBPase (Nucle- During our research, higher resistance to infection with ar Localization Signal — NLS) is the sequence KKKGK in B. thuringiensis kurstaki within insects exposed to elevated position 203-207, which is present in primary structures of temperature was observed. Ratio of survival was higher all known muscle FBPases.To define the role of KKKGK in heat-shocked animals in comparison to control group. motif in the nuclear import of the enzyme we have used Higher levels of humoral antimicrobial activities were ob- site-directed mutagenesis and constructed nuclear locali- served in parallel experiments, that could explain higher zation signal mutants harboring point mutation: K203/E, survival rates. In order to investigate the significance K204/E, K205/E and K207/E as well as quadruple mutant of Hsp’s in immunological response, we employed 17- with all lysines within the region 203-207 replaced with DMAG — specific inhibitor of Hsp90 – one of the mostly glutamic acid (4xKE) or with alanine (4xKA). These mu- represented heat shock proteins of Galleria mellonella. Sur- tants were expressed in E. coli, then isolated, purified to prisingly, we obtained increased levels of humoral antimi- homogeneity and labeled with FITC. Subsequently they crobial activities after inhibitor administration. The level were introduced into HL-1 cardiomyocytes using Proteo- of proteolytic activity that usually appears after Bacillus Juice reagent. The intracellular distribution of the mu- infection descended in the presence of inhibitor tants was checked and compared with the localization of wild-type (WT) muscle FBPase.Transfection of HL-1 cells with the mutants revealed that even a single amino acid change (K to E) within potential NLS motif results in sig- nificant decrease of nuclear accumulation of FBPase and that the most pronounced effect on cytoplasmic reten- tion of FBPase seems to exert mutation of K at position 204. Targeted mutations substituting all four lysines for alanines or glutamic acid in the KKKGK motif completely abolished its capacity to mediate nuclear import of the enzyme. These experiments demonstrated critical role of the KKKGK motif in the nuclear localization of FBPase, although functional significance of the enzyme presence in nucleus is yet to be clarified. 92 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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99mTc-HYNIC-TOC and 99mTc-HYNIC- Influence of Tavamin on the amino TATE – synthesis, labeling and biological acids metabolism in brain cortex with activity useful in medical diagnosis morphine withdrawal syndrome Barbara Janota, Renata Mikołajczak, Edyta Mikhail Kurbat, Vlodimer Lelevich Oonk, Robert Lipka, Elżbieta Jakubowska Department of Biochemistry, Grodno State Medical Institute of Atomic Energy Radioisotope Centre Polatom, University, Grodno, Belarus Otwock – Swierk, Poland e-mail: Kurbat Mikhail e-mail: Barbara Janota Several studies indicate that same amino acid, in particu- Somatostatin receptors have been identified in different lar, excitatory (glutamate and aspartate) and inhibitory kinds of tumours such as neuroendocrine tumours and (glycine and gamma-aminobutiric acid) participate in tumours of the central nervous system, breast, lung and the expression of opioid withdrawal behavior. However, lymphatic tissue. These observations have served as the their role in the morphine-induced alterations in brain biomolecular basis of the clinical use of radiolabelled so- metabolism completely is not known. The goal of the matostatin analogs that, at present, are of great interest present study was to assess changes of these amino acids in nuclear medicine for diagnostic and peptide receptor in the brain cortex of rats in dynamic of morphine with- radionuclide therapy applications. The natural soma- drawn (MW) and their correction by amino acids compo- tostatin was discovered in 1973 as cyclic 14 amino acid sition “Tavamin”. White inbred mail rats (initial weight peptide with a potent biological activity, mostly inhibi- 180–200 g) were used in this experiment. 1% solution of tory in a wide variety of organs. Somatostatin has a very morphine hydrochloride was administered chronically short biological half-life of several minutes, hampering i.p. in increases doses (from 10 mg/kg daily to 40 mg/kg sufficient receptor binding to allow adequate imaging. In daily) for 7 days. In next 7 days animals were not taken our laboratory we started synthesis of new 8-amino acid any drags and withdrawn syndrome were appeared. The analogs of somatostatin. These somatostatin derivatives: combined action of “Tavamin” (i.p. 500 mg/kg b.w. in 3 (I) Tyr3-Octreotide and Tyr3-Octreotate have a biological or 7 (II) days of MW) and withdrawal syndrome stabilize half-life of about 2 hours, D-Phe at the N-terminus and an concentration of glutamine (I), leucine (I), serine (I and II) amino acid or amino-alcohol at the C-terminal protects and methionine (I). Level of GABA was increased in all the molecule against degrading enzymes. experimental groups. Concentration of isoleucine (II) was Methods: HYNIC – Tyr3-octreotide (HYNIC – TOC) and increased as compared with intact rats as well as with HYNIC – Tyr3 – Octreotate (HYNIC – TATE) were synthe- animals under condition of MW duration 3 days. sized in our laboratory on solid phase using the Fmoc strat- egy. The overall yields were between 14% and 20%. Wet 99mTc-labelling of HYNIC-TOC and HYNIC-TATE were performed to optimize the amount and concentration of reagents, temperature and reaction time which was then transferred to HYNIC-TOC and HYNIC-TATE dry kit for- mulation. The kit contains two vials: the first vial: 20 mg

HYNIC-TOC or HYNIC-TATE, 40 mg SnCl2, 50 mg tricine, 10 mg mannitol, the second vial: 10 mg EDDA. Radiola- belling was carried out by the addition 1 ml of generator eluate (20–40 mCi) to kit and 0.5 ml EDDA followed by 30 min incubation at 80oC. Radiochemical purity of 99mTc- HYNIC-TOC and 99mTc-HYNIC-TATE, controlled by TLC and HPLC methods showed over 90% radiochemi- cal yield and percentage of unbound free 99mTc-pertech- netate as well as colloidal forms of 99mTc was in the range of 2–3%. The stability of these kits was determined for 1 year. Stability of the obtained kits allowed extensive clinical studies of 99mTc-HYNIC-TOC and 99mTc-HYNIC- TATE. Hynic-TOC and Hynic-TATE conjugates obtained in our laboratory were successfully labelled with Tc-99m with the yields over 90%. To make labelling procedure easier, dry kits were produced with Tricine and EDDA as co-ligands. The in vitro and in vivo features of the 99mTc- Hynic-Tyr3-octreotide and 99mTc-Hynic-Tyr3-octreotate confirmed their diagnostic potential. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 93

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Hemolimpha of endromis versicolora can prevent Structural and functional analysis ethanol-induced hyperhomocysteinaemia of Maf1, the general regulator of the RNA polymerase III transcription Yury E. Razvodovsky1, Yevgeny M. Doroschenko1, Aleksander V. Naumov 1, V. M. Sheibac1 Anna Gajda1,2, Damian Graczyk1, Christine Conesa2, Malgorzata Legowiak1, Marlena Grodno State Medical University, Grodna, Bielarus Maciak1, Olivier Lefebvre2, Magdalena Boguta1 e-mail: Razvodovsky Yury 1Department of Genetics, Institute of Biochemistry and Biophysics PAS, Warszawa, Poland; 2iBiTec-S, CEA, Gif sur Background: Homocysteine is a toxic sulfur-containing Yvette, France amino acid that in recent years has become the subject e-mail: Anna Gajda of great interest. Excessive alcohol consumption is asso- ciated with elevations in the plasma homocysteine con- During life yeast cell encounters several different situa- centration and hyperhomocysteinaemia may be an im- tions that demand a rapid adaptation of cellular meth- portant underlain factor of organ impairment in chronic abolism to changing life conditions. One of the first steps alcoholics. of the events leading cell to handle with an unexpected Aims: This study was designed to examine the effect of situation occures inhibition of the transcription depend- supplementation with hemolimpha of Endromis versicol- ent to RNA polymerase III (Pol III). The Maf1 protein was ora on plasma homocysteine level in experimental model identified in our laboratory as a negative regulator of of alcoholism. the Pol III apparatus in Saccharomyces cerevisiae yeast Method: The rats were randomized into three groups: cells. Maf1 plays a central role in coupling different sig- control group; alcohol treated group were administered nal transduction pathways to Pol III transcriptional ma- ethanol during one week according to Majchrowizc; the chinery. Secretory pathway defects, nutrient limitation, alcohol + hemolimpha of Endromis versicolora group re- DNA damage, low nutrients availability, cell growth rate, ceived a hemolimpha of Endromis versicolora intragastri- oxidative stress and chemical treatment with different cally concomitantly with ethanol administration. drugs influence repression of the Pol III transcription ac- Results: One week alcoholization according to Majchrow- cording to the Maf1 activity. Maf1 is a phosphoprotein izc was associated with marked increase plasma homo- that in favourable growth conditions is phosphorylated cysteine level. Hemolimpha of Endromis versicolora sup- and its presence is visible in cytoplasm. Different stress plementation, when co-administered with alcohol lead conditions, change to nonfermentable carbon source and to statistically significant decrease plasma homocysteine approaching stationary phase by cell cause dephosphor- level in comparison to alcohol treated group. ylation of Maf1 and its relocalization to the nucleus. De- Conclusion: The results of this study suggest that hyper- phosphorylated Maf1 interacts directly with N-terminal homocysteinaemia caused by alcohol consumption can region of the largest subunit of Pol III leading to repres- be prevented by administration of hemolimpha of En- sion of transcription. dromis versicolora. Nevertheless the exact sequence of events appearing dur- ing action of the Maf1 remains not clear. The Maf1 pro- tein is conserved through evolution, suggesting that the regulatory mechanisms involving Maf1 may be similar from yeast to man.We have created several mutants of the Maf1 in order to distinguish if the phosphorylation status affects localization of the Maf1 and if this phenomenon may occure either in cytoplasm or nucleus. We will define characteristics of mutated Maf1. Our approach concerns also link between structure and activity of Maf1. The fam- ily of eukaryotic Maf1 share highly conserved aminoacid sequence with easily recognizable two regions called A and BC domain. The function of each of the domain is un- known. With the 2H yeast system we were able to prove their physical interaction and define the minimal region of A domain involved. This interaction is supported by the screen for suppressor mutants of a single point muta- tion in A domain. We have found Maf1 mutants carry- ing additional mutations localized in the BC domain that suppress phenotype of the single point mutation in A domain. The result of our researches will be widely pre- sented on the poster. 94 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Inhibitors of allosteric human The effects of N-terminal part modification prostatic acid phosphatase of arginine vasopressin analogues with cis-1- amino-4-phenyl-cyclohexane carboxylic acid: a Magdalena Górny, Natalia Hutyra, highly potent oxytocin receptor antagonists Ewa Luchter-Wasylewska Anna Kwiatkowska1, Dariusz Sobolewski1, Chair of Medical Biochemistry, Jagiellonian University Małgorzata Śleszyńska1, Jiřina Slaninovà2, Medical College, Kraków, Poland Bernard Lammek1, Adam Prahl1 e-mail: Magdalena Gorny 1Faculty of Chemistry, University of Gdansk, Gdańsk, Poland; Human prostatic acid phosphatase (PAP), synthesised 2Institute of Organic Chemistry and Biochemistry, Academy and secreted by a prostate gland, is built of two identi- of Sciences of the Czech Republic, Prague, Czech Republic cal subunits. The monomeric subunit is composed of e-mail: Adam Prahl two domains of various size. The active center, with his12 covalently phosphorylated during the reaction course, is Arginine vasopressin (AVP) is a cyclic nonapeptide with located in a large open cleft between domains. PAP ca- multiple functions. The main peripheral physiological talyses hydrolysis of many phosphoesters (phosphosug- roles of AVP are the regulation of water balance, the con- ars, nucleotides, phospholipids and phosphoproteins on trol of blood pressure, and the release of adrenocortico- phosphoserine, phosphothreonine, and phosphotyrosine tropin hormone (ACTH). Moreover, AVP also exhibits to residues). PAP also displays phosphotransferase and some extent typical oxytocin (OT, a closely related neuro- peptidase catalytic activities. PAP dephosphorylates re- hypophyseal peptide) activities such as the galactogogic ceptor cErbB2 in prostate cells but LPA and semenogelins and the uterotonic effects.Many of vasopressin agonists in a seminal fluid. Additionally PAP proteolytically cleav- and antagonists have been designed and synthesized in ages semenogelins. Human prostatic acid phosphatase the course of extensive investigation of structure – activ- exhibits positive cooperativity in substrate binding. The ity relationship. We decided to check how substitution of substrate saturation curves are sigmoidal, thus the sub- position 2 with bulky cis-1-amino-4-phenyl-cyclohexane strates are positive homotropic effectors (activators) of carboxylic acid (cis-Apc) would reflect in the values of PAP and PAP belongs to the regulatory enzymes. The ex- biological potency of the analogues. All the synthesized tent of cooperativity grows when enzyme concentration peptides have the following structures: [cis-Apc2]AVP is increased.The studies on the inhibition of phenyl phos- (I), [Mpa1,cis-Apc2]AVP (II), [cis-Apc2,Val4]AVP (III), phate (FP) hydrolysis, catalysed by human prostatic acid [Mpa1,cis-Apc2,Val4]AVP (IV). The activities of the new phosphatase, have been performed where two inhibitors: analogues were determined in the in vitro rat uterotonic vanadate (substrate analogue) and L(+)-tartrate (tran- test in the absence of magnesium ions, the rat pressor test, sient-state analogue) have been used at selected enzyme and in the antidiuretic assay the conscious rats were used. concentrations (10, 25 and 50 nM). The experimental data Compounds (I-III) modified at position 2 with cis-Apc have been fitted to the Hill rate equation in order to deter- were highly potent oxytocin receptor antagonists. Addi- mine the values of catalytic constant (kcat), as well as half tionally, all four peptides exhibited moderate antidiuretic saturation constant (K0.5) and Hill cooperation coefficient activities with prolonged action. The results presented (h).L(+)-tartrate and sodium vanadate have been found here confirm previous knowledge about the significant to be non-linear competitive inhibitors of PAP-catalysed role of position 2 in AVP sequence on pharmacological hydrolysis of FP. In conclusion, when concentration of properties. Summing up, our studies provide new, use- inhibitors is increased, the values of half saturation con- ful information about structure-activity relationships stants (K0.5) rise but the values of turnover number (kcat) and open up new possibilities for designing potent OT remain constant. The Hill cooperation coefficient (h) is antagonists. decreased when L(+)-tartrate concentration is increased, thus L(+)-tartrate diminishes the cooperative character of PAP. Sodium vanadate, at growing concentration, does not change the cooperativity in substrate binding exhib- ited by PAP. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 95

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Protein oxidative modification: iodination Analogues of trypsin inhibitor SFTI- 1 modified in P position by non-natural Liliya I. Nadolnik, Darya A. Goreva, Sergey 1 aromatic amino acid residues V. Lupachyk, Sergey S. Chumachenko Anna Łęgowska, Dawid Dębowski, Department of Bioregulators, Institute of Pharmacology and Adam Lesner, Krzysztof Rolka Biochemistry of the National Academy of Sciences of Belarus, Grodno, Belarus Faculty of Chemistry, University of Gdansk, Gdańsk, Poland e-mail: Liliya Nadolnik e-mail: Anna Łęgowska

Oxidative stress induces protein damage and modifica- Serine proteinases are widely distributed in nature and tion characterized by oxidation of thiol groups, nitra- are responsible for many physiological processes. Their tion of tyrosine radicals, aggregation and fragmentation uncontrolled activity may be dangerous for the organism of proteins and impairment of their functional activity. and cause several critical pathological conditions. There- Thyroglobulin (TG) is the basic iodinated protein in the fore, serine proteinase inhibitors which help to control ac- thyroid, a matrix for synthesis of thyroid hormones, their tivity of these enzymes are a promising class of therapeu- storage and dosed release. Aim: to study the effect of oxi- tic agents. In 1999 Luckettet al. [1] isolated from sunflower dative stress on the thyroid content of iodinated proteins, seeds the trypsin inhibitor SFTI-1, the smallest one among the extent of their iodination and spectral characteristics. the most potent inhibitors of the Bowman-Birk family. Experimental studies on Wistar rats show that excess io- SFTI-1 is a bicyclic peptide and its primary structure is dide intake as well as exposure to stress and external -ir- shown bellow: &1Gly-Arg-Cys(&2)-Thr-Lys-Ser-Ile-Pro- 2 1 ’ radiation increase the extent of TG iodination. Pro-Ile-Cys(& )-Phe-Pro-Asp& The reactive site P1-P1 of Highly purified TG was isolated from thyroids of control the SFTI-1 inhibitor is located between residues Lys5 and rats, those treated with high doses of KI and rats irradi- Ser6. Our group has already reported [2], that replacing ated in combination with administration of 10 diurnal the Lys residue in position P1 by l-phenylalanine yielded doses of KI. Thyroids from controls demonstrated pro- a highly potent chymotrypsin inhibitor with the value of 10 –1 nounced heterogeneity of iodinated proteins which en- association equilibrium constant (Ka) 10 M . Moreover, hanced during increased iodine consumption and under we have shown that among the chromogenic substrates irradiation. The extents of thyroid iodination amounted modified in position P1 by derivatives of Phe, the highest to:1.1 μg/mg protein for controls, 2.9 μg/mg protein for activity as expressed by specificity parameter (Kcat/Km) animals treated with 10 daily doses of KI and 5.4 μg/mg displayed the one with p-nitrophenylalanine (Phe(NO2) protein for irradiated rats treated with 10 KI daily doses. [3]. Taking into consideration our previous results, we Modified TG was characterized by a shift in absorption have designed and synthesized series of monocyclic ana- maximum to a more long wave region and by appearance logues of SFTI-1 with non-natural aromatic amino acid of an additional absorption arm in the region of 300–370 residues introduced in position P1. Our aim of this work nm. Under the action of ultrasound during 15 min in the was to select peptidomimetic, potent proteinase inhibi- presence of potassium iodide, the extent of iodination of tors. We expect that similarly to the recently published re- TG from control rats increased up to 9.9 μg/mg protein. sults the monocyclic peptomeric analogues [4] should be Experimental evidence has been obtained both in vivo and also proteolytic resistant.The peptides were synthesized in vitro for probability of nonenzymatic oxidative iodina- manually by solid-phase method using Fmoc chemistry. tion of TG. Modification of the key thyroid protein can To evaluate the inhibitory activity of the compounds syn- impair thyroid hormone biosynthesis. Taking into con- thesized, their values of Ka with bovine α-chymotrypsin sideration the fact that TG is the main thyroid antigen, we and cathepsin G were determined along with hydrolysis can suggest that oxidative modification of TG may induce rates (khyd). development of autoimmune thyroiditis. References: 1. Luckett S, Santiago Garcia R, Barker JJ, Konarev AV, Shewery PR, Clarke AR, Brady RL (1999) J Mol Biol 290: 5252. 2. Zabłotna E, Jaśkiewicz A, Łęgowska A, Lesner A, Rolka K (2007) J Pept Sci 13 7493. 3. Wysocka M, Łęgowska A, Bulak E, Jaśkiewicz A, Miecznikows- ka H, Lesner A, Rolka K (2007) Mol Divers 11: 934. 4. Łęgowska A, Bulak E, Wysocka M, Jaśkiewicz A, Lesner A, Dębowski D, Rolka K (2008) Bioorg Med Chem Apr 1.

Acknowledgements: This work was supported by the University of Gdańsk (BW/8000- 5-0125-8). 96 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Distinct roles of AnxA6 isoforms in bone Involvement of the Trametes versicolor mineralization by osteoblast-like Saos-2 cells proteasome in metabolism and aging Agnieszka Strzelecka-Kiliszek1, Cyril Magdalena Staszczak, Dorota Thouverey2, Rene Buchet2, Slawomir Pikula1 Jańczyk, Mirosław Śmietański 1Department of Biochemistry, Nencki Institute of Department of Biochemistry, Maria-Curie Skłodowska Experimental Biology PAS, Warszawa, Poland; 2University University, Lublin, Poland Lyon 1, ICBMS CNRS UMR, Villeurbanne, France e-mail: Magdalena Staszczak [email protected]> The ubiquitin-proteasome system represents the major Bone is a dynamic form of connective tissue composed of pathway for intracellular proteolysis in eukaryotes and three cell types (osteoblasts, osteocytes and osteoclasts), serves to control many important proteins including key extracellular matrix providing tensile strength and hy- metabolic enzymes, cyclins and transcription factors. The droxyapatite giving mechanical resistance. Osteoblasts efficiency of quality control in protein turnover declines and osteocytes produce matrix proteins and release ma- with age. Proteins tagged with a polyubiquitin chain trix vesicles (MVs) where the initial steps of mineraliza- are recognized and degraded by the 26S proteasome, an tion occur. Little is known about the mechanisms regulat- approximately 2.5-MDa ATP-dependent multisubunit ing mineralization. It has been reported that the release of complex. The 26S proteasome is assembled from the 20S MVs is correlated with changes in cellular actin distribu- proteasome and one or two 19S regulatory complexes. tion and regulated by Src phosphorylation. In addition, The 19S regulatory complex confers ubiquitin and ATP annexins, a family of Ca2+- and phospholipid-binding dependence, and the 20S proteasome provides the pro- proteins, were suggested to be involved in the Ca2+ home- teolytic activities. The 20S proteasome is composed of ostasis of mineralizing cells and the Ca2+ influx into Vs.M four stacked 7-membered rings, with two inner rings of We selected osteoblast-like Saos-2 cells, spontaneously re- β-type subunits and two outer rings of α-type subunits. leasing MVs, as a model to determine the roles of AnxA6 Each β-ring contains three distinct proteolytically active isoforms, actin network and Src phosphorylation in bone sites, which are classified as: ‘chymotrypsin-like’ (CHTL), mineralization. In this respect, the transfection of Saos- ‘trypsin-like’ (TL), and ‘caspase-like’ (peptidylglutamyl- 2 cells with EGFP-AnxA6 isoform cDNA was performed peptide hydrolyzing, PGPH) as defined by cleavage after and the mineralization process stimulated with ascorbic hydrophobic, basic and acidic amino acids, respectively. acid and b-glycerophosphate was monitored. Calcium In our previous studies, 26S proteasomes have been sepa- nodule detection by Alizarin Red-S (AR-S) staining and rated from mycelial extracts of the ligninolytic fungus T. phase contrast showed distinct influence of AnxA6 isofor- versicolor. Moreover, we have recently demonstrated the ms on cellular morphology and mineral formation. After involvement of the proteasome-mediated pathway in the stimulation, AnxA6-2-overexpressing cells were round regulation of laccase activity in T. versicolor upon nutrient in shape and surrounded by a high amount of mineral, deprivation and in response to Cd2+ exposure. Laccase, whereas AnxA6-1-overexpressing cells had fibroblast- a major ligninolytic enzyme of this fungus, is biotechno- like shape and released MVs with mineral phase inside. logically important due to its capacity to degrade a broad Spectrophotometric determination of AR-S concentration range of diverse aromatic pollutants. Secretion of laccase confirmed that AnxA6 isoforms are responsible for differ- can be enhanced by a number of environmental stimuli, ent phases of mineralization. After stimulation, AnxA6- including certain aromatic compounds. Production of 2-transfected cells produced 3.2-fold higher amount of laccase is thought to be regulated mostly at the transcrip- mineral within 3 days whereas 1.3-fold lower amount tional level. of mineral within 6 days than AnxA6-1-transfected cells. In the present study we investigated effects of several Immunofluorescence microscopy analysis of AnxA6 dis- phenolic compounds on laccase activity levels in T. ver- tribution revealed an enrichment of both AnxA6 isoforms sicolor cultures in the presence of the proteasome inhibi- in plasma membrane-bound vesicles in cells stimulated tor, MG 132 (Z-Leu-Leu-Leu-al). Our results indicate that for mineralization. In the case of stimulated cells over- inhibition of proteasome function significantly affects expressing AnxA6-1, F-actin was localized in the focal laccase activity in cultures supplemented with potential contacts which rearrangement is necessary for MV bio- laccase inducers. We also investigated proteasome activi- genesis and/or mineral formation, whereas in the case of ties and assembly during aging of T. versicolor. CHTL, TL, AnxA6-2-expressing cells, F-actin accumulated under the and PGPH activities were examined by monitoring cleav- membrane of released MVs containing minerals. We also age of three different fluorogenic peptides. Proteasome demonstrated that cytochalasin D (inhibitor of actin po- assembly was detected by native PAGE and fluorogenic lymerization) and PP2 (inhibitor of Src phosphorylation) peptide overlay. We found age-dependent changes in enhanced the AnxA6 isoforms effects on mineralization proteasome assembly and activities. process. Acknowledgements: This work was supported by a grant N301 025 32-1120 from PM- SHE. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 97

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Penetration of the selected protein and The influence of prolactin upon bones peptide substances through pericardium mineral density (bmd) and some biochemical markers of rat females after ovariectomy Barbara Dolińska, Florian Ryszka, Anna Pawlik-Gałczyńska, Janusz Pluta Florian Ryszka, Aleksandra Suszka-Świtek, Barbara Dolińska Medical University of Silesia, Sosnowiec, Poland; ”Biochefa” Pharmaceutical Research and Production Plant, Sosnowiec, ”Biochefa” Pharmaceutical Research and Poland; Medical University of Wroclaw, Wrocław, Poland Production Plant, Sosnowiec, Poland; Medical e-mail: Barbara Dolińska University of Silesia, Katowice, Poland

Analysis of penetration of such substances through bio- e-mail: Florian Ryszka logical membranes shall allow, among others, for deter- mination of their possible application in medicine. Peri- The model of post menopause osteoporosis was used cardium in defrosted state has been used for examination in the research. Starting 107 days after ovariectomy purposes. Nowadays, the biotechnological methods us- the animals were administered 1.0 I.U. of prolactin/kg ing natural membranes are more and more often used of body mass. or 1.0 I.U. calcitonin/kg of body mass for scientific researches purposes. The above results from subcutaneous during seven days. in one dose a day. improvement of these methods. In vitro model has been The medicament was administered again 80 days af- used to examine penetration of protein and peptide sub- ter the first administration. After 194 days the animals stances through pericardium. Penetration of the selected were put to sleep, their sample bloods were taken and protein and peptide hormones through pericardium have both lumbar and femoral bones (L2–L4) were prepa- been analyzed. Results of the penetration of the selected rated. The obtained material underwent densitometric substances presentation in table 1. estimation of bones mineral density. With rat females after ovariectomy loss of bone tissue were observed Substances Total Entire pen- Critical Critical Availabil- Ava- only in lumbar bone. Applying prolactin to rat fe- quan- etration concen- time — ity AUC bility males after ovariectomy caused increase of bone min-

tity that process tration tk [h] [0–30 h] level eral density to BMD value equal to the control group permeated rate — — C [μg/ml/h-1] –EBA k where the ovaries were maintained (healthy control -1 in time V [μg/h ] [ug/ml] [%] group). The results presentation in Table 1. 0–30 h [%]

GnRH 52.80 35.20 252.20 2.18 2383.04 84.05 Table 1. Lumbar and femoral bone density – BMD (g/cm2) Calcitonin 103.57 56.50 506.10 2.40 5488.41 193.58 Cortico- 97.07 64.71 281.50 2.37 5862.14 206.76 Group Parameters trophin Prolactin 29.51 19.67 175.10 4.00 1347.82 47.54 Lumbar bone Femoral bone Lutropin 51.72 34.48 234.40 2.90 2269.07 80.03 SHAM control – females with 0.239±0.007 0.213±0.010 Insulin 103.47 68.98 750.90 3.76 2835.23 100.00 maintained ovaries Tryp- 66.09 44.06 401.70 4.57 2862.81 100.97 tophan OVX control – females with ex- 0.236±0.010 ** 0.165±0.014 Albumin 35.89 23.93 100.30 2.50 2654.50 93.63 tracted ovaries Lysozyme 16.37 10.91 33.40 1.92 1083.04 38.20 OVX + Prolactin 0.239±0.014 ns/?? 0.212±0.028

OVX + Calcitonin 0.234±0.007 ns/?? 0.210±0.010 ns/?? The currently obtained results suggest that despite of sig- Statistical significance according to the SHAM control: **P<0.01; Instatistical signifi- nificant differences in structure and physical and chemical cance in relation to OVX control: ??P<0.01; ns – no significance properties of the examined substances, they are all able to penetrate through the pericardium. Penetration process The similar results were observed after applying calcitonin. of these substances is related probably to their systemic Decrease of osteocalcine concentration, activity of alkaline effect. The above may constitute for further researches on phosphatase isoenzyme (BAL) and acid one (TRAP) was their application in medicine. It has been recently stated stated as well. Results are presented in Table 2. that many hormones shows wide spectrum of systemic and therapeutic effects. Therefore protein and peptide substances have a great opportunity for their novel ap- plication in medicine. Acknowledgements: Scientific research supported from the 2006–2008 science funds as research project No. N405 019 31/0875. 98 The Congress of Biochemistry and Cell Biology — Abstracts 2008

Table 2. Osteocalcin concentration, BALP and TRAP activity P5.26 in serum of the females of the groups: SHAM, OVX, OVX + Prolactin, OVX + Calcitonin Prolactin (PRL) slows the release of Group Parameters aminotransferases from an isolated pig’s liver Osteocalcin (μg/l) BALP (U/l) TRAP (U/l) SHAM control 3.98±0.97 122.25±19.95 7.66±2.33 Florian Ryszka, Barbara Dolińska, Grzegorz — females with Budziński, Lech Cierpka, Zdzisław Smorąg, Aneta maintained ovaries Ostróżka-Cieślik, Artur Caban, Beata Szulc-Musioł OVX control – fe- 5.95±0.25 * 162.50±17.60 * 17.51±3.80 *** males with extracted Medical University of Silesia, Katowice, Poland; “Biochefa” ovaries Pharamceutical Research and Production Plant, Sosnowiec, OVX + Prolactin 4.52±0.35 ns/? 101.03±23.61 ns/? 8.75±3.35 ns/? Poland OVX + Calcitonin 4.93±0.46 ns/? 113.58±17.83 ns/? 8.16±2.19 ns/? e-mail: Aneta Ostróżka-Cieślik

Statistical significance according to the SHAM control: *P<0.05; ?P<0.05 statistical sig- This study aimed at determining the effect of prolactin nificance in relation to OVX control: *P<0.05 ns – no significance (PRL) on the release of asparaginate aminotransferase Acknowledgements: (AspAT) and alanine aminotransferase (ALAT) from a Scientific research supported from the ”Biochefa” Pharmaceuti- pig’s liver during its 24-hour preservation. The aim was cal Research and Production Plant. achieved by determining the release rate and amount of the enzymes released from an isolated liver to pres- ervation solution (HTK) with or without an addition of PRL. The pig’s livers isolated ex vivo were preserved for 24 hour in HTK solution with and without an addition of 100 I.U. PRL. The effectiveness of preservation was re- flected by the amounts of enzymes — AspAT and ALAT – released to the preservation solution. The amount of re- leased enzymes increased in the solutions of both groups. However, much less was released to the solution with PRL. The amount of released ALAT increased from 8.0 U/l + 1.4 to 35.0 U/l + 0.1 in the solution without PRL. The amount of ALAT released to the solution with PRL in- creased from 7.0 U/l + 0.1 do 20.5 U/l + 4.9 (P=0.051). Simi- lar release from hepatocytes was observed for AspAT. In the case of the liquid without PRL, the amount of released enzyme increased from 286.5 U/l + 2.1 to 619.0 U/l + 25.5, and to the solution with PRL – from 254.5 U/l + 17.0 to 325.5 U/l + 82.7 (P=0.041). ALAT was released much fast- er to the solution without PRL (k=-0.1230 [U/l/h-1]), and slower to the solution with PRL (k=-0.0895 [U/l/h-1]). By adding PRL to the HTK solution, the rate of ALAT release was decreased by the factor of 1.4. The time of secreting 10% of the enzyme was 52 minutes in the case of solu- tion without PRL and 72 minutes – for the solution with PRL. Similar results were obtained when the release rate of AspAT was analysed. AspAT was released faster to the solution without PRL (k=-0.0642 [U/l/h-1]), and slower to the solution with PRL (k=-0.0205 [U/l/h-1]). The time of se- creting 10% of AspAT h to the solution without PRL was 1.64 and to the solution with PRL – 5.12 h. The transfer of the enzymes from hepatocytes to preservation solution is a proof of hepatocytes cytolysis, which results in liver de- struction.The inhibited release of aminotransferases from hepatocytes is a proof of a lower damage to the liver’s integrity during its preservation. This may be an indica- tion of hepato-protective properties of PRL. These results suggest, that PRL can protect liver cells. Acknowledgements: Scientific research supported from the 2006–2008 science funds as research project No. Nr N 405 002 31/0124. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 99

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Influence of kinetin and putrescine on human Fhit protein, known as dinucleoside skin fibroblasts growth, proliferation, total triphosphatase, behaves also as adenylylsulfatase protein content and collagen biosynthesis and nucleoside phosphoramidase Agata Jabłońska-Trypuć, Romuald Andrzej Guranowski1, Anna M. Czerpak, Walentyn Pankiewicz1 Wojdyła1, Małgorzata Pietrowska- Borek2, Paweł Bieganowski3 1Białystok Institute of Cosmetology and Health Care, Białystok, Poland 1Department of Biochemistry and Biotechnology, 2Department e-mail: Agata Jabłońska-Trypuć Poland; 3International Institute of Molecular and Cell Biology, Warszawa, Poland Kinetin (N6-furfuryladenine), a cytokinin plant growth e-mail: Andrzej Guranowski hormone was identified as a natural component of DNA in plant extract, calf thymus DNA, fresh DNA prepara- Fhit (fragile histidine triad) protein has been found in all tions from human cell culture, and in human urine. It was types of eukaryotic cells. Since 1996 Fhit has been known shown that kinetin delays the onset of several cellular and as a typical dinucleoside triphosphate hydrolase (EC biochemical characteristics associated with cellular aging 3.6.1.29) that liberates nucleoside 5′-monophosphates in human fibroblasts cultures. preferably from dinucleoside triphosphates, e.g. diad- Putrescine (aliphatic amine) is natural compound, ubiq- enosine 5′, 5′′′-triphosphate and less effectively from di- uitously present in its polycationic form in eukaryotic nucleoside tetraphosphates and dinucleoside pentaphos- and prokaryotic cells. It may have different functions in phates. We show that both human and plant (Arabidopsis) cell physiology, however the most important is partici- Fhits liberate AMP from such natural metabolites as pation in cell growth, proliferation and differentiation. adenosine 5′-phosphosulfate and adenosine 5′-phospho- It was also shown that putrescine has a specific role in ramidate thus behaving as adenylylsulfatases (EC 3.6.2.1) skin physiology; it’s overaccumulation in the skin leads to and nucleoside 5′-phosphoramidases (so far without EC early loss of hair due to disturbed cells differentiation. number), respectively. Some kinetic parameters of the In this study we tested the influence of kinetin and pu- novel reactions catalyzed by these Fhit proteins are pre- trescine on normal human skin fibroblasts. The fibrob- sented and implications of this novel capability of Fhit lasts cell line derived from a patient was maintained in proteins for basic metabolism and their anti-oncogenic DMEM medium supplemented with 10% foetal bovine role are discussed. o serum at 37 C in a humidified atmosphere of 5%2 CO in the air. Suspensions of cells (1×105 cells/ml) in 2 ml of culture medium were incubated with or without the test compounds in tissue culture 6-well plates. We tested cell proliferation at 10–3–10–7 concentration of kinetin and 10–3–10–7 putrescine. Cell numbers were counted in haemacytometer acc to Bürker, double ruling by colouring dead cells with methylene blue. Total pro- tein content was measured according to the method of Lowry. Collagen content in the medium was measured using the Picro-Sirius Red method. We observed stimula- tion of cells proliferation at 10–3 and 10–4 putrescine and 10–5 and 10–6 kinetine. We also observed changes in the cells morphology. Both kinetin and putrescine stimulated cells proliferation, total protein biosynthesis and collagen biosynthesis. Kinetin stimulated cells proliferation the most at 10–5–10–6M and putrescine at 10–3–10–4M. At these concentrations we also observed the biggest stimulation of total protein and collagen biosynthesis. Kinetin was more efficient than putrescine. Further experimentation is necessary to understand the mechanisms of kinetin and putrescine action in cells and their influence on protein biosynthesis and connected enzymes activity. 100 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Obtaining monoclonal antibodies anti- Characterization of UBP1.3 enzyme UBP 1.3 protease with the aim to elaborate and research on its stability ELISA test detecting residues of UBP 1.3 Agnieszka Kowalska1, Grażyna Płucienniczak2, protease in recombinant proteins Iwona Sokołowska2, Alina Marciniak-Rusek2, Anna Porębska, Marcin Zieliński, Iwona Marcin Zieliński2, Anna Wójtowicz-Krawiec, Anna Sokołowska, Alina Marciniak-Rusek, Natalia Mazurkiewicz-Pisarek, Natalia Łukasiewicz2 Łukasiewicz, Grażyna Płucienniczak, Anna 1Centre for Biotechnology, Warsaw University of Technology, Wójtowicz-Krawiec, Andrzej Płucienniczak Warszawa, Poland; 2Department of Bioengineering, Institute Department of Bioengineering, Institute of Biotechnology and of Biotechnology and Antibiotics, Warszawa, Poland Antibiotics, Warszawa, Poland e-mail: Marcin Zieliński e-mail: Marcin Zieliński Ubiquitin is a highly conserved protein present only in Expression of polipeptides and proteins joined with ubiq- eukaryotic cells either free or covalently joined to a va- uitin gave start of technology called ubiquitin fusion tech- riety of proteins. Fusing proteins to ubiquitin can cause nology. Benefits of this technology is higher expression a significant increase in yield, what is often used to ob- level of heterologous proteins equal in prokaryotic and tain high expression levels of desired proteins. Such fu- eukaryotic expression system. Naturally synthesized fu- sion can be afterwards cleaved after the Gly76 of ubiqui- sion proteins including ubiquitin are fast and precisely tin by specific proteases called DUBs (deubiquitinating cut by specific ubiquitin’s proteases. Bacteriums lack enzymes). UBP1, a Saccharomyces cerevisiae enzyme, is a ubiquitin and ubiquitin’s metabolizing enzymes. There- member of this family. It cleaves linear fusions to ubiqui- fore heterologous proteins expressed in Escherichia coli tin irrespective of their size or the presence of an amino- stays not cleaved. In majority case presence of ubiquitin terminal ubiquitin extension. With the aim of applying interferes with final protein product and should - bede this method in large scale biotechnological processes leted. For this purpose it is possible to use recombinant several changes in the gene sequence were introduced ubiquitin protease. Using such of enzymes in technologi- resulting in an UBP1 analogue — UBP1.3. This analogue cal process causes necessity to measure residues of the consists of yeast ubiquitin fused to an N-terminal amino- enzymes in final products. Immunoenzymatic test ELISA acid of modified UBP1 gene (modifications: deletion of gives capability to measure level of ubiquitin’s protease 98 residues from the amino terminus, a substitution of impurities. This method is approved by Pharmacopoeia glutamine at position 754 for leucine, codon changes ac- to control quality of recombinant proteins. On account of counting for the requirements of expression in Escherichia unique character of antigens and lack of commercial avail- Coli, 6xHis tag at the C-terminus). That allowed for suffi- able antibodies against of UBP 1.3 protease it was need to cient levels of expression and purification. 1. Research on obtain such of antibodies. In Department of Bioengineer- stabilityUBP1.3 has been purified using two methods: Ion ing IBA obtained clons of mouse hybridoma cells produc- exchange chromatography (combination of DEAE and SP ing monoclonal antibodies against UBP 1.3 protease. An- columns) and Affinity chromatography (Ni-NTA column) tibodies were purificated on protein-G sepharose 4B fast Part of the DEAE/SP and NI-NTA material was dialyzed flow chromatography column and will be used to elabo- against pure phosphate buffers to test the influence of ap- rate ELISA test detecting residues of UBP 1.3 protease in plied supplements on stability. To research the impact of recombinant proteins. changes in pH level, the DEAE/SP enzyme pH level was modified. Listed variants were stored in different- tem peratures. Enzyme activity was examined over time, in reaction with substrate (ubiquitin — growth hormone), and analyzed using SDS/PAGE electrophoresis. Part of the modified material was rejected at the beginning due to an instantaneous loss of activity. The best results were observed for the DEAE/SP purification method, with en- zyme stored in –70oC. 2.Research on enzyme activity in various conditions and influence of supplements in the course of reactionEffect of various pH levels and- tem perature on the activity was verified. The highest activity was obtained for the 7–7.5 pH level and temperatures in the 30–37oC range. Additionally the influence of series of protease inhibitors was researched. Enzyme activity was verified in reaction with substrate and analyzed using SDS/PAGE electrophoresis. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 101

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Development of a method for purifying Studies on the desulfuration of recombinant human interferon-γ (rhIFN-γ) 5’-o-phosphorothioylated nucleosides from transformed Escherichia coli cells catalyzed by hint-1 hydrolase Agnieszka Romanik1, Grażyna Płucienniczak2, Magdalena Ozga, Agnieszka Krakowiak, Iwona Sokołowska2, Alina Marciniak-Rusek2, Renata Kaczmarek, Wojciech J. Stec Marcin Zieliński2, Natalia Łukasiewicz2, Lena Department of Bioorganic Chemistry, Centre of Molecular and Wójcik2, Luiza Chojnacka-Puchta, Piotr Baran2 Macromolecular Studies PAS, Łódź, Poland 1Warsaw University of Life Sciences,Warsaw, Poland; e-mail: Magdalena Ozga 2Department of Bioengineering, Institute of Biotechnology and Antibiotics, Warszawa, Poland The histidine triad binding protein 1 (Hint-1) e-mail: Marcin Zieliński is an enzyme of the first branch of the HIT superfamily that catalyses the hydrolysis of P-N bond in AMP-lysine, Interferon-γ (IFN-γ) is a cytokine which is secreted by T AMP-alanine, AMP-morpholidate substrates with the lymphocytes and natural killer (NK) cells stimulated by rate of ca.~ 1 nmol/min/mg and adenosine-5’-monophos- antigens or mitogen. Natural human IFN-γ is basic glyco- phoramidate (AMP-NH2) with the rate of 1.71 nmol/min/ protein composed of 143 amino acid and does not contain mg [1]. Recently we reported that in the cleavage reaction any cysteines. Biologically active IFN-γ is a homodimeric catalyzed by the wt Hint-1 of the P-diastereoisomers of protein, which induces antiviral, immunomodulatory 5’-O-[N-(tryptophanylamide)phosphoramidothioate, the and antiproliferative effects on target cells. Recombinant hydrolysis of P-N bond was accompanied by a parallel human interferon-γ (rhIFN-γ) is not glycosylated, has loss of the sulfur from the resulting AMPS [2]. We have metionine on the N-terminal instead of pyroglutamic demonstrated that the wt Hint-1 hydrolyzes the AMPS acid and C-terminal deletion of three amino acid. Cur- with the rate of ca. 0.2 nmol/min/mg resulting in forma- rently IFN-γ has therapeutic efficacy on chronic granulo- tion of AMP and H2S as final products. We also noticed matous disease (CGD) and malignant osteopetrosis, and that another ribonucleoside phosphorothioate (GMPS) a great potential application in many other disease.Our was hydrolysed with even higher rate than AMPS. The aim was to purify recombinant human interferon-γ from aim of this study is to establish if the remaining 5’-O- the inclusion bodies produced in genetically transformed phosphorothioylated ribonuclesides (UMPS, CMPS) and Escherichia coli cells. We used two-steps Ion Exchange corresponding deoxyribonucleosides are also substrates Chromatography (first anion-echange resin, then cation- for Hint-1 hydrolase. We have found that desulfuration exchange resin) to obtain pure protein. The first step was process is not only ribonucleotide specific although the isolation of inclusion bodies from E. coli cells, which in- rate of P-S bond cleavage in 5’-O-phosphorothioylated cluded cell disruption by sonication, washing inclusion deoxyadenosine, deoxyguanosine, deoxycytidine is low- bodies using buffer with Triton X-100 and low concen- er than for the corresponding ribonucleotides analogs. tration of urea, and separated insoluble aggregations by Since the Hint enzyme has been shown to have its homo- high-speed centrifugation. Pure rhIFN-γ inclusion bodies logues in all forms of life, these observation can lead to were then solubilized in phosphate buffer containing urea the further elucidation of the metabolism of nucleoside and centrifuged. The clear solution was passed through 5’-O-phosphorothioates that are primary products of a DEAE Sepharose Fast Flow column equilibrated with degradation of antisense oligonucleotide phosphorothio- phosphate buffer pH 9.0 containing urea. The column was ates. The enzyme Hint-1 could be the possible candidate washed with the same buffer and two major peaks was responsible for their metabolism [1]. observed, each containing rhIFN-γ. The fraction obtained References: from DEAE Sepharose Fast Flow column was diluted in 1. Bieganowski P et al. (2002) J Biol Chem 277: 10852–10860. the refolding buffer for 24 h, at 4°C without stirring. Af- 2. Krakowiak A et al. (2007) Chem Comm 2089: 2163. ter that rhIFN-γ solution was concentrated by salting out, Acknowledgements: and precipitate was redissolved in phosphate buffer at This project is financially assisted by the grant 2PO4A07929 (to pH 6.0. After dialysis protein solution was loaded onto a A.K.) from Polish Ministry of Science and Higher Education and SP Sepharose Fast Flow column equilibrated with phos- by the MolMed program for PhD students (for M.O.). phate buffer pH 6.0. Pure rhIFN-γ was eluted as a broad peak with linear gradient of 0–1.0 M NaCl. The purity of the product was demonstrated by single principal band in the electrophoregram. There were made also control tests, necessary for the clinical use of this cytokine, in- cluding examine by peptide mapping, definition molecu- lar mass of protein by mass spectrometry and other. The overall yield of purified rhIFN-γ from the inclusion bod- ies of E. coli was more than 30% (> 70 mg protein per liter E. coli culture), which is high final result. 102 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Allele-specific silencing of Presenilin 1 gene GABA-shunt enzymes activity in with 392 L/V mutation by RNA interference PC12 and GH3 cell lines Alina Paduszyńska, Małgorzata Antoni Kowalski, Elżbieta Rębas, Sierant, Barbara Nawrot1 Ludmiła Żylinska Department of Bioorganic Chemistry, Centre of Molecular and Department of Molecular Neurochemistry, Medical Macromolecular Studies PAS, Łódź, Poland University of Lodz, Poland e-mail: Alina Paduszyńska e-mail: Elżbieta Rębas

Presenilins (PS1 and PS2) are polytopic proteins that, Glutamate and gamma-aminobutyric acid (GABA) are together with Nicastrin, Aph-1 and Pen-2, form a high the major neurotransmitters in the central nervous- sys molecular weight protein complex called γ-secretase. γ- tem. A metabolic pathway by which GABA is synthesized Secretase exerts its proteolytic function towards several from glutamate and then catabolized to succinic semial- substrates, e.g. the Notch receptor and amyloid precursor dehyde and succinic acid is called GABA-shunt. There protein (APP). Under normal conditions, APP processing are three enzymes involved in this process: glutamate results in a soluble shorter form of the amyloid-β peptide decarboxylase (GAD), GABA-aminotransferase (GABA- (Aβ) at tightly controlled concentration. In amyloidog- T) and succinic acid dehydrogenase (SSA-DH). In brain enic pathway, APP cleveage products are longer (40/42 GABA-shunt is about 17% of TCA cycle, and it is safe en- amino acids) and exhibit tendency for self-aggregation ergetic process, in which energy is generated from gluta- into β-sheet fibrils. PS1 mutation-dependent alternations mate without production of toxic ammonia. There is close are the most common mutations identified in Familiar dependence between GABA-shunt and neurotransmit- Alzheimer’s disease (FAD). Currently, more than 100 dif- ters release. The aim of our work was to investigate the ferent mutations of PS1 gene are known and almost all of activity of GAD, GABA-T and SSADH in PC12 and GH3 them lead to an increased Aβ40/42 production and early cell lines. Both lines belong to the group of secretory cells, onset of the disease. and possess the complete set of GABA-shunt enzymes, as Complete knockdown of PS1 gene can evoke severe side well as the receptors for gamma-aminobutyric acid. Both effects. Therefore, for these studies we selected - thetar used lines originate from cancer cells, but are the suitable get PS1 gene with a point mutation (1174 C→G) leading model for study of molecular mechanisms of secretion.. to the substitution of leucine 392 to valine (392 L/V) in We have compared the mRNA level of aforementioned the mature protein. Our aim was to use synthetic siRNAs enzymes using RT-PCR analysis in examined cell lines for allele-specific silencing of the mutated gene. The se- The activity of glutamate decarboxylase we measured in quence of the siRNA guide strand was fully complemen- cytosol fraction using spectrofluorimetric assay based on tary to the mRNA of the mutant protein. To evaluate the detection of condensated ninhydrin with GABA. GABA- silencing efficiency of the designed siRNAs, we cloned transaminase and succinic acid dehydrogenase activities the gene of the wild type PS1 as well as its 1174 C→G mu- were assayed in mitochondrial fraction and the enzyme tant in fusion with the enhanced green fluorescent protein activities were estimated by measuring of NADH forma- (GFP) cDNA in the pEGFP-C1 plasmid. Red fluorescent tion. Our results indicate on diverse function of GABA- protein (RFP) expressed from pDsRed-N1 plasmid was shunt in GH3 and PC12, suggesting the differences in used as a control. Both EGFP-PS1 and EGFP-392 L/V PS1 energetic metabolism. fusion genes, together with the RFP reporter gene, were Acknowledgements: expressed in HeLa cells. This model was used to screen The study was supported by the Medical University of the siRNA activity towards mutant allele of PS1 by de- Lodz grants No 502-16-810 and 503-6086-2. termination of the lowering of the EGFP fluorescence in comparison to the fluorescence signal originated from the reference RFP. First experiments demonstrate that activ- ity of the test siRNA towards PS1 mutant gene is signifi- cantly higher in comparison to the wild type PS1. Acknowledgements: These studies are financially supported by the grant from the Ministry of Science and Higher Education number PBZ-MNiSW- 07/I/2007. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 103

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Interaction of cytokinins with CK2-mediated phosphorylation – the novel proteins investigated by fluorescence mode of thymidylate synthase regulation? correlation spectroscopy Konrad Kubiński1, Maciej Masłyk1, Tomasz Paweł Zawadzki1,2, Genowefa Ślósarek2, Frączyk2, Wojciech Rode2, Ryszard Szyszka1 Przemysław Wojtaszek1 1Department of Molecular Biology, The John Paul II Catholic 1Department of Molecular and Cellular Biology, Faculty of University of Lublin, Lublin, Poland; 2Laboratory of Biology, 2Department of Molecular Biophysics, Faculty of Comparative Enzymology, Nencki Institute of Experimental Physics, Adam Mickiewicz University, Poznań, Poland Biology PAS, Warszawa, Poland e-mail: Pawel Zawadzki e-mail: Konrad Kubiński

Cytokinins are essential plant hormones that are involved Thymidylate synthase (TS) is folate-dependent enzyme, in cell division, shoot initiation, leaf and root differen- which is essential for DNA synthesis. It is dimeric protein tiation, photomorphogenesis and senescence. Cytokinin localized in cytoplasm and nucleus. Nowadays thymi- binding proteins (CBPs) have been identified in many dylate synthase is one of the key targets in cancer chemo- species, from mosses to higher plants. Unfortunately, the therapy. There are several evidences that higher level of biological function of these proteins is still unclear, with the synthase is observed in cancer cell lines and malig- the exception of CRE1 which has been recognized as cyto- nant tumors. Its expression can be regulated at various kinin receptor. Identification and characterization of new levels, including gene amplification, transcription, post- CBPs is important to better understand the mechanism of transcription, translation and post-translational events. cytokinin action. Our investigations are focused on reversible phosphor- Fluorescence correlation spectroscopy (FCS) is a method ylation as the mechanism of thymidylate synthase regu- which combine high resolution of confocal microscopy lation. Recently we have shown that human thymidylate with sensitivity of fluorescence spectroscopy. It allows synthase undergoes CK2-mediated phosphorylation the investigation of ligand-receptor interactions at the in vitro. Here we present further results, which provide level of single molecule in a confocal volume of about 1fL additional evidences for physiological meaning of such (10–15L). Single fluorophore is exited with a focused laser posttranslational modification in case of thymidylate syn- beam and the autocorrelation analysis of the fluorescence thase. fluctuation provide information about diffusion- coeffi Beside human synthase, its homologs from three other cient. In case of fluorescent ligand interactions with much organisms are also phosphorylated, what may suggest bigger protein, the signal from slow and fast diffusing conservation of this process. The highest efficiency of molecules can be differentiated and kintics of intraction phosphorylation was found in case of human synthase. can be calculated. We synthesized fluorescently labeled Moreover high phosphorylation was detected only when cytokinin and investigated specificity of interaction with free catalytic subunits of CK2 (α and α’) were utilized. Sig- CBPs. To confirm specificity of these interactions with nificant loss of TS phosphorylation (up to 97 percent) was proteins, we performed analysis of binding of fluores- observed after addition of CK2 regulatory β subunits to cently labeled cytokinin to accidental proteins. Lysosyme, reaction. The influence of β subunits on synthase phospho- BSA, chymotrypsine, and ovoalbumine do not bind fluo- rylation was confirmed with the use of recombinant human rescent cytokinin. Next, we asked if proteins which binds CK2 holoenzyme. Finally, the effect of C-mediated phospho- naturally occurring cytokinins would bind fluorescently rylation on synthase activity was examined. Initial results labeled cytokinin. To answer this question we investi- show that phosphomodification of thymidylate synthase gated interaction of fluorescent cytokinin with proteins: can change its enzymic properties towards dUMP and PR-10 and VrCSBP, known to bind biologically active mTHF, while dUMP does not affect CK2-mediated phos- cytokinins. In our method, PR-10 and VrCSBP strongly phorylation. bind labeled cytokinin. Specificity of this interaction was This is probably the first notice aboutin vitro modification confirmed by competition assay. We calculated kinetics of thymidylate synthase by any protein kinase. Further of interaction of fluorescent cytokinin with either PR-10 experiments including investigation of TS–CK2 protein or VrCSBP. We also calculated kinetics of interaction be- interaction and influence of TS phosphorylation on abil- tween synthetic cytokinins and VrCSBP, our data agree ity to its own mRNA binding, will broaden our knowl- very well with previously published results. edge upon this possible mode of TS regulation. Acknowledgements: Acknowledgements: This work was supported by Ministry of Science and Higher This work was supported by scientific resources for the years Education grant N N302 430034. 2005–2008 as an ordered research project PBZ-MIN-014/P05/2005 from The Ministry of Science and Higher Education in Poland.

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Thymidylate synthase – an Yeast protein kinase CK2a mutants atypical field of CK2 action defective in specific inhibitors binding Maciej Masłyk1, Konrad Kubiński1, Ewa Sajnaga, Ryszard Szyszka Tomasz Frączyk2, Wojciech Rode2, Ulf Hellman3, Ryszard Szyszka1 Department of Molecular Biology, John Paul II Catholic University of Lublin, Lublin, Poland 1Department of Molecular Biology, The John Paul II Catholic University of Lublin, Lublin, Poland; 2Laboratory of e-mail: Ewa Sajnaga Comparative Enzymology, Nencki Institute of Experimental Biology PAS, Warszawa, Poland; 3Ludwig Institute for CK2a is highly conserved member of the family of serine/ Cancer Research, Sweden threonine eukaryotic protein kinases. CK2a monomers e-mail: Maciej Masłyk are able to bind CK2b dimers forming heterotetrameric enzyme that plays a key role in various cellular processes Protein kinase CK2 is highly conserved, second messen- like proliferation, differentiation, apoptosis, tumorigene- ger-independent Ser/Thr kinase that utilizes both ATP sis, stress response and cell survival. A number of specific and GTP as phosphate donor. This ubiquitously distrib- and potent ATP competitive inhibitors of protein kinase uted in eukaryotes, constitutively active enzyme is en- CK2 are available to date. The structural basis for their gaged in wide range of cellular processes, including the selectivity is provided by hydrophobic pocket adjacent to regulation of cell cycle, apoptosis, transformation and tu- the ATP/GTP binding site, which in CK2 is smaller than morigenesis. Two catalytic (α and α’) and two regulatory in the majority of the other protein kinases.We investi- β subunits can form three tetrameric structures (αα’ββ; gated the importance of the hydrophobic interaction in

α2ββ; α’2ββ). Furthermore, naturally occurring free mon- the binding of specific inhibitors by mutational analysis omers of catalytic subunits may function independently. of yeast CK2a residues whose side chains contribute to CK2 can phosphorylates serine or threonine residues that reduce the internal size of the hydrophobic pocket. Site- are proximal to acidic amino acids. At least 300 substrates directed mutagenesis was used to replaced of Val67 and of CK2 have been identified to date. This include tran- Ile213 (the homolog of Val66 and Ile174 in human CK2) scription factors, signaling molecules, and proteins affect- by Ala. The kinetic properties of the individual mutants ing the structure and function of DNA and RNA. CK2a Val67Ala, CK2a Ile213Ala and the double mutant Thymidylate synthase (TS) is the enzyme that catalyzes CK2 Val67Ala Ile213Ala were studied with respect to ATP, the conversion of dUMP to dTMP used in DNA synthesis competitive inhibitors TBBt and TBBz and Elf1 protein as pathway. The second known function is that ST can form a substrate. The replacement of hydrophobic residues complex with its own mRNA and directly regulate ex- by alanines gave rise to catalytically active kinases. The pression of its self and some other proteins (eg. p53). Fur- phosphorylation efficiencyk ( kat) of the double substitut- thermore, higher level of TS expression has been found in ed CK2a mutants were the highest, little surpassing that case of several cancers (eg. breast, head, neck and colon of parental kinase. Km of the analysed mutants for ATP cancers). were not changed or very close to those of the parental Here we present thymidylate synthase as a new atypical kinase. In contrast, all analysed CK2a mutants displayed target for CK2-mediated phosphorylation in vitro. Using markedly higher Ki values towards inhibitors TBBz i TBBt computer analysis, mass spectrometry study and auto- comparing to recombinant wild-type subunits. We also radiography we identify Ser124 within the sequence of found that analysed mutants were much more resistant L121GFSTREEGD130 as the phosphoacceptor site for CK2. to TBBt than TBBz. Moreover, the site being phosphorylated differs from Acknowledgements: minimum consensus which is recognize by CK2 found in This work was supported by scientific resources for the years other protein substrates. We compared catalytic proper- 2005-2008 as an ordered research project PBZ-MIN-014/P05/2004 from the Ministry of Education and Science. ties such as Km, kcat and Vmax for α and α’ towards TS. Whereas the Km and Vmax values demonstrate no signifi- -3 -3 cant differences, the catk constants are 1.5×10 and 0.63×10 [1/s] for α and α’ respectively. Additionally we check an influence of β subunit on α and α’ activity towards thymi- dylate synthase. It seems that CK2β has strong inhibitory effect on both catalytic subunits activities what was con- firmed by CK2 holoenzyme phosphorylation. This results indicate connection and regulatory effect on Thymidylate synthase activity in the cell. Acknowledgements: This work was supported by scientific resources for the years 2005-2008 as an ordered research project PBZ-MIN-014/P05/2004 from the Ministry of Education and Science.

Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 105

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Cloning and expression of a new recombinant Protein kinase CK2 is engaged in phosphate thrombolytic and anthithrombotic sensing in yeast Saccharomyces cerevisiae agent – a staphylokinase variant. Elżbieta Kochanowicz, Kamila Marek Kowalski1, George Brown1, Magdalena Filipiak, Ryszard Szyszka Bieniasz1, Katarzyna Oszajca1, Ewa Department of Molecular Biology, The John Paul II Catholic Chabielska2 , Tadeusz Pietras3, Zofia Szemraj1, University of Lublin, Lublin, Poland Jacek Bartkowiak1, Janusz Szemraj1 e-mail: Elżbieta Kochanowicz 1Department of Medical Biochemistry, 3Department of Pneumology and Allergy, Medical University of Lodz, Łódź, Protein kinase CK2 is a multisubstrate and highly con- Poland; 2Department of Biopharmacy, Medical University of served, serine/threonine phosphotransferase present Białystok, Białystok, Poland in all eukaryotic organisms from yeast to human. As a e-mail: Janusz Szemraj holoenzyme, yeast kinase is composed of two catalytic (α, α’) and two regulatory (β, β’) subunits but it can also To develop a more potent antithrombin agent with throm- exists as a free catalytic subunit. It participates in majority bolytic and antiplatelet properties new SAK variant was of signaling cascades linked to gene expression, cell cycle constructed. The Kringle 2 domain (K2) of tissue type- and survival events. plasminogen activator (t-PA), containing a fibrin-specific One of the cellular processes of our interest, in which binding site (i), the RGD sequence (Arg-Gly-Asp) for the CK2 is engaged, is phosphate-responsive signaling path- prevention of platelet aggregation and (ii) the antithrom- way (PHO). It was reported that deletion of CK2 subunits botic agent – hirulog (iii) was assembled to the C-terminal causes repression of genes encoding phosphate transport- part of recombinant staphylokinase (r-SAK). The cDNA ers and phosphate supplying phosphatases. for hybrid protein SAK-RGD-K2-Hirul was cloned into PHO pathway is mediated by about 30 proteins. One of Pichia pastoris pPICK 9K yeast expression vector. The in- them is Spl2 which is a negative regulator of low-affinity troduction of K2 t-PA, RGD sequence and hirulog into the phosphate uptake in yeast S. cerevisiae. It may directly or C-terminus r-SAK molecule did not alter the staphyloki- indirectly downregulate proteins engaged in this signal- nase activity. We observed a higher clot lysis potency of ing event (Pho87 and Pho90). It is a small protein (17 kDa) SAK-RGD-K2-Hirul as evidenced by a faster and more which reveals homology to the Pho81 (component of the profound lysis of 125I-labeled human fibrin clots. The po- cyclin-CDK regulatory complex). SPL2 is a phosphate re- tency of thrombin inhibition by the hirulog C-terminal sponsive gene. part of the recombinant fusion protein SAK-RGD-K2- Spl2 undergoes CK2-mediated phosphorylation in vitro. Hirul was almost identical to that of r-Hir alone These We found that there are two possible phosphoacceptor results suggest that the SAK-RGD-K2-Hirul construct can sites within its amino acid sequence (Ser142 and Ser143) be a more potent and faster acting thrombolytic agent which fulfill the minimal consensus recognized by CK2. with better antithrombin and antiplatelet properties com- In this report we provide the evidence that both C-termi- pared to r-SAK and SAK-RGD-K2-Hir. nal serines in Spl2 (Ser142 and Ser143) are phosphorylat- ed. Using directed mutagenesis we replaced Ser142 and Ser143 by alanines. We have obtained two mutant proteins named M1 for Spl2 without Ser143 and M2 for Spl2 with- out Ser142. We observed that mutated proteins (neither M1 nor M2) were not phosphorylated, while native Spl2 is strongly phosphorylated. It may indicate that presence of both serines is necessary for phosphorylation by CK2. This peculiar type of interaction between CK2 and its substrate needs further experiments 106 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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The influence of cryoprotectors and Stimulation of soluble or particulate guanylyl acetamide on the β-N-aceltylglucosaminidase cyclase differently affects the activity of NF-κB activities from rainbow trout (Oncorhynchus in rat peripheral blood mononuclear cells mykiss) and Siberian sturgeon (Acipenser Małgorzata Mitkiewicz, Ewa Kurowska, baeri) milt plasma and spermatozoa Marianna Kuropatwa, Wojciech A. Gorczyca Beata Sarosiek, Jan Glogowski Laboratory of Signaling Proteins, L. Hirszfeld Institute of Institute of Animal Science and Food Researches of Polish Immunology and Experimental Therapy, PAS, Wrocław, Academy of Sciences, Department of Molecular Andrology, Poland Olsztyn, Poland e-mail: Małgorzata Mitkiewicz pan.wroc.pl>

Sturgeon spermatozoa are significantly different from Guanylyl cyclases (GC) are a family of enzymes which freshwater teleost fish (rainbow trout) sperm. These dif- catalyze the conversion of GTP to cGMP. The family com- ferences concern presence of acrosom and acrosomal prises cytosolic (soluble, sGC) and membrane-bound enzymes, ex. acrosin, arylsulfatase, β-N-acetylglucosami- (particulate, pGC) isoforms. Soluble GCs are activated nidase. But in spite of lack of acrosome, β-NAGase is by nitric oxide, whereas pGCs by natriuretic peptides, present in rainbow trout milt (in contrast to acrosin and guanylins or bacterial enterotoxins. The aim of this study arylsulfatase). The cryopreservation methods are estimat- was to determine whether and how synthesis of cGMP by ed from many years, however optimizations of technol- various GCs affects an activity of nuclear factor-κB (NF- ogy are still needed. Important parameter for cryopreser- κB) in rat peripheral blood mononuclear cells (PBMC). vation include types of extenders and cryoprotectants. In We found that in rat PBMC synthesis of cGMP increased this work we checked the influence of some cryoprotect- in response to sodium nitroprusside (SNP), the activator ants like 10% DMA (dimethyl acetamide), 10% DMSO of sGC as well as in response to atrial natriuretic peptide (dimethyl sulfoxide), 10% methanol, 30% glycerol, 0.9M (ANP) and C-type natriuretic peptide (CNP), the activa- sucrose and 1M acetamide (β-NAGase inhibitor) on the β- tors of particulate GC-A and GC-B, respectively. Using N-aceltylglucosaminidases activities from rainbow trout electrophoretic mobility shift assay (EMSA), we found and sturgeon milt plasma and spermatozoa extract. The that in these cells SNP caused increase in NF-κB activ- sturgeon and rainbow trout milt we obtained in aquacul- ity. Applying specific inhibitors, we further found that ture facilities, then after transport to the laboratory on ice stimulatory effect of NO/cGMP was mediated by cGMP- (+4oC), milt plasma was separated from spermatozoa by dependent protein kinase (PKG I). At the same time, ANP centrifugation. Frozen spermatozoa were thawed at room and CNP did not influence an activity of NF-κB. How- temperature (in 2 ml of 0,01 M imidazole HCl, pH 6.8) ever, the activity of this transcription factor induced by for 1 hour and centrifuged. The milt plasma and obtained bacterial endotoxin (LPS) was significantly decreased in supernatant were mixed with all above mentioned solu- the presence of ANP but not SNP or CNP. These observa- tions at 1:1 ratio, and the β-NAGase activity was meas- tions were additionally supported by changes in expres- ured after 0, 3 and 24 hours. In our experiment wees- sion of NF-κB-dependent IL-1β, IL-6, TNF-α and iNOS as tablished that methanol is the one cryoprotectant which shown by RT-PCR. Taken together, our results indicate slightly increased the β-NAGase activities in all sources that cGMP affected the activity of NF-κB, but differently of enzymes (without sturgeon milt plasma, where the dipending on the isoform of GC involved in its synthe- methanol addition had no effect). The glycerol and- su sis. crose supplementation had no effect on the rainbow trout Acknowledgements: milt plasma and spermatozoa enzymes in the contrast Supported by the grant No. N N301 4158 33 from the Ministry of to DMA and DMSO which decreased these activities up Science and Higher Education, Poland. 11 to 22%. Acetamid caused 35% and 51% reduction of rainbow trout sperm extract and milt plasma β-NAGase activities respectively. In the sturgeon milt plasma and spermatozoa extract we observed that DMA and DMSO addition caused about 50% loss of β-NAGase activities. Glycerol decreased enzymatic activities up 5 to 20%, like sucrose 12–23%. The acetamide was the strongest inhibi- tor and caused about 70 and almost 90% loss of sturgeon milt plasma and sperm extract activities respectively.The time of incubation had no effect, we observed no statisti- cal differences between the β-NAGase activities after 0, 3, and 24 hours in both species. Acknowledgements: This study is supported by Ministry of Science and Higher Edu- cation Grant No N 308 018 31. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 107

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Analysis of interactions within the Effect of cGMP/cAMP-dependent ClpB/DnaK chaperone system pathway on the activity of NF-κB in monocytic cell lines U937and THP-1 Izabela Guenther, Andrzej Taranta, Sabina Kędzierska-Mieszkowska Ewa Kurowska, Małgorzata Mitkiewicz, Dorota Hyd, Marianna Kuropatwa, Wojciech A. Gorczyca Department of Biochemistry, University of Gdansk, Gdańsk, Poland Laboratory of Signaling Proteins, Ludwik Hirszfeld Institute e-mail: Izabela Guenther of Immunology and Experimental Therapy PAS, Wrocław, Poland Bacterial heat-shock protein, ClpB, member of the AAA+ e-mail: Ewa Kurowska ing effects of protein inactivation and aggregation that arise after extreme heat stress. It exists as a hexameric Cyclic nucleotides (cGMP/cAMP) are key messenger ring that contains two conserved nucleotide-binding sites molecules in various cellular processes. They are also im- per monomer. It is known that ClpB cooperates with the portant modulators of the immune response. However, DnaK chaperone system in efficient solubilization and mechanisms of their action remain still unclear. The aim reactivation of strongly aggregated proteins in vivo and of our studies was to determine the role of cGMP/cAMP- in vitro, however the roles of the individual chaperones dependent signaling pathways in the inflammatory proc- in disaggregation and the functional interplay between ess. As a model we used the monocytic cell lines U937 ClpB and DnaK are not yet fully understood. Earlier stud- and THP-1. It was determined that in THP-1 cells cGMP ies showed that ClpB from E. coli binds at the substrate was synthesized by both soluble (sGC) and particulate binding site of DnaK and it is stimulated by ADP. We (NPR-A) guanylyl cyclases, while in U937 cells only by tested whether this interaction plays essential role in pro- NPR-A. Both cell lines expressed cGMP hydrolyzing or teins’ reactivation by the ClpB/DnaK chaperone system. cGMP-dependent phosphodiesterases (PDE) belonging We examined the complex formation of ClpB and DnaK to families PDE1, PDE3, and PDE9. Additionally, U937 in vitro in the presence of aggregated substrate and DnaJ cells expressed PDE2. Stimulation of sGC with nitric ox- protein (DnaK co-chaperone), and nucleotides ADP or ide donors or stimulation of NPR-A with atrial natriuretic ATP.We used the glucose-6-phosphate dehydrogenase peptide (ANP) did not affect activity of transcription (G6PDH) as a model system to generate stable popula- factor NF-κB in THP-1 cells. At the same time activity tions of protein aggregates comprising controlled ranges of NF-κB induced by LPS was significantly decreased of particle size.When aggregated substrate was incubated in the presence of ANP. Similar effects were observed in with ClpB and ADP, or DnaK and ADP, and then chro- case of U937 cells. It was also found that in both cell lines matographed on a gel filtration column, only DnaK was the membrane-permeable analog of cAMP (8Br-cAMP) found in fractions corresponding to the aggregates. The caused similar decrease in the NF-κB activity induced by same result was observed when both ClpB and DnaK LPS. Effects of ANP and 8Br-cAMP were abolished in the were incubated with the G6PDH aggregates in the pres- presence of an inhibitor of cAMP-dependent protein ki- ence of ADP. This indicates that the affinity of ClpB for nase (PKA) suggesting that common cGMP/cAMP signal- its aggregated substrates is low in the ADP-bound state ing pathway might be involved in this process. and that the complex of a substrate with both DnaK and Acknowledgements: ClpB is not populated in the presence of ADP.In the case Supported in part by the grant No. N N301 4158 33 from the when aggregated G6PDH was incubated with ClpB and Ministry of Science and Higher Education, Poland. ATP, or DnaK and ATP, either chaperone was found in fractions corresponding to the aggregates, however the DnaK protein bound to the G6PDH with lower affinity than ClpB. This result indicates that both ClpB and DnaK recognize an aggregated substrate in the presence of ATP. The same situation was observed when both chaperone proteins were incubated with G6PDH.It is possible that a ternary complex of a substrate with the ClpB/DnaK bi- chaperone system was formed in the presence of ATP.The additional presence of DnaJ protein didn’t stimulate the G6PDH-ClpB-DnaK complex formation. 108 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Inhibitors of phosphorylation of NS5A protein Biochanin A is a potent modulator of multidrug as potential anti-hepatitis C virus agents transporter Cdr1p mutant of Candida albicans Mariusz Krawczyk1, Monika Liszewska1, Kamila Środa, Marcin Kołaczkowski, Zygmunt Kazimierczuk2 , Anna M. Krystyna Michalak Boguszewska-Chachulska1 Department of Biophysics, Wrocław Medical University, 1Institute of Biochemistry and Biophysics PAS, Warszawa, Wrocław, Poland Poland; 2Warsaw University of Life Sciences, Warszawa, e-mail: Kamila Środa Poland e-mail: Anna M. Boguszewska-Chachulska many human candidiasis in people with immunodefi- ciency syndromes. The phenomenon of multidrug resist- Hepatitis C virus (HCV) is a (+) RNA virus of the Flaviv- ance (MDR) is a serious obstacle in antifungal therapy. iridae family. HCV infects about 3% of the world popula- Overexpression of drug efflux pumps belonging to the tion and it is the major reason of liver cancer as well as liv- ATP-binding cassette (ABC) superfamily of transport- er transplants in the developed world. To date, no vaccine ers is an important mechanism of resistance acquisition. against HCV has been adopted although several clinical Among ABC transporters, Cdr1p has been shown to play trials have been started. The newly developed dual thera- a key role in resistance to azole in Candida albicans. This py with pegylated interferon and ribavirin is effective on resistance can be reversed by variety modifiying agents average only in about 60% of the cases, depending on the called MDR reversal modulators. genotype of the virus and the duration of the treatment. Here we have examined the modulatory effect of flavo- There is an urgent need for a new therapy that would be noids on azole resistance mediated by the wild type and less expensive and more effective against the prevailing mutants of Cdr1p overproduced in the strain of bakers genotypes. Two non-structural HCV proteins, NS2 and yeast Saccharomyces cerevisiae devoid of endogenous drug NS5A, are phosphorylated by casein kinase 2 (CK2) and/ efflux pumps. Flavonoids are compounds known to mod- or related kinases. NS5A plays an important regulatory ify the multidrug resistance. In our studies Biochanin A role in HCV RNA replication and modulates host intrac- occurred to be one of the most effective inhibitor of yeast ellular signaling pathways. Phosphorylation and hyper- cells growth among all tested flavonoids. Biochanin A phosphorylation of NS5A appear to be highly regulated showed also a significant drug resistance modyfying and involve several cellular protein kinases. Inhibition effect. Here we have also demonstrated that Cdr1p is of NS5A phosphorylation affects RNA replication, while strongly glycosylated membrane protein. This transport- complete prevention of formation of the hyperphospho- er is only N-glycosylated and this posttranslational modi- rylated form leads to total arrest of replication.We over- fication may play an important role in the plasma mem- expressed the NS5A protein in bacteria to study the effect brane expression and in functional activity of Cdr1p. of protein kinase inhibitors, especially those belonging to the group of CK2 inhibitors. The His-tagged protein was purified under denaturing conditions using - guani dine hydrochloride and Ni-NTA resin, then submitted to renaturation by dialysis. The protein was purified with the yield of 17 mg per 1 liter of culture. Recognition of NS5A phosphorylation sites by CK2 was confirmed us- ing a standard radioactive CK2 assay and by a novel ap- proach, based on luminescence, in which the phospho- rylation efficiency is assessed indirectly by measuring the amount of ATP left after the reaction. Therefore high concentration of NS5A and low concentration of ATP are required. Initial results of testing of CK2 inhibitors using the NS5A substrate will be presented.In parallel, a direct effect of kinase inhibitors on viral RNA replication was studied with the use of HCV subgenomic replication system in the Huh-7 (human hepatoma) cell line. Experi- ments with several groups of inhibitors were carried out, with a special emphasis on halogenated benzimidazoles. Most of the compounds under study failed to show any significant inhibitory effect but some compounds exerted an influence on viral replication without being highly cy- totoxic. Acknowledgements: This work was supported by the MNiSW grant PBZ-MIN-014/ P05/2004. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 109

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Antibacterial activity in the haemolymph of Differences in immune response Galleria mellonella larvae after infection with of Galleria mellonella to yeast and Pseudomonas aeruginosa and Escherichia coli filamentous fungi infection Mariola Andrejko, Magdalena Mizerska- Agnieszka Zdybicka-Barabas, Dudka, Teresa Jakubowicz Małgorzata Cytryńska, Mariola Andrejko, Magdalena Mizerska-Dudka, Department of Invertebrate Immunology, Institute of Biology, Monika Koziej, Teresa Jakubowicz UMCS, Lublin, Poland e-mail: Mariola Andrejko Department of Invertebrate Immunology, Maria Curie- Skłodowska University, Lublin, Poland Important element of innate immune response in both e-mail: Małgorzata Cytryńska bial peptides (AMP’s). Naturally occurring peptides are small, amphipatic molecules (12–50 amino acid residues). Insects, which lack adaptive immunity, have very efficient Most of them possess net positive charge because of ex- innate mechanisms of humoral and cellular response. cess number of cationic amino acids. AMP’s exhibit direct Our previous studies have shown that immune system antimicrobial activity but in some cases they modulate of Galleria mellonella can distinguish bacterial and fun- immune system or neutralize endotoxins such as LPS. gal infections and responds specifically to these classes Antimicrobial peptides are found and described in al- of microorganisms. To further characterize immune re- most all invertebrates taxa, especially different species of sponse of G. mellonella to fungal pathogens, we used dif- insect are widely examined in this area. It is confirmed ferent species of budding yeasts and filamentous fungi that each insect species can express individual set of an- as immunogenes. Capsule formation and changes in timicrobial peptides and proteins in response on invad- haemocyte morphology were observed in haemolymph ing microorganisms. In haemolymph immune challenged of larvae injected with fungal spores but not yeast cells. Galleria mellonella larvae (model organism to study innate Moreover, considerable differences in level and kinetics immunity and to test pathogens), twelve inducible anti- of antifungal and antibacterial activity in haemolymph microbial peptides were identified. of G. mellonella larvae were detected when yeast and fila- In this study we tested the antimicrobial activity of mentous fungi were used for immune challenge. It was haemolymph from G. mellonella larvae infected with found that antifungal activity appeared 24h and 48h after entomopathogenic strain Pseudomonas aeruginosa or challenge of larvae with yeast cells and filamentous fungi, non-pathogenic bacterium Escherichia coli D31 (known respectively. Electrophoretic analysis of haemolymph ex- immune response elicitor). Antimicrobial activity in cell- tracts of immune-challenged larvae revealed differences free haemolymph (obtained in different time points after in the pattern of appearing antimicrobial peptides which infection) was measured by diffusion well assay and in were dependent on the immunogen used. Incubation of situ by bioautography. We observed that the antimicro- yeast cells with immune haemolymph extracts contain- bial activity appeared shortly after infection with both P. ing defense peptides and apolipophorin III (apoLp-III) aeruginosa and E. coli, reaching maximum 18 h after in- caused cell aggregation and formation of pseudohyphae. fection. The activity induced by E. coli sustained on high In the case of filamentous fungi, inhibition of spore ger- level until the end of experiment — 48 h after infection, mination and changes in hyphae morphology were ob- while in the case of infection with P. aeruginosa only trace served. It is known that apoLp-III constitutively present of antimicrobial activity was detected after 30 h. We also in haemolymph of G. mellonella, plays an important role noted that the antimicrobial activity level induced by in immunity. Recent studies have shown that apoLp-III is non-pathogenic bacterium was higher in comparison to able to bind to lipopolysaccharides, lipoteichoic acids and that measured after infection with entomopathogenic P. β-1,3-glucans and therefore may act as a pattern recogni- aeruginosa. Above results were confirmed by Tris-tricine tion molecule for foreign invaders. In addition, apoLp-III SDS/PAGE electrophoresis. We observed appearance of can act as an activator of cellular reactions (phagocyto- two polypeptide bands with molecular mass below 6.5 sis) and stimulate antibacterial activity of haemolymph. kDa after infection with both P. aeruginosa and E. coli. Immunoblotting analysis with anti apoLp-III antibodies These bands represent inducible antimicrobial peptides showed that this protein bound to the surface of yeast G. mellonella larvae. cells and fungal spores, suggesting that apoLp-III is en- gaged in antifungal activity of G. mellonella haemolymph. Our results suggest that immune system of G. mellonella can distinguish yeast and filamentous fungi infection. 110 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Transformation of methoxyphenylpropanoid Binding of self-assembling dyes to albumin. compounds by Nocardia corallina Barbara Stopa, Janina Rybarska, Leszek Konieczny and Nocardia opaca Chair of Medical Biochemistry, Medical College, Jagiellonian Marzanna Paździoch-Czochra, Weronika University, Kraków, Poland Komorowska, Elżbieta Malarczyk e-mail: Barbara Stopa Department of Biochemistry, Maria Curie-Skłodowska University, 20-031 Lublin, Poland Congo red (CR) and related dyes self-assemble in water e-mail: Marzanna Paździoch-Czochra pable of interacting with proteins (e.g. antigen-complexed antibodies). While most ligands bind as single molecules, Phenylpropanoid compounds are widely widespread in CR preserves its supramolecular organization in protein– plants. Most of them (p-cumaric, ferulic and cinnamic ac- dye complexes. This makes this group of dyes a possibly ids) are precursors of lignin, but others (flavonoids, iso- good system for carrying different stains or drugs, which flavonoids, anthocyjanins, phytoalexins) are used as part may be incorporated by intercalation into the supramo- of plant responses to various stress factors (biotic and lecular assembly that binds to suitable carriers (e.g. anti- abiotic) [1]. Many phenylpropanoids are pharmacologi- bodies). The effect of plasma albumin on such immuno- cally active or are important aromatic flavour compounds targeting system requires elucidation. The key question is used in food and perfumes [2]. Microbial metabolism of whether albumin binds CR as independent molecules, or phenylpropanoids is important element of global carbon if it can bind supramolecular moiety with carried drugs cycle and due to its biodegradation is investigated in vari- included. In such a case, albumin could support the car- ous microorganisms as well as fungi (depolimerization rying of drugs by supramolecular systems. The supramo- of lignin) and bacteria (degradation of simpler aromatic lecular character of dye ligands bound to albumin was compounds). Aerobic metabolism of methoxyaromatic demonstrated by indicating the complexation of dye mol- compounds is initiated by oxidative O-demethylation re- ecules outnumbering the binding sites in albumin and by sulting in hydroxyaromatic compounds, formaldehyde. measuring the hydrodynamic radius of albumin which Such systems was described in Nocardia and Rhodococcus increases upon binding of self-assembling dyes (Congo with reference to methoxybenzoic acids [3]. The reaction red, Evans blue) in contrast to dyes lacking this property proceeded according to the scheme proposed by Ribbons (Trypan blue, ANS). It was also shown that CR can intro- [4] and is accompanied by consumption NADH, oxygen duce into a complex compounds which itself are unable of uptake and release HCHO. The present paper demon- binding to albumin (Janus green), but can be incorporat- strates relationship between demethylase activity and ed into the CR supramolecular assembly. This confirms, production of formaldehyde in cultures Nocardia corallina that self-assembling dye forms the complex with albumin and Nocardia opaca grown on methoxyphenylpropanoids: as a supramolecular entity and not as a collection of in- dimethoxycinnamate (DMC), ferulate (FER) and isoferu- dividual dye molecules. Congo red binding makes albu- late (IFER) as sole carbon source. Demethylase activity min less resistant to conformational changes induced by was measured towards DMC, FER, IFER and caffeic acids low pH, as concluded from the facilitated N–F transition, as well as in the presence effectors ATP, GTP, GSH, HCHO observed in studies based on the measurement of hydro- added before and after phenolic compounds according to dynamic radii. The specific differences were observed in Malarczyk [5]. The studies examining induction pattern susceptibility of albumin and CR-albumin complex to of the demethylase were conducted with early-, mid- and trypsin digestion. Results suggest that the self-assembled late-log phase growth. The study showed the growth- dye penetrates the central crevice of albumin molecule, phase dependence for demethylase activity. It was found which is easy to be penetrated by the large polymolecu- that Nocardia opaca induced higher activity than Nocardia lar assembly and contains the long, nonhelical fragment corallina, particularly in the presence DMC and IFER. probably representing the receptor element for binding Growth on DMC induced maximal activity towards FER the dye. To study the stability of CR-albumin the kinetics and IFER. In contrast growth on FER and IFER induced of the exchange of ligands (Congo red, Evans blue, pal- comparable levels activity against FER and IFER. All these mitic acid) was analyzed. Fluoresceinated dodecylamine changes strongly depended on type of effectors. The level was used as an easily detectable fatty acid analog. Quan- of HCHO was determined using fluorimetric method and titative measurements of albumin ligands exchange show was correlated with the demethylase activity. competition between CR supramolecular ligand and fatty Refereneces: acids. 1. Solecka D (1997) Acta Physiol Plant 19: 257–268. 2. Rosazza JP, Huang Z, Dostal L, Volm T, Rousseau B (1995) J Ind Microbiol 15: 457–471. 3. Paździoch-Czochra M, Malarczyk E, Sielewiesiuk J (2003) Cell Biol Intern 27: 325–336. 4. Ribbons DW (1970) FEBS Lett 8: 101–104. 5. Malarczyk E (1989) Acta Microbiol Polon 38: 45–53. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 111

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Binding of Congo red to antibodies and Biochemical analysis of Bacillus its effect on the interaction with C1q subtilis PrpE protein phosphatase Barbara Piekarska, Anna Jagusiak, Arletta Nowodworska, Joanna Karczewska, Leszek Konieczny Adam Iwanicki, Michał Obuchowski Chair of Medical Biochemistry, Medical College Jagiellonian Intercollegiate Faculty of Biotechnology, University of Gdańsk University, Poland and Medical University of Gdansk, Division of Molecular e-mail: Barbara Piekarska Bacteriology, Gdańsk, Poland e-mail: Joanna Karczewska Congo red is a dye which self-assembles in water solu- tions forming supramolecular entities that bind to anti- Protein phosphorylation and dephosphorylation are im- bodies in immune complexes, leading to the stabilization portant in controlling a variety of biological processes of antigen-antibody interaction with simultaneous inhibi- such as metabolism, cell differentiation and proliferation, tion of the complement activation. Model studies indicate gene expression, transport and memory. These modifica- that Congo red supramolecular assembly is bound to the tions are catalyzed by protein kinases and protein phos- antibody V domain in a cavity generated as a result of phatases, respectively. structural destabilization resulting from distortion that Presented results concern our studies of the Bacillus sub- accompanies the binding of bivalent antibody to random- tilis PrpE protein. The protein was characterized in our ly distributed antigenic determinants. The interaction of laboratory as a PPP phosphatase removing phosphate the dye with constant domains is also possible. Binding groups from phosphotyrosine, and not being able to re- of Congo red to rabbit polyclonal anti-TNP antibodies move phosphate groups from phosphoserine and phos- and its influence on the interaction with C1q was stud- phothreonine. Moreover, our protein shows asymmetri- ied using both insoluble antigen (TNP-derivatized CM- cal Ap4A hydrolase activity and ATPase activity. Sephadex beads) and the soluble immune complexes The aim of the study was the evaluation of amino acid with bivalent synthetic antigens. The number of dye mol- composition of the PrpE active centre. For this purpose ecules attached to antibodies was determined and the lo- six mutants in the prpE gene were constructed. Mutation calization of the binding site in the Fab region confirmed. sites were chosen to introduce amino acid substitutions of The aim of the study is to elucidate if Congo red inhibits residues reported as essential for dephosphorylation and complement activation by interaction with antibody, by Ap4A hydrolysis catalysis. All six PrpE protein variants blocking the specific binding sites in C1q head domains, were checked for the ability to dephosphorylate peptides, or if it affects further steps of the complement cascade. hydrolyse Ap4A molecules, and for ATPase activity. The The analysis of the inhibitory effect of Congo red on com- results allowed us to calculate kinetic parameters for all plement activation performed with guinea pig sera as a three types of enzymatic reactions (maximum velocity, source of complement proteins did not give equivocal re- Michaelis-Menten constant, catalytic constant). Data ob- sults, because of the interaction of the dye with plasma tained show, that four out of six amino acid substitutions albumin. The effect of Congo red on C1q-antibody inter- influenced all three PrpE enzymatic activities, either by action was studied using the ELISA system with TNP- decreasing maximum velocity or by decreasing enzyme- derivatized maleic anhydride activated plates, polyclonal substrate affinity. One amino acid substitution increased rabbit anti-TNP antibodies, purified C1q and anti-C1q maximum velocity of dephosphorylation reaction, and antibodies. The inhibitory effect of Congo-red on the C1q another one completely abolished any enzymatic activity binding was observed, although the number of antibod- of the studied protein. ies bound to the antigen in presence of Congo red was Taken together our results let us define amino acid con- increased. The explanation of the mechanism of Congo tent of the PrpE active site, as well as make assumptions red binding to antibodies will lead to understanding of on the role of particular amino acid residues in catalysis. the conformational changes that accompany the forma- In further studies we aim to confirm our results byin vivo tion of immune complexes and the role of these changes studies on mutants devoid of selected enzymatic activity. in effector activation. 112 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Cloning and expression analysis The synthesis of catalytically active NTPDase2 fragment of insulin receptor substrate from Arabidopsis thaliana in bacteria with the in red mason bee (Osmia rufa L.) use of two-component expression system Marek Skrzypski1, Zdzisław Wilkaniec2, Dorota Ściesińska, Paulina Wardęcka, Dawid Szczepankiewicz1, Małgorzata Michał Komoszyński Ciupa2, Karol Giejdasz2, Monika Biochemistry Department, Nicolaus Copernicus University, Fliszkiewicz2, Krzysztof W. Nowak1 Torun, Poland 1Department of Animal Physiology and Biochemistry, e-mail: Dorota Ściesinska University of Life Sciences in Poznań, Poznań, Poland; 2Department of Useful Insects Breeding, University of Life NTPDases are commonly used in biotechnology and Sciences in Poznań, Poznań, Poland medicine because they hydrolyze 5’-, di- and triphos- phate nucleotides (mainly ATP and ADP). The most in- e-mail: Ewa Pruszyńska-Oszmałek blood conservation, nucleic acid sequencing or as anti- coagulant reagents. The mechanism of catalysis of NTP- Insulin is the hormone from peptide hormone family that Dases is still unknown. Crystallization of proteins, which includes relaxins/insulin like peptides (ILPs) and insulin like allows to describe their tertiary structure, is one of the growth factors (IGFs). According to development of novel main conditions to solve this mechanism. The generation molecular techniques, genes coding peptides similar to in- of sufficient amount of proteins for the crystallization sulin were also discovered in invertebrates. Many data indi- from plant material using standard biochemical methods cate that signal transduction system of ILPs and ILPs is very is a long-term and low efficient process. The aim of this conservative. One of major protein involved in cellular trans- research was to obtain pure and active from of apyrase duction system is insulin receptor substrate (IRS). Mutation from Arabidopsis thaliana using two-component bacterial in the IRS gene displays reduced cell number and cell size expression system (E. coli). in drosophila melanogaster. Recently IRS gene was cloned Bacteria Escherichia coli BL21(DE3) were first transformed in honey bee, were it is involved in development and cast with a pG-KJE8 plasmid carrying the genes of DnaK, determination. Actually, there is no data about expression DnaJ, GrpE, GroES and GroEL chaperones. Subsequent- and IRS gene sequence in red mason bee. In this study we ly, cells were transformed with vector pET 28a, carrying focused on cloning and expressing IRS in course of ontogen- shortened form of the gene of NTPDase2 from A. thaliana esis using real time PCR method. Total RNA was isolated by (without the sequence of transmembrane domain). use of RNeasy Mini kit. Reverse transcriptase reaction was In the first variant of bacteria cultivation E. coli were in- prepared with VerteKit. Primers for PCR were designated duced, one by one, to chaperones and apyrase synthesis. based on already known sequence of IRS in honey bee. PCR Both types of proteins were overexpressed in bacteria product was separated on 1% aggrose gel. Obtained PCR cells. However, overproduced and catalytically inactive product was isolated from gel and purified using PCR puri- NTPDase was accumulated in inclusion bodies. fication kit. PCR product was sequenced by IBB Oligo. Ob- The second variant of bacteria cultivation based on two- tained sequence was analyzed by comparison with honey step cultivation procedure. Bacteria E. coli were induced bee sequence. Both sequences showed approximately 90% to simultaneous synthesis of both types of proteins (like similarity. Expression of cloned fragment was analyzed by in the first variant). After overnight cultivation cells were using real time PCR with LightCycler FastStart DNA Master transfered to the fresh medium supplemented with tet- SYBR Green. As reference gen we used 8S rRNA (primers racycline and the temperature of the cultivation was was prepared based on sequence of honey bee). Expression reduced to 25oC. At the presence of co-expressed chap- was measured during the individual development period: erones, some part of accumulated in inclusion bodies in the egg, larva (1st, 3rd, 6th and 12th day of develop- NTPDase was refolded. Catalytically active protein was ment stage), spinning larva, prepupa, pupa, and male’s and then purified using affinity chromatography methods. female’s imago stages in head and abdomen. Results ob- tained in our experiments showed that IRS gene expression increased during development. In the egg and larva (1st, 3rd, 6th day of development) expression level was similar. Higher level of expression was observed in 12th day’s larva. We found the highest level of IRS expression in the spinning larva and prepupa. In the pupa expression was smaller but higher than at the beginning of the development. In imago stages maximal expression was observed in female’s heads. In abdomen we was not observed significant changes be- tween males and females. Our results confirmed that insulin transduction pathway in invertebrates is very conservative. We suggest that insulin receptor substrate is very essential in red mason bees regulation of development. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 113

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Kinetic analysis of nematode DcpS catalyzed The role of proteins released from hydrolysis of mRNA cap analogues platelets during activation-dependent platelet adhesion to endothelial cells Anna Wypijewska1, Elżbieta Bojarska1, Janusz Stępiński1, Marzena Jankowska- Jakub M. Kryczka, Marta Stasiak, Urszula Kralisz anyszka2, Edward Darżynkiewicz1 Department of Molecular and Medical Biophysics, Medical 1Department of Biophysics, Warsaw University, Warszawa, University of Lodz, Poland Poland; 2Department of Chemistry, Warsaw University, e-mail: Jakub kryczka Warszawa, Poland e-mail: Anna Wypijewska It becomes increasingly evident that blood platelets do not only exert important functions in hemostasis and Hydrolysis of the cap structure plays a pivotal role in eu- thrombus formation but are also involved in atheroscle- karyotic mRNA turnover. Decapping scavenger enzymes rotic vascular disease. The intact, nonactivated endothe- (DcpS) are specific hydrolases catalyzing the cleavage of lium normally prevents platelet adhesion to the arte- the cap structure resulting from the exosom-mediated di- rial wall. Inflamed endothelial cells become adhesive for gestion of deadenylated transcript. In nematodes mRNA platelets. During the adhesion process, platelets become metabolism and cap-interacting proteins must deal with activated and release potent inflammatory and mitogenic two populations of mRNAs, containing trimethylguano- substances into the local microenvironment, thereby al- 2,2,7 7 sine (m3 G) and monomethylguanosine (m G) caps. tering chemotactic, adhesive, and proteolytic properties Nematode DcpS from C. elegans readily hydrolyzes both of endothelial cells. Activation of platelets and endothe- cap structures.The aim of the present study was to give lial cells result in the formation of increased levels of insight into the molecular mechanism of recognition and microparticles, highly proinflammatory and proathero- degradation of structurally different mRNA cap - ana sclerotic bodies distributing substantial amounts of im- logues by the C. elegans DcpS. In the current study we munologically active substances between cells promoting focused on dinucleotide triphosphates cap structures inflammation. The evolving inflammatory reactions are modified in the nucleoside’s base or ribose moiety. For instrumental in the initiation of atherosclerotic plaques quantitative determination of enzymatic hydrolysis we and their destabilization. 7 m 7 chose the following cap analogues: m Gp3G , m Gp3A, In the present study we attempted to study the adhesion 7 6 2,2,7 7,2’O 7,3’O m Gp3m A, m3 Gp3A, m2 Gp3G and m2 Gp3G. of platelets to cultured human umbilical vein endothe- The kinetic measurements were performed by means of lial cells (HUVEC) and immortalized endothelial cell the emission fluorescence spectroscopy. The observed line E.Ahy 926. We have investigated whether the plate- increase of fluorescence intensity after the cleavage of let derived microparticles (PMP) influence the adhesion triphosphate bridge in dinucleotide was used to deter- of platelets to HUVEC and E.Ahy 926. Isolated platelets mine the kinetic parameters Km and Vmax, using the ini- were activated with collagen or thrombin. Endothelial tial velocity method.Hydrolysis studies of selected cap cells (HUVEC and E.Ahy 926) were activated with tumor analogues catalyzed by nematode DcpS showed that all necrosis factor (TNF) or phorbol 12-myristate 13-acetate investigated compounds are specifically degraded by this (PMA). Platelet adhesion was measured using platelets enzyme. Substrate activity studies of adenine-, guanine-, labeled with calcein AM. Activation of platelets resulted 7 2,2,7 ribose-unmodified m G and m3 G-containing cap ana- in the increase in their adhesion to endothelial cells. A logues towards nematode DcpS indicated that these ana- significant increase in adhesion was also observed after logues have similar hydrolytic susceptibility to the en- activation of endothelial cells. Incubation of platelets zyme. The obtained low Km values (below 10 μM), reflect with PMP induced an increase in the platelet adhesion to high specificity to the DcpS from C. elegans.However, the endothelial cells. Activation of endothelial cells induced 7,2’O 7,3’O anti-reverse cap analogues (m2 Gp3G and m2 Gp3G) also the increase in the adhesion of calcein AM labeled subjected to the enzymatic hydrolysis were strongly dis- PMP to endothelial cells. criminated in favour of ribose-unmodified 7m G-contain- We have found that proteins such as RANTES (Regulated 7,2’O ing caps. Especially m2 Gp3G has significantly higher upon Activation Normal T-cell Expressed and Secreted), CD40L Km value in comparison with the standard compound. In (Ligand CD40), and interleukin IL-1b were released from conclusion, we propose that the presence of methyl group platelets during adhesion of platelets to endothelial cells. at 2’-O position in m7G moiety of the cap analogue, steri- These proteins play an important role in activation-de- cally hinders interaction of the DcpS enzyme with a py- pendent platelet adhesion to endothelial cells. rophosphate bridge. This finding may represent a new and important aspect in the mechanism of cap analogues hydrolysis catalyzed by nematode DcpS. Acknowledgements: Supported by Polish Ministry of Science (National Science Sup- port Project 2008-2010, No.PBZ-MNiSW-07/I/2007) and the grant from Howard Hughes Medical Institute to ED (No. 55005604). 114 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Purification of antimicrobial Defining network of interactions for Ruk/ peptides from potato pulp CIN85 adaptor protein by LC-MS/MS Agnieszka Barnyk, Włodzimierz Przewodowski, Serhiy Havrylov, Jolanta Redowicz Jerzy Lewosz, Krzysztof Treder Nencki Institute of Experimental Biology PAS, Warszawa, Department of Potato Protection and Seed Science at Bonin, Poland Plant Breeding and Acclimatization Institute at Radzików, e-mail: Serhiy Havrylov Radzików, Poland e-mail: Agnieszka Barnyk Mammalian adaptor molecule Ruk/CIN85 has been im- plicated in several cellular processes including receptor Antimicrobial peptides (AMPs) have been found in all endocytosis, PI 3-kinase signalling, cytoskeletal rear- groups of organisms. It is commonly accepted that AMPs rangements and apoptosis. To gain new insight into forms the most ancient evolutionary defense mechanism molecular functions of Ruk/CIN85 we have conducted of higher organisms (plants, insects, higher animals and search for novel interaction partners for this protein using humans) against microbial pathogens. Many scientists simple high throughput approach combining GST pull- consider them as a potential new antibiotic class due to down assays and tandem mass spectrometry analyses. their minimal propensity for inducing antimicrobial re- With this approach we identified sets of proteins binding sistance and broad spectrum of action including antibi- to different protein interaction modules of Ruk/CIN85. otic-resistant bacterial strains, viruses, and fungi. AMPs Most of newly found Ruk/CIN85 binding candidates are are usually short, positively charged peptides exibiting membrane and actin regulators. By clustering proteins both pore-forming and intracellular killing activities on involved in membrane and actin remodeling, Ruk/CIN85 a broad range of microorganisms. Common character- probably acts at the interface of membranous and cy- istics of AMPs is ability to form amfipatic conformation toskeletal structures in different membrane trafficking where groups of hydrofobic aminoacids are spatially events both at the cell periphery and in the cell interior. separated from positively charged aminoacids. In small Acknowledgements: group of AMPs this conformation is stabilised by disul- Supported by the Polish Network for Mechanisms of Cell Motil- phide bridges. However most of AMPs occurs in linear ity (Mobilitas.pl). form and rearangment to amphipatic conformation is in- duced only by interactions with cell membranes. Potato tuber contains many different peptide groups rich in cysteines capable to form disulphide bridges. The most known of them are protease inhibitors. Tuber is a storage organ and resides in soil accompanied by milions of mi- croorganisms. Thus it has to posses efficient defence sys- tem. Indeed, several different AMPs were found in tuber juice. Additionally we have detected antimicrobial pep- tides in tuber cell walls. The potato pulp, a waste product of starch industry is mainly composed from cell walls. Therefore we decided to check if it can be a source for purification of antimicrobial peptides. Our preliminary results shows that pulp contains active AMPs which can be purified by combination of high salt extraction, cross- flow fractionation, membrane ion-exchange chromatog- raphy and finall polishing by RP-HPLC. Obtained pep- tides exibited antimicrobial activity against Clavibacter michiganensis ssp. michiganensis, Clavibacter michiganen- sis ssp. sepedonicus, Ervinia carotovora ssp. carotovora and Pseudomonas syringae. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 115

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Expression and purification of ELP-GUSplus Changes of visfatin mRNA and protein fusion protein from transgenic tobacco plants level in white adipose tissue during growth and aging in the rat Piotr Łuchniak, Tomasz Kowalczyk, Katarzyna Hnatuszko-Konka, Andrzej K. Kononowicz Maciej Sassek, Ewa Pruszyńska-Oszmałek, Dawid Szczepankiewicz, Marek Skrzypski, Iwona Department of Genetics and Plant Molecular Biology and Hertig, Piotr Ogrodowicz, Paweł Maćkowiak Biotechnology, University of Lodz, Łódź, Poland e-mail: Piotr Łuchniak Department of Animal Physiology and Biochemistry, University of Life Sciences, Poznań, Poland Elastin-Like Polypeptides (ELP) possess the ability to un- e-mail: maciej sassek dergo a reversible inverse phase transition in response to the changes in the solution parameters (i.e. tempera- Fat tissue is an endocrine organ which produces many ture, ionic strength, pH). This transition from a soluble biological active peptides (called adipokines). These in- polymer to insoluble aggregates is fully reversible upon clude adiponectin, tumor necrosis factor-alpha, leptin and returning the solution parameters to the initial state. The resistin. In 2005 visfatin, as a new member of the adipok- phenomenon described above can be useful in recom- ine family, was added to the list of adipose hormones. binant protein production by fusion of an ELP domain to Visfatin has insulin-mimetic properties such as enhancing the protein of interest, as such recombinant protein can glucose uptake by cultured adipocytes and muscle cells. also undergo inverse phase transition. High selectivity Moreover visfatin affects liver metabolism suppressing of this process provides high purity of isolated protein glucose release by hepatocytes and blocking gluconeo- without cost building chromatographic steps. Transgenic genesis. Literature data suggest also that visfatin plays a N.tabacum lines expressing an ELP—GUSplus fusion pro- role in the pathophysiology of obesity and type 2 diabetes tein were obtained using an A.rhizogenes mediated plant mellitus. transformation methodology. This model peptide was The aim of this study was to determine visfatin expres- purified, proving feasibility of such an approach in plant sion in different depots of white adipose tissue during expression system. The details of the procedure are de- development of rat. Furthermore changes of visfatin con- scribed. centration in serum blood were detected. Acknowledgements: The experiment was carried out using female Wistar This research was supported by the Ministry of Science and rats with an initial body weight 180±20 g. Animals were Higher Education (grant No 2 P04B 029 29) and the University of housed under standard condition, with a 12 h : 12 h light- Lodz (grant No 506/040996). dark cycles, at 22±1oC, with unlimited access to water and food. Animals were divided into four groups at different ages: 4 weeks, 2, 6 and 12 months. After sacrificed by -de capitation blood and subcutaneous, mesenteric, epididy- mal and perirenal pieces of white adipose tissue (WAT) were taken. Fat samples were frozen in Trizol solution and stored in –80oC until analysis. Reverse transcription (RT) were performed using Promega reagents. Expression of visfatin was performed by the use of Real- Time PCR with primers designed for visfatin gene and standard gene HPRT — hypoxanthine-guanine phos- phoribosyltransferase. Visfatin concentration in serum was measured with radioimmunological kit (Phoenix Peptides). The experiment was performed according to rules ac- cepted by the Local Ethical Commission for Investigation on Animals. The presented studies demonstrate changes of visfatin concentration in serum blood during rat development. We also observed differences of visfatin expression in white adipose tissue between experimental groups. Acknowledgements: Supported by the Polish Ministry of Science and Higher Educa- tion grant No N303 085 32/2760. 116 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Preliminary study of visfatin changes Biologically active protein and peptides derived during pregnancy in the rat from European mistletoe (Viscum album L.) Ewa Pruszyńska-Oszmałek, Maciej Sassek, Regina J. Frączek1, Elżbieta Kostyra1, Dawid Szczepankiewicz, Marek Skrzypski, Iwona Małgorzata Mierzejewska1, Małgorzata Hertig, Piotr Ogrodowicz, Paweł Maćkowiak Nowak1, Dominika Świątecka2 Department of Animal Physiology and Biochemistry, 1Department of Biochemistry, University of Warmia University of Life Sciences, Poznań, Poland and Mazury, Olsztyn, Poland; 2Institute of Animal e-mail: ewa pruszyńska Reproduction and Food Research, Olsztyn, Poland

Visfatin, the newly characterized hormone secreted by e-mail: Regina Frączek adipose tissue, is adipokine that work synergistically with insulin by binding to the insulin receptor (at site sep- Mistletoe is a semi-parasitic plant that lives symbiotically arated from insulin). Visfatin, like insulin, induces phos- with several tree species, including conifer and decidu- phorylation of signal transduction proteins and activate ous trees. Mistletoe species grow on a wide range of trees intracellular kinase cascades. Furthermore, like insulin, around the world. The primary chemical constituents of visfatin stimulates glucose uptake by cultured adipocytes European mistletoe has been found to very according to and muscle cells and suppressed glucose release by hepa- the host plant, but typically include lectins, viscotoxins, tocytes. alcaloids, amines, flavonoids, organic acids, terpenes Pregnancy maternal adipose tissue is metabolically active and phenolic compounds. Wiscotoxins and lectins exert and produces adipocytokines in higher concentration in cytotoxic and immunostimulatory activities, that is why comparison to normal metabolic status. In the presence extracts form the mistletoe are often a key component of study we explored changes of visfatin level in serum dur- anti-cancer preparations (e.g. Iscador, Helixor). ing pregnancy and the first 24 hours after delivery in the Electrophoretic separation of the protein fraction extract- rat. Moreover we determined visfatin expression in white ed from mistletoe was according to Laemmli (1970) and adipose tissue. Additionally we investigated lipid and showed 17 fractions. Three of the fractions 34 kDa, 40 kDa carbohydrate metabolism changes. and 53 kDa were indentified as glycoprotein by the use of The experiment was carried out using female Wistar rats PAS method. The separation of purified peptide extract with an initial body weight 180±20g. To obtain pregnant on Tris-Tricine electrophoresis (Schäggera, 1987) showed rats, females were kept with males overnight. Mating was fractions from 6 to 67 kDa. The fraction about 6 kDa were indicated by the presence of sperm in the vaginal spears. purified by the use the ultrafiltration method with 5 kDa Moreover, a non-pregnant control group of rat was kept. centrifugal filter. At days 4, 13 and 18 of pregnancy and at the first day All bacterial strains used in the experiment were resistant after delivery animals were killed by decapitation. The to protein extract from mistletoe. The influence of peptide control females were sacrificed in the diestrus phase of extract and purified fraction weight 6 kDa on microbial the estrous cycle. Visfatin, leptin and insulin concentra- grown. The same zone of inhibition were observed in the tions in serum were measured with radioimmunological case of full peptide extract and 6 kDa fraction in concen- kit. Blood glucose and lipid concentrations were assayed tration 3.3 mg/ml and 5 mg/ml on following strains: Ente- by colorimetric methods. rococcus faecalis, Staphylococcus saprophyticus, Pseudomonas Total RNA was isolated from white adipose tissue sam- aeruginosa, Enterobacter aerogenes. The stronger inhibition ples by use of Trizol solution. Reverse transcriptase re- effect on microbial grown was determined for the puri- action was prepared with Im-Prom II System (Promega). fied peptide fraction than the crude extract. In the case Expresssion was executed by use of real time PCR meth- of 6 kDa fraction an effect in concentration 1.7 mg/ml has od with LightCycler FastStart DNA Master SYBR Green, been observed when this concentration was too low to according to manufacturer’s protocol with our modifica- give any inhibition zone. All tested fungus species were tion. Genes were standardized against the HPRT house- resistant to both peptide’s extracts (crude and purified). keeping gene. The Caco-2 cells growing in 24-well plates were incubated Results obtained in the experiment indicate that pregnan- 24 hours with investigated extracts. All extract was highly cy influenced on the level of serum hormones. The lowest cytotoxic to the Caco-2 cell line. Decrease of intestinal epi- concentration of blood glucose was noticed on days 18 of thelium cell monolayer viability for 10, 40 and 100 ug of gestation. There were also differences of lipid parameters purified peptide extract was 73%, 50% and 29% compar- between experimental groups. ing to control sample. Acknowledgements: Supported by the Polish Ministry of Science and Higher Educa- tion grant No N303 085 32/2760.

Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 117

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The significance of fibronectin adsorption Obtaining and structural examination and arrangement in cell adhesion of the A/B region of the nuclear receptor HR 38 from Drosophila melanogaster Małgorzata Nowak-Wyrzykowska1, Robert Kołos2, Jacek Dobkowski2, Hanna M. Kowalczyńska1 Agnieszka Dziedzic, Grzegorz Rymarczyk, Tomasz M. Kapłon, Andrzej Ożyhar 1Department of Biophysics, Medical Centre for Postgraduate Education, Warszawa, Poland; 2Institute of Physical Department of Biochemistry, Wroclaw University of Chemistry PAS, Warszawa, Poland Technology, Wrocław Poland e-mail: Marzena Juszczak e-mail: Agnieszka Dziedzic Cell adhesion to extracellular matrix (ECM) proteins, such as fibronectin (FN), is essential in organogenesis, Molecular mechanism of the orphan nuclear receptor development, wound healing, tumor invasion and metas- HR38 activity is still not obvious. It is known that this tasis. The interaction of cells with FN provides signals, homologue of rat NGFI-B is one of the key mediators of which affect the morphology and cell shape, motility, ecdysteroid-induced processes in insects. HR38 competes gene expression and survival of adherent cells. Adhesion against ecdysteroid receptor for its dimerization with is mediated through interactions of cell membrane recep- ultraspiracle and is responsive to some ecdysteroids al- tors–integrins with proteins present in the ECM. FN ad- though it has not classical ligand binding pocket. HR38 sorption onto different surfaces results in conformational exhibits characteristic for nuclear receptor modular organ- changes that affect its biological activity. In this study we ization and amino acid sequence of its A/B region reveals examine the early phase of adhesion of L1210 leukaemia some typical features of intrinsically disordered proteins cells to human plasma FN adsorbed on unmodified (non- (IDPs). In this project the A/B region of HR38 nuclear sulfonated) and sulfonated polystyrene (PS) surfaces — receptor from Drosophila melanogaster (dHR38-AB) was in order to understand possible conformational changes studied. We have developed and optimized procedure adopted by FN on these surfaces. We observed a six-fold for efficient expression and purification of homogenous increase of cell adhesion to FN adsorbed on polar surfac- dHR38-AB. The purification method is based on immo- es compared to nonpolar onces. Our results revealed that bilized metal affinity chromatography and size exclusion the cell adhesion to FN adsorbed on sulfonated PS sur- chromatography. Obtaining the target recombinant was face proceeds via the interaction between α5β1 integrins confirmed by immunoblotting and by ESI MS. The gel and the GRGD peptide sequence within the cell-binding filtration experiments exhibited extended conformation domain of FN. In the case of FN on nonsulfonated sur- of dHR38-AB in solution, showing Stokes radius of 31 Å, faces, RGD seems to be masked in the adopted protein which is much higher than the Stokes radius predictable structure, hindering the integrin-ligand interaction. A for a globular protein of the molecular mass of dHR38-AB separate mechanism, which does not involve interactions (24.2 kDa). This observation along with in silico examina- with the α5β1 integrin, has to be responsible for the cell tions signify for the first time that the A/B region of the adhesion to FN on nonpolar substrates. Our image analy- dHR38 displays properties of IDPs. sis method suggests that the pattern of F-actin organiza- Acknowledgement: tion in cells adhering to FN on these surfaces (contrary to This work was supported by Polish Ministry of Science and cells adhering to FN on sulfonated surfaces) is typical for Higher Education grant 2827/P01/2007/32 and by Wrocław Uni- a nonspecific adhesion. 125I radioisotope assays directed versity of Technology. towards FN, as well as ELISA measurements of adsorbed FN (using monoclonal antibodies directed against cen- tral cell binding FN domain) were also carried out. These results revealed the multilayer FN adsorption and indi- cated that more FN molecules are adsorbed on nonpolar than on polar PS. This suggests an unfolded conforma- tion adopted by FN on sulfonated PS, while on nonsul- fonated ones the conformation is likely to be much more compact (globular). Acknowledgements: This work was supported by CMKP grant No. 501-1-1-23-10/05. 118 The Congress of Biochemistry and Cell Biology — Abstracts 2008

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Computer-aided prediction of taste on Starmaker is an extended monomeric protein the example of cytisine derivatives Tomasz Kapłon, Andrzej Ożyhar Dariusz Kikut-Ligaj1, Anna K. Przybył2, Department of Biochemistry, Wroclaw University of Jan Jasiczak1, Agnieszka Skolik1 Technology, Wrocław, Poland 1Faculty of Commodity Science, University of Economics, e-mail: Tomasz Kapłon University, Poznań, Poland e-mail: Dariusz Kikut-Ligaj Fish otoliths are the dense crystals composed of calcium carbonate and an organic matrix. As a biominerals, oto- The molecular mould of the bitter taste receptor imposes liths are primarily involved in gravity sensing and in the a certain specific spatial orientation of the agonist in the perception of sound. The Starmaker (Stm) is a protein, receptor. The computer projections of cytisine derivatives which was the first shown to be capable of influencing on into the molecular mould permitted to rank the bitter the process of biomineralization of otoliths. Stm dictates taste compounds. This observation confirms, that with the shape, size and selection of calcium carbonate poly- increasing size of the substituent at N12 the taste indices morph, in a concentration-dependent manner. characterising these compounds should decrease. The To facilitate understanding of the molecular role Stm pro- results of attested (corroborate) sensory analysis showed tein plays in biomineralization, we have obtained large the bitter taste intensity.The results obtained have con- amounts of pure Stm for in vitro studies. The identity of firmed, that at the border of the sectors A and B of the purified recombinant non-tagged Stm was confirmed by molecular mould are localised on the bipolar subsite of ESI MS showing MW of 64498±2 Da. However, subsequent electron-donor and proton-donor character. gel filtration and dynamic light scattering experiments -re vealed extended conformation of Stm in solution, show- ing Stokes radius of 78.6 Å, which is much higher than expected for a globular protein of the molecular mass of Stm. Monomeric state for Stm was confirmed by native discontinuous PAGE at different concentrations of acryla- mide showing MW of 60 kDa. Circular dichroism experi- ments revealed low content of secondary structures for Stm, what is consistent with its extended conformation. Gel filtration experiments performed in denaturing con- ditions proved, that Stm posses properties of unfolded protein irrespective to concentration of denaturant. A biochemical and biophysical analysis of the recom- binant Stm indicate for the first time that the Stm is a monomer that exhibits properties of intrinsically disor- dered proteins. Moreover, preliminary experiments re- vealed slight change in Stm dimensions upon small Ca2+ ions concentration. Acknowledgements: This study is supported by Polish Ministry of Science and Higher Education grant 2827/P01/2007/32 and by Wrocław Univeristy of Technology. Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 119

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Preliminary purification of bacteriocin Application of DNA electrochemical biosensor from Propionibacterium freudenreichii for toxic compounds present in food culture – effect on antibacterial activity Joanna Jasnowska-Małecka, Marta Ligaj, Daniela Gwiazdowska Mariusz Tichoniuk, Marian Filipiak Chair of Biochemistry and Microbiology, University of Chair of Biochemistry and Microbiology, University of Economics, Poznań, Poland Economics, Poznań, Poland e-mail: Daniela Gwiazdowska ae.poznan.pl>

Bacteriocins are antimicrobial protein or peptides pro- The high quality of food and its health safety requires duces by both bacteria Gram-positive and Gram-nega- the application of appropriate analytical methods during tive. Many of these metabolites are active towards closely food processing, which enable highly sensitive and selec- related species but some of them are able to inhibit bacte- tive detection of particular compounds or microorgan- ria not related with bacteriocin producer including food isms. Such expectations might be fulfilled by biosensors, pathogens such as Listeria monocytogenes or Clostridium analytical devices that exploit biological molecules such botulinum. The most studied bacteriocins are those pro- as enzymes or nucleic acids and antibodies. The biomol- duced by lactic acid bacteria, but there is observed an ecules act as analyte detectors and are in close proxim- increased interest of bacteriocin synthesized by propioni- ity into transducer, which transform effect of biological bacteria in last years. recognition into measurable signal. The use of double The objects of this research comprised bacteriocin ob- stranded DNA as biomolecule gives ability to detect com- tained from Propionibacterium freudenreichii ssp. shermanii pounds which presenting high affinity to DNA helical 2435 culture. Biosynthesis of active protein was conduct- structure and are potentially toxic. ed on MRS and casein medium. Bacteriocin was isolated This paper presents electrochemical DNA biosensor for from culture by ammonium sulfate precipitation and toxic compound which can interact with deoxyribonucle- dialysis. Extracts were preliminary purified by anion ex- ic acid and may be present in food e.g. for heavy metals change and gel filtration chromatography. Every step of and heterocyclic aromatic amines. Heterocyclic aromatic purification was monitored by antimicrobial activity as- amines (HAA) are mutagenic and carcinogenic com- say and SDS-PAGE electrophoresis. Antibacterial activity pounds formed at nanogram levels in protein-rich food was tested by the spot on lawn technique using 24-cells during cooking. These aromatic planar compounds can plates and by adding crude and purified extract to liquid intercalate double helix of DNA. Metal ions can interact medium. P. freudenreichii ssp. freudenreichii 109, P. freuden- with DNA by electrostatic interaction with negatively reichii ssp. shermanii 41 and Aeromonas hydrophila were charged backbone. DNA electrochemical biosensor con- used as indicator strains. sisting of double stranded DNA (dsDNA) immobilized Crude bacteriocin extracts showed inhibitory activity onto carbon paste electrode (CPE) surface. DNA is elec- against examined indicators only in liquid medium. It troactive and on CPE represents two peaks, one at +1V of concerned extracts obtained from propionibacteria cul- guanine and second at +1.3V of adenine oxidation. The tures on both MRS and casein medium. Active proteins method relies on detection of the changes in guanine oxi- caused aggregation of cells, their morphological changes dative signal caused by interacting compound. and inconsiderable decrease of cell number. Inhibition Biosensor signal (the signal of guanine) was dependent was not observed when spot on lawn technique was used. on concentration of both analytes: metal ions (Cu2+) and Crude extract was next purified on DEAE-cellulose col- HAA (2-amino-3-methylimidazo[4,5-f]quinoline — IQ). umn preequilibrated with 10 mM Tris-Cl (pH 8.2). Pro- In the case of copper determination the DNA biosensor teins were eluted from column using linear gradient of signal was decreasing with the concentration of Cu2+ in

NaCl in the same buffer. Separation of protein revealed the range of 0.1 to 100 μM CuSO4. The strongest guanine six fraction. Inhibitory activity against all examined signal reduction to 44% was caused by Cu2+ ions concen- strains showed two last fractions. It suggest that bacte- tration exceeding 1mM. In the case of intercalating HAA riocin produced by P. freudenreichii ssp. shermanii 2435 is the guanine signal was decreasing within the range of 0,1 negatively charged, what is not typical for bacteriocins. to 500 μM 2-amino-3-methylimidazo[4,5-f]quinoline in After first step of purification antibacterial activity- in examined sample. Higher IQ concentrations caused total creased and clear zone of inhibition were observed using guanine reduction. spot on lawn technique. Active fractions were pooled and Noteworthy was that both analytes were determinated used in next purification step on Sephadex G-50 column. beyond the examined sample, e.g. in bulky electrolyte Proteins were separated in two fraction and both of them what can be used for simplifying the analytical process. were active against indicator strains. The reason of that The analytical process lasted 10 min. The presented DNA result was probably tendency to aggregation of bacteri- biosensor provides promising features towards construc- ocin, what was earlier noticed. The research on further tion of simple and fast analytical tool for sensing of food purification and more detailed characteristics of active constituents which can interact with DNA and are poten- protein produced by P. freudenreichii ssp. shermanii 2435 tially toxic for human health. are continued. 120 The Congress of Biochemistry and Cell Biology — Abstracts 2008

P5.69 in 1 ml of hybridization solution containing target DNA in the amount of 1.5 μg. The electrochemical detection Electrochemical determination of of genetically modified RR soybean provided promis- genetically modified soybean Roundup ing features towards construction of similar analytical tools for GMOs sensing. Ready by DNA hybridization biosensor Mariusz Tichoniuk, Marta Ligaj, Joanna Jasnowska-Małecka, Marian Filipiak Chair of Biochemistry and Microbiology, University of Economics, Poznań, Poland e-mail: Mariusz Tichoniuk

The fast and reliable detection of genetically modified organisms (GMOs) is one of the most desirable goals in contemporary food quality assessment. Monitoring of genetic modifications in food components has been de- veloped with the progress in molecular techniques. Usu- ally, GMOs are detected by polymerase chain reaction (PCR), but it is time-consuming, laborious and expensive approach. The sensing of genetically modified food via hybridization of specific DNA sequences with oligonucle- otide probe is very promising technique to implement it in routine tests. Therefore DNA hybridization biosensor is anticipated to be very useful analytical device in this area. Comparing the different combinations of single stranded DNA probe immobilized on the transducer surface the electrochemical sensors characterize very useful abilities. They are relatively simple devices, that enable appropri- ately sensitive and selective detection of target DNA frag- ments. With the further development of nanotechnology they could be easily applied in routine practise. The analytical properties of biosensor are strictly as- sociated with the performance of its detection layer. In order to provide efficient duplex formation, in this work, the probe was immobilized on the gold elec- trode surface by one-point covalent binding through molecular self assembly. The self-assembled monol- ayer of cysteamine provided linker between the gold support and nucleic acid strands. Such immobilized DNA probes formed biosensor detection layer highly accessible for target nucleic acid fragments. The elec- trochemical detection of genetically modified Roundup Ready (RR) soybean by the electrochemical biosensor was accomplished in two ways. In both approaches nu- cleic acid probes with different sequences were used. The first one was specific for 35S promoter, the second – for nos terminator. The identification of one of these regulatory sequences in examined soya DNA sample indicated the presence of RR soybean. The monitoring of hybridization reaction between the probe and target nucleic acid fragments was carried out by voltammetric measurement of methylene blue (MB) accumulated in the biosensor detection layer. The electrochemical in- dicator interacted with exposed guanine bases in DNA strands, which provided higher accumulation rate of MB (and more intensive signal) on the probe than on DNA duplex immobilized on the gold electrode. Sam- ples of DNA isolated from soybean Roundup Ready and from non-genetically modified soybean as a ref- erence material were examined. The identification of samples isolated from RR soybean was accomplished Vol. 55 43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology 121

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Evaluation of hybridization ability of DNA samples isolated from foods by electrochemical biosensors Marta Ligaj, Mariusz Tichoniuk, Joanna Jasnowska-Małecka, Marian Filipiak Chair of Biochemistry and Microbiology, University of Economics, Poznań, Poland e-mail: Marta Ligaj

The DNA electrode interface provides a unique platform for specific DNA sequences identification with the use of electrochemical biosensors. The process of DNA hy- bridization taking place on the detection layer surface is a critical moment of such analysis. Many studies are con- centrated on biosensors construction, but sensitive and selective device for specific DNA sequences detection re- quire suitably prepared sample of isolated DNA contain- ing a target sequence. Damaged nucleotides applied for hybridization can possess the low ability to base-pairing. This may cause a false results of analysis. In our research we applied electrochemical sensors to evaluate conditions of preparation of DNA samples used for hybridization e.g. denaturation and storing param- eters. Investigation was based on the analysis of square wave voltammograms obtained for DNA in acetate buffer solution (pH 4.7) with the use of carbon paste working electrode. Analyzed DNA samples were isolated from foods and denatured before electrochemical examina- tion. Achieved DNA voltammograms revealed two sig- nals: one of guanine (G) at +1.1 V (vs. Ag/AgCl) and sec- ond of adenine (A) at +1.3 V. Thymine and cytidine are significantly more difficult to oxidize, and they were not detected in applied parameters. Sometimes square wave voltammograms revealed additional signal at +0.8 V. It was observed in samples, which were denatured in boil- ing water longer than 5 minutes and current of this peak increased with denaturation time extension. This signal was also observed for DNA stored at -20oC longer than 3 months and in samples repeatedly frosted before electro- chemical analysis. Observation of signal at +0.8 V probably indicated DNA damage. Those DNA samples possessed lower ability for base-pairing, what was monitored with 3+ the use of Co(bpy)3 as hybridization indicator. The pres- ence of typical G and A signals on DNA voltammograms was considered as indication of high quality of isolated samples. Obtained results suggested possibility of electrochemical sensors application for evaluation of hybridization ability of isolated DNA samples. Such information is very im- portant to obtain reliable results of performed analyses.