Abstracts Presented at the 12Th International Congress on Amino Acids, Peptides and Proteins

Amino Acids (2011) 41 (Suppl 1):S1–S86 DOI 10.1007/s00726-011-0955-6

ABSTRACTS

Abstracts presented at the 12th International Congress on Amino Acids, Peptides and Proteins

Beijing, China August 1–5, 2011

Presidents: Dacheng He and Jianguo Ji, Beijing, China

Acknowledgment: We are highly indebted to the Red Bull company for sponsoring the meeting

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data were used to perform a database search using the Mascot Analysis algorithm which led to the identification of major peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6. Particularly, we detected A hydrophobic interaction chromatographic media the APMs DLAFPGSGEQVEKL, SARQQWELQGDRRCQS, prepared from poly (N-isopropylacrylamide-co-methyl RSVNELDLPIL and KRELMNLPQ for Ara h 1, Ara h 2, Ara h 3 and Ara h 6, respectively. These data suggest that enzymatic methacrylate) treatment during EAAE did not eliminate the presence of peanut allergens, however, as a rich source of peptides the protein extracted Junmin Yuan, Jing Li, Chaozhan Wang* through EAAE may have potential for nutritional applications (e.g. in sports nutrition, enteral and infant formulas). Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, China *College of Chemistry and Materials Science, Northwest University, Analysis, characterization and separation of N-acyl 229 Tai Bai North Road, Xi’an 710069, People’s Republic of China, derivatives of 2,6-diaminopimelic acid by capillary zone Tel: +86-29-88302604, [email protected] electrophoresis and micellar electrokinetic chromatography A series of poly (N-isopropylacrylamide-co-methyl methacrylate) with different ratios of N-isopropylacrylamide to methyl methacrylate were synthesized, and the polymers were grafted to silica gel Miloslava Vı´tovcova´, Jan Hlavaćˇek, Jan Pıćha, Vaćlav Vaneˇk, to prepare hydrophobic interaction chromatographic media. The Jirˇ´ı Jiraćˇek, and Vaćlav Kasˇicˇka separation efficiency of the prepared media was tested by using a standard protein mixture, it was found that the media has quite Institute of Organic Chemistry and Biochemistry, Academy good performance for protein separation. Effects of temperature on of Sciences of the Czech Republic, 166 10 Prague, Czech Republic protein separation and retention were investigated, the results showed that the hydrophobicity of the media increased gently with Recently, the mono-N-protected derivatives of 2,6-diaminopimelic increasing the ratio of methyl methacrylate in the grafted polymer. acid (DAP) were prepared to function as competitive inhibitors of For a given prepared media, the retention of proteins increased the mono-N-succinyl-DAP hydrolysis catalyzed by dapE enzyme, when the column temperature was elevated, and the resolution of with aim to interrupt the pathways leading to development of bac- proteins changed dramatically with varying temperature. The pre- teria. In this work, capillary zone electrophoresis (CZE) and pared chromatographic media was used for purification of micellar electrokinetic chromatography (CMEKC) were employed myoglobin from pig heart as well as lysozyme and ovalbumin from for determination of the electrophoretic purity degree, limit of hen egg white. detection (LOD) and limit of quantification (LOQ) of twelve mono- This work was supported by the National Natural Science Foun- N-acylated derivatives of DAP, using an HPCE analyzer equipped dation in China (No. 20705028). with fused silica capillary (50/375 lm id/od, 400/290 mm total/ effective length), UV-photometric detector operating at 206 nm and high-voltage power supply (30 kV, 200 lA). The DAP derivatives were characterized by effective electrophoretic mobilities in several Allergens presence in peanut proteins extracted classical and isoelectric buffer-based acidic and alkaline background by enzymatic aqueous extraction: analysis by mass electrolytes within a pH range 2.18–8.64. A new procedure has been spectrometry developed for correction of effective mobilities to the reference temperature of 25°C. The best separation of the mixtures of DAP

1* 2 1 1 derivatives was achieved by CMEKC in acidic background elec- S. Latif , J. Pfannstiel , H. P. S. Makkar and K. Becker trolyte (500 mmol/L acetic acid, pH 2.54) using anionic surfactant

1 60 mmol/L SDS as a constituent of the micellar pseudostationary Institute for Animal Production in the Tropics and Subtropics (480), phase. University of Hohenheim, 70593, Stuttgart, Germany 2 Acknowledgments: Supported by the Czech Science Foundation Life Science Center Core Facility, University of Hohenheim, (203/08/1428; 203/09/0675) and the Academy of Sciences of 70593 Stuttgart, Germany the Czech Republic (GAV A400550614; Research Project AV040550506). Enzyme-assisted aqueous extraction (EAAE) is a promising green alternative for simultaneous oil and protein extraction from peanut. Furthermore, enzymatic treatment is considered to eliminate some food allergens. On the basis of allergen peptide markers (APMs), a Chemical ligation: an effective tool to characterize proteomic-based approach was used to evaluate the allergens pres- the interaction between Nedd4L WW domains ence in the protein extracted through EAAE. Recoveries of 92% oil and the Smad3 linker region by NMR and 83% protein were obtained on subjecting peanuts to EAAE with alkaline protease derived from Bacillus licheniformis under an optimized set of conditions. The protein fraction extracted through Nina Goerner EAAE was subjected to matrix-assisted laser desorption/ionization- time of flight mass spectra (MALDI-TOF/MS) which revealed it as Smad proteins are the mediators of the TGF-b signalling pathway a rich source of peptides. Peptides (107 in number) with molecular that controls cell growth and specific cell differentiations. Defects masses ranging from m/z 700.39–2,369.08 Da were identified. In in this pathway can lead to rapid cell growth resulting in cancer order to get detailed information on peptide sequences, the protein diseases. Smad proteins interact with other proteins through their fraction was further analyzed by nano-LC–ESI-MS/MS on a high linker region that connects a DNA binding domain named MH1 and resolution mass spectrometer (LTQ-Orbitrap XL). The ESI-MS/MS a TGF-b-receptor-binding domain known as MH2 domain. The

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Smad2/3 linker region consists of four phosphorylation sites and a Enantioselective separation of branched amino acids PPxY (PY) motif that is recognized by WW domains. WW domains using various separation systems/modes in HPLC are small binding domains present in around 170 proteins of variable function, which are involved in many cellular processes, such 1 2 4 as transcription, splicing or protein degradation to mention a few. Kveˇta Kalı´kova´ , Pavel Repko , Marta Kowalczyk , 2 ˇ 3 1 We have characterized in detail the interaction of Nedd4L and a Zuzana Bosa´kova´ , Juraj Sevcˇ´ık , and Eva Tesarˇova´ fragment of Smad3 linker including the PY site and also combi- 1 nations of the phosphorylated residues. To obtain the structural Department of Physical and Macromolecular Chemistry, information we have used protein constructs containing pairs of Faculty of Science, Charles University in Prague, Albertov 2030, 128 43 Prague 2, Czech Republic domains and a segmental labelling approach that requires the use of 2 a chemical ligation strategy. Department of Analytical Chemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 128 43 Prague 2, Czech Republic 3Department of Analytical Chemistry, Faculty of Science, Palacky University in Olomouc, Czech Republic Chiral discrimination of amino acids using achiral 4Department of Analytical Chemistry, Faculty of Science, University electrophoresis separation hyphenated with mass of Warsaw, Poland, [email protected] spectrometry Enantiomers of branched amino acids (BAAs), namely alanine, Vaćlav Ranc1, Radim Knob1, Jan Petr2, Vitezslav Maier2, valine, leucine and isoleucine, are present in fresh food. Their Eva Tesarova3, and Juraj Sevcik1 D-/L enantiomeric ratio can give information about origin of the food product and/or can be used for indication of the quality of food. In 1Department of Analytical Chemistry, Faculty of Science, addition BAAs are important components of food supplements, in Palacky´ University in Olomouc, 17. Listopadu 12, 77146 Olomouc, which just L-enantiomers should be found. Czech Republic Separation and determination of enantiomers of amino acids can 2Regional Centre of Advanced and Technologies, Department be achieved in various separation systems. In HPLC mostly chiral of Analytical Chemistry, Faculty of Science, Palacky´ University stationary phases are employed. in Olomouc, 17. Listopadu 12, 77146 Olomouc, A problem in the branched amino acids determination can cause Czech Republic low sensitivity of UV detection. Therefore, derivatization of these 3Department of Physical and Macromolecular Chemistry, amino acids is usually required. Faculty of Science, Charles University in Prague, Albertov 2030, In this work, enantioseparation of BAAs was performed in various 128 40 Prague 2, Czech Republic, separation systems employing different chiral stationary phases in various separation modes. The best enantioseparation was achieved The study of chiral body constituents like amino acids (AAs) repre- on a teicoplanin based column in reversed phase mode or in hydro- sents one of the frequent tasks of modern analytical chemistry. This philic interaction liquid chromatography (HILIC). increasing demand tension for a development of chiral methods is In order to improve sensitivity of detection two derivatization given by a large number of physiological processes involving studied procedures, in which dansyl chloride and fluorenylmethyl chlorofor- compounds. As analytical tools, chromatographic or electrophoretic mate were used as derivatization agents, were tested and compared. In approaches are usually employed. Mass spectrometry could present both cases derivatization of BAAs resulted in decreasing LOD and an interesting alternative. LOQ values, approximately two orders of a magnitude, as compared This work deals with a mass spectrometric behavioral study of to underivatized BAAs. amino acids, where their ability to form host–guest clusters with chiral host is evaluated. This pilot demonstration is based on the chiral discrimination of selected target analytes: D,L-Ala, D,L-Phe, and EXAFS and XANES analysis of the Fe and Zn D,L-Val, where L-Trp was chosen as the chiral host. environment in tissue samples at different growth stages Firstly, the target amino acids were separated by foregoing electrophoresis using 0.75 M formic acid as a background elec- trolyte. After the separation step, they AAs were injected to the Sunil Dehipawala, Todd Holden, E. Cheung, Robert Regan, MS part using electrospray ion source. Sheath liquid (water– P. Schneider, G. Tremberger Jr, D. Lieberman, and T. Cheung methanol 1:1, v/v) contained host compound (total concentration = 1 9 10-3 M). City University of New York, Queensborough Community College, Chiral discrimination was calculated using relative intensities 222-05 56th Ave, Bayside NY 11364, USA of protonated host molecule and cluster containing host and guest molecule. It was shown that the ratio of these two relative The zinc and iron environments in different growth stages have been intensities is proportional to the chiral composition of initial studied with EXAFS and XANES with Brookhaven Synchrotron sample. Light Source. Tissue samples included meat, organ, vegetable, bean, Quantification of chiral composition (for a determination of opti- leaf, and yeast. The EXAFS data was background subtracted and cal purity) is based on five point calibration curves containing 0, 25, standard Fourier component analysis was used. The XANES spectra 50, 75 and 100% of corresponding D-enantiomer for a determination were analyzed with standard methods including the use of a linear of optical purity. Represented correlation coefficients are 0.986 for combination of XANES standard spectra. Ala, 0.995 for Phe and 0.997 for Val, respectively. The Zn nearest neighbor bond length deduced from EXAFS and This work was supported by the Research Project of the Ministry XANES are found to be associated with aging in bean, leaf, yeast of Education of the Czech Republic No. MSM6198959216, by a grant samples. Zn signal in chayote pith is higher than those in the seed agency of the Palacky University PRF_2011_025 and by Operational and skin. Edible chicken tissues show that the liver has the Program Research and Development for Innovations–European smallest Zn signature while thigh tissue has the highest as com- Social Fund (project CZ.1.05/2.1. 00/03.0058). pared to drumstick, neck, breast, and wing tissues. Duck embryo

123 S4 12th International Congress on Amino Acids, Peptides and Proteins tissues from an edible source (balut) show that the heart has the peptide and protein chemistry. However, the polar analytes occur smallest Zn signature as compared to liver, brain, stomach, intes- mostly in aqueous media and need to be converted into volatile, tine, and thigh. The accompanying yolk and white show negligible thermally stable derivatives prior to GC. Two decades of extensive zinc and would be consistent with the early utilization of zinc research have proved alkyl chloroformates highly attractive for this during embryo growth. The Fe nearest neighbor bond length purpose. The reagents react immediately with most amino acid analysis show similar results, but is less striking as a bio-marker functional groups in aqueous matrices and the process can easily when compared to Zn. Nevertheless the Fe environment changes in be coupled with liquid–liquid extraction of the resulting less polar calf liver after cooking as was detected in XANES data. The derivatives into immiscible organic phase. Here a novel, simple results suggest an effective synchrotron probe for the study of protocol for in situ derivatization of amino acids with heptaﬂuo- cellular changes in tissue samples as well as the effect of microbe/ robutyl chloroformate (HFBCF) is described followed by virus in diverse environments such as bio-energy production. subsequent nonchiral as well as chiral GC/MS (mass spectrometric) Extension to cobalt in vitamin B12 will be discussed. analysis on a respective nonpolar fused silica and an enantioselective Chirasil-Val capillary column. The novel amino acid assay is presented by both nonchiral GC/MS analysis of more than ﬁfty amino acids in aqueous matrices and, simultaneously, by chiral Non-destructive analysis of coffee by NMR investigations of more than 25 resolved enantiomeric antipodes in spectroscopy peptide hydrolysates, human serum and environmental samples. Acknowledgments: The Czech Science Foundation, projects No. Feifei Wei, Kazuo Furihata, Fangyu Hu, Takuya Miyakawa, 203/09/2014 and P206/10/2401. Masanori Koda and Masaru Tanokura

Modern nuclear magnetic resonance (NMR) spectroscopy with Phylloseptin-2 conformational analysis by solution dramatically improved resolution and sensitivity has been widely applied in food science to achieve a direct and comprehensive NMR at different pH values observation of foods in a non-destructive way, as it is capable in a rapid, single experiment of simultaneously detecting multiple Dorila Pilo´-Veloso*, Bruno Aldrin Assunçaõ, Naira de Oliveira Toˆrres, components without destruction of the food. By identifying the Rodrigo Moreira Verly, and Jarbas Magalha˜es Resende different spin systems from appropriate two-dimensional NMR spectra, comprehensive analysis with NMR makes it possible to Departamento de Quı´mica/ICEx/UFMG/Brazil identify and quantify the components of foods as complex mixtures [email protected] without physically separating them. Coffee is one of the most consumed beverages in the world. The analyses on the chemical Keywords: Peptide antibiotics, Peptide structure, Amphipathic compositions of coffee are still necessary since it is closely related a-helix, Solution NMR, Conformational analysis to the quality, security and nutrition of coffee. The cationic peptide Phylloseptin-2 (PS-2) has been isolated from A complex mixture analysis by one- and two-dimensional NMR the skin secretion of the tree-frog Phyllomedusa hypochondriasis that spectroscopy was carried out for the identification and quantifica- inhabits Brazilian tropical forests.2 It is naturally amidated at C-ter- tion of organic compounds in green and roasted coffee without any minus, is composed of nineteen amino acid residues, of which three separation. A detailed NMR signal assignment, as well as quanti- possibly charged histidines are at positions 7, 17 and 18. Antimi- fication, coupled with multivariate statistical chemometric methods crobial assays demonstrate that it exhibits activity against both Gram- offers a powerful new approach for coffee bean quality control and positive and Gram-negative bacteria.1,2 assessment. As an important result of this study, amino acids can We have previously described the NMR structure of PS-2 in TFE- 2 be used as significant marker compounds to distinguish the species d2:H2O (60:40, v/v) at pH 7.0 (phosphate buffer). In this work it is of green coffee beans. presented the conformational study of this peptide in TFE-d2:H2O (60:40, v/v) at pH 5.6 and 8.6. TOCSY, NOESY, 1H-13C HSQC and 1H-15N HMQC experiments were acquired at 20°C on a Bruker AVANCE-III 800 MHz spectrometer equipped with a triple resonance Nonchiral and chiral gas chromatographic: mass (1H/13C/15N) 5 mm gradient probe. spectrometric analysis of amino acids in aqueous The assignment of backbone atoms was performed by the solutions. Application of a novel heptafluorobutyl sequential assignment methodology.3 The NMR spectra were pro- chloroformate sample preparation approach cessed with NMRPipe and then analysed using NMRVIEW. The obtained NOE intensities were converted into semi-quantitative dis- to investigations in amino acid and peptide chemistry tance restrains and then 200 structures were calculated using Xplor- NIH. The quality of the lowest energy structures was validated by Petr Sˇimek*, Helena Zahradnıćˇkova´, and Petr Husˇek PROCHECK-NMR. The display, analysis, and manipulation of the 3D structures were performed with MOLMOL. These structures were Department of Analytical Biochemistry, Biology Centre, Academy compared with the published results.2 of Sciences of the Czech Republic, Branisˇovska´ 31, 37005 Cˇ eske´ As a result the most stable structures indicate that PS-2 main- Budeˇjovice, Czech Republic, [email protected] tains an amphipathic distribution of its residue side chains within the helical segment, albeit the presence or absence of a Nonchiral and chiral gas chromatographic (GC) amino acid anal- positive charge on the His residues leads to important structural ysis has been an important research tool in the field of amino acid, differences.

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Preparation and characterization of a hydrophobic inter- Quantitative and qualitative proﬁling of mitochondrial action chromatographic media with a polymeric ligand DNA in antlers individuals

Hua Fan, Xiaohui Tang, Lili Wang, and Chaozhan Wang* Young Hwa Kima, Jae Woong Leea, Byong Seob Koa, Seung-Eun Ohb, Mi Young Leea,* Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry aAging Research Center, Korea Institute of Oriental Medicine, and Materials Science, Northwest University, Xi’an 710069, China Daejeon 305-811, Korea, Tel: +82 42 868 9508, *College of Chemistry and Materials Science, Northwest University, Fax: +82 42 868 9301, [email protected], 229 Tai Bai North Road, Xi’an 710069, People’s Republic of China. bDepartment of Biological Sciences, Konkuk University, Tel: +86-29-88302604, Email: [email protected] Seoul 143-701, Korea, [email protected]

A series of temperature responsive polymer with different lower critical The analysis of mitochondrial DNA sequence variation is of great solution temperatures were synthesized by using chain transfer free interest in medical, forensic, and population genetics. Quantitative radical polymerization. And a novel hydrophobic interaction chromato- and qualitative analysis of mitochondrial DNA length for performed graphic media, with one of the synthesized polymers as ligand, was using agarose gel electrophoresis, capillary electrophoresis, quanti- prepared for protein separation. The prepared media was used to separate tative real-time PCR, and allelic discrimination by sequence-specific a protein mixture containing seven proteins, and was compared with two based antlers. Specific primers and fluorescence tags were designed normal hydrophobic interaction chromatographic media, with phenyl by using mitochondrial D-loop genes for various Cervus subspecies and PEG 600 as ligands, respectively, under various temperatures and and Rangifer tarandus. When primers that differentiated Cervinae solution compositions. It was found that the newly prepared chromato- and Rangifer antlers were used to amplify mtDNA, a 466 bp frag- graphic media has much better performance for protein separation than ment that was observed for both Cervinae and Rangifer antlers media with phenyl and PEG 600 as ligands. Moreover, it is more sensitive served as a positive control, while a 270 bp fragment was specific for to temperature, and selectivity for proteins can be easily modulated by Rangifer antlers. Allele discrimination was used to differentiate varying temperature. With temperature increasing, its hydrophobicity is Cervinae and Rangifer antlers on the basis of the amplification of increasing correspondingly. Therefore, a series of hydrophobicity could specific alleles for both types of antlers. These PCR-based assays can be obtained on one column packed with the prepared media, which is very be used for quantitative and quantitative analyses of Cervinae and important for separation of very complex protein samples. Rangifer antlers in a single step without any sequence information. This work was supported by the National Natural Science Foun- In addition, multiple PCR-based assays are more accurate and dation in China (No. 20705028), National Fund for Talent Training in reproducible than a single assay for species-specific analysis and Basic Science (No. J0830417). especially in this study for the identification of deer products Cer- vinae from Rangifer antlers.

Purification of cytochrome c from pig heart by using frontal hydrophobic interaction chromatography Specific modification of amino acids to induce separation Rong Chang, Xiangqin Chen, and Chaozhan Wang*

Key Laboratory of Synthetic and Natural Functional Molecule Yinglai Teng, Elinor L. Scott and Johan P. M. Sanders Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, China Amino acids (AA’s) are interesting raw material as the feedstocks *College of Chemistry and Materials Science, Northwest University, for the chemical industry due to the functionalities they contained. 229 Tai Bai North Road, Xi’an 710069, People’s Republic of China. This makes them similar to conventional petrochemicals and pro- Tel: +86-29-88302604, [email protected] vides the possibility to circumvent process procedures, additional reagents and energy. They could be obtained as a mixture by Protein separation and purification is one of the most important steps hydrolysis of potentially inexpensive protein sources such as agri- during production of protein products, especially protein drugs. culture waste or the side stream of biofuel productions. Still, Cytochrome c is a small heme protein found loosely associated with separation is required in order to carry out further transformation the inner membrane of the mitochondrion, it is an essential component into the desired products. Theoretically it could be achieved by of the electron transport chain, where it carries one electron. In this electrodialysis (ED). To increase the efficiency of separating AA’s work, cytochrome c was extracted from pig heart, and ammonium with similar charge behavior, specific modification, preferably to the sulfate precipitation was used as the first step for cytochrome c puri- products with industrial interest, is required. This could be achieved fication, in which cytochrome c was precipitated, and frontal by enzymatic reaction such as decarboxylation. Here we focus on hydrophobic interaction chromatography (FHIC) was employed to the specific modification for the separation of basic AA mixture further purify cytochrome c to homogenous. Various concentrations of containing L-arginine (Arg) and L-lysine (Lys). L-lysine decarbox- ammonium sulfate in the mobile phase of FHIC were investigated, and ylase (LDC) was applied to convert Lys to 1,5-pentanediamine a suitable concentration under which cytochrome c was not retained on (PDA) in the presence of Arg to obtain products with different 1 the column but impurities retarded was selected. The cytochrome charge so as to be separated by ED. Immobilization of LDC was c precipitated by ammonium sulfate was solubilized in this concen- performed to enhance the operational stability. Though product tration of ammonium sulfate and was pumped onto the column to be inhibition was seen, the enzyme is highly specific and at 30°C the purified by using FHIC. Cytochrome c with a purity of 99% was presence of Arg has little effect. Based on these, a potential process obtained finally, almost all cytochrome c was recovered during FHIC. using a mixture of Arg and Lys was designed and the cost was This work was supported by the National Natural Science Foun- estimated based on the volumetric productivity, raw material price dation in China (No. 20705028). and transformation costs.

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Structure determination at molecular resolution using found to be 1:1 ratio by the Job’s and straight line methods atomic force microscopy: focusing on artifacts between donors and acceptor. The observed data were discussed in terms of equilibrium constant (K ), molar extinction coefficient and reproducibility CT (CT), thermodynamic standard reaction quantities (DG°, DH° and DS°). Different variables affecting the reaction were studied and Yang Gan optimized. At the optimum reaction conditions Beer’s law was obeyed in a concentration limit of 1–18 lgml-1 for taurine. The School of Chemical Engineering and Technology, Harbin Institute developed method was applied successfully for the determination of Technology, Harbin, Heilongjiang 150001, People’s Republic of taurine in drug preparation, and relative standard deviation of of China, [email protected] the method was 0.58–2.63%. Percentage recoveries ranged from 98.5 to 102.8%. Despite the enormous success and wide application of Atomic Force Keywords: Taurine, 7,7,8,8-Tetracyanoquinodimethane (TCNQ), Microscope (AFM) in various areas including biology and chemistry, Charge transfer complex, Spectrophotometry many researchers have still been conservative about the capability of AFM to achieve atomic and molecular resolution in ambient conditions. This scenario was formed, historically, due to numerous inappropriate declarations of obtaining so-called atomic and molec- Study on enzyme kinetics of glutamic pyruvic ular resolution AFM topographs. In the last 15 years, reports of true transaminase and amino acids conversion by microchip atomic and molecular resolution in ambient conditions were by no electrophoresis means scarce; nevertheless, some have been overlooked and are rarely known, even to an AFM specialist! The AFM community could 1,2 1, 1 1,2 hardly benefit from this overlook because an AFM user, trained with Xiaoyu Mu ,LiQi *, Juan Qiao , Haizhi Zhang this biased philosophy, might be frustrated and regard the challenge of 1 achieving trustworthy high resolution as an impossible task. Instead, Key Laboratory of Analytical Chemistry for Living Biosystems, one needs to appreciate the power of AFM as an atomic and Institute of Chemistry, Chinese Academy of Sciences, 100190 Beijing, China molecular scale analytical technique that is capable of producing 2 reliable and reproducible topographs with various operation modes; at Graduate School, Chinese Academy of Sciences, the same time, one needs to learn that AFM, as a local probe tech- 100049 Beijing, China, Tel: +86-10-82627290, nique, is particularly vulnerable to artifacts. An experienced AFM Fax: +86-10-62559373, [email protected] user should be able to identify artifacts, avoid artifacts, and live skeptically with artifacts if necessary. In this work, we firstly presented an effective microchip electropho- This presentation, based on my review article, will review critically resis-laser induced fluorescence (MCE-LIF) technique to analyze and timely the achievements molecular resolution in ambient condi- amino acids (including L-Glu and L-Ala) with ethylene oxide/propyl- tions by AFM. The concept of resolution is discussed after a brief ene oxide block copolymer as the surfactant by using the micellar introduction of various AFM operation modes. Various types of tip- electrokinetic chromatography (MEKC). Then a new method based on surface forces, particularly the forces prominent in liquid and in air, the determination of the variation of the concentrations of L-Glu and are introduced. Different viewpoints on the conditions for achieving L-Ala was developed to assay the glutamate pyruvate transaminase molecular resolution are discussed. The important issues of repro- (GPT) activity directly. Both the forward catalyzed reaction and the ducibility and artifacts are discussed in depth, with some examples reverse catalyzed reaction were investigated to study the kinetics of taken from the latest biology literature. Finally, the challenges of AFM GPT by monitoring the concentration changes of L-Glu and L-Ala, and K V L as a trustworthy high resolution technique are discussed. the m and max were 3.6 mM, 0.2 mM/min for -Glu and 10.6 mM, 0.5 mM/min for L-Ala, respectively. Furthermore, this MCE-LIF system was applied in the study of GPT activity in serum. The results not only could help us to understand the clinical mechanisms of grip- Study of spectrophotometric characteristics ping hepatic diseases, but also showed the potential of exploring other of the charge transfer reaction of taurine transaminase activities simply by the new method. with 7,7,8,8-tetracyanoquinodimethane

Shengyun Li The advantages of performing interaction assays in complex matrices, particularly native membranes Department of Chemistry, Taiyuan Normal University, Taiyuan, Shanxi, People’s Republic of China Darryl J. Bornhop1, Amanda Kussrow1, Michael Baksh2, M. G. Finn2, 3 3 3 Charge transfer (CT) complex of taurine drug as electron donor Paige E. Selvy , H. Alex Brown , and Craig W. Lindsley with 7,7,8,8-tetracyanoquinodimethane (TCNQ) as electron accep- 1 tor has been studied in Britton-Robinson buffer solution. The Department of Chemistry and The Vanderbilt Institute for Chemical spectrum obtained for taurine/TCNQ system shows the maximum Biology, Vanderbilt University, Nashville TN, 37235, USA 2 absorption band at wavelength of 420 nm, which is not due to the Department of Chemistry and The Skaggs Institute for Chemical absorption of any of the reactants. This band is characteristic of an Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., intermolecular charge transfer involving the overlap between the La Jolla, CA, 92037, USA 3 lowest unoccupied molecular orbital (LUMO) of the acceptor with Department of Pharmacology, Chemistry and The Vanderbilt the highest occupied molecular orbital (HOMO) of the donor. The Institute for Chemical Biology, Vanderbilt University Medical results reveal that the interaction between the donor and acceptor is Center, Nashville, TN, 37232, USA due to n–p* transitions by the formation of radical ion pairs. The formation of such complex was conﬁrmed by both infrared and 1H Though membrane-associated proteins are ubiquitous within all living NMR measurements. The stoichiometry of the complex was also organisms and represent the majority of drug targets, a general

123 12th International Congress on Amino Acids, Peptides and Proteins S7 method for the direct, label-free measurement of membrane-bound Biochemistry protein–ligand binding within the native bilayer environment has not been reported. In this presentation it will be shown that backscattering interferometry (BSI) is a viable tool for quantifying binding affinities in such systems. BSI relates minute changes in the refractive index of Analysis of skin secretions from the Lithobates a solution to equilibrium binding constants over a range of several pustulosus frog using high-resolution mass orders of magnitude. Values obtained by BSI for small- and large- molecule interactions with receptors in vesicles derived from syn- spectrometry thetic lipids and from cell extracts were found to be in good agreement with those previously obtained by indirect methods. The Emmanuel Rıós, Erika Patricia Meneses, Lorena Hernańdez possibility of using BSI as a generally applicable methodology for and Cesar V. F. Batista quantifying membrane-associated protein–ligand interactions of biochemical and pharmacological interest, over a wide range of binding Laboratorio Universitario de Proteo´mica, Instituto de Biotecnologıá, affinities, will be discussed. UNAM Av. Universidad, 2001, Col. Chamilpa, CP 92210, Further, we show that is has been possible to measure changes in Cuernavaca, Mor., Me´xico www.ibt.unam.mx interfacial and kinetic parameters on in vitro reconstituted extruded lipid vesicles and purified protein with BSI. These studies charac- The lack of efficacy of conventional antibiotics against an increasing terized the molecular mechanism of inhibition for a recently number of pathogenic microorganisms constitutes a serious public identified class of potent and isoform-selective small molecules that health problem. One of the strategies applied in order to overcome block phospholipase D (PLD) activity and compared activity to microorganism resistance resides in the search for novel antimicrobial other known small molecule inhibitors of mammalian PLD. Our agents. Antimicrobial peptides (AMPs) are widespread in living preliminary results confirm that BSI is a useful nanoscale technique organisms and constitute an important component of innate immunity to measure Ki and Kd, indicates these new compounds disrupt against microbial infections. Amphibians are known to produce a large interfacial binding and bulk lipid binding disruption (Ks) can be number of amphipathic peptides which manifest a broad range of quantified with BSI. pharmacological activities. They have been shown to inhibit the growth of numerous species of bacteria, fungi, protozoa, and also some types of tumor cells without evidence of significant hemolytic activity. Litho- bates pustulosus is an endemic Mexican frog which inhabits the deciduous forest of Morelos State and to the best of our knowledge, no data is available in the current literature regarding the composition of Using SR-IMS technique with synchrotron for imaging its skin secretions. At present, liquid chromatography coupled with molecular chemistry of protein high-resolution mass spectrometers constitute the most powerful analytical procedure for studying complex mixtures of peptides and proteins. Here we used a LC–MS system composed of a nanoflow Peiqiang Yu liquid chromatography coupled with an Orbitrap Velos mass spectrometer to determine the molecular mass of all components and the College of Agriculture and Bioresources, The University primary structure of most of these. Primary structure comparison was of Saskatchewan 51 Campus Drive, Saskatoon, SK, S7 N 5A8, searched against a public data bank showing that L. pustulosus skin Canada, [email protected] secretion is mainly composed of ranatuerin, brevinin, esculetin and bradykinin related-peptides. Analysis of large proteins by gel electro- The cutting-edge synchrotron-radiation infrared microspectroscopy phoresis was also performed and many tryptic peptides were de novo (SR-IMS) is able to study cell or living cell biochemistry at cel- sequenced using CID and HCD dissociation methods. lular and molecular levels. The objective of this synchrotron-based research program was to use the advanced synchrotron-based SR- IMS technique as a novel approach to image the molecular chemistry of protein and image cell architecture in plant-based feed Anti-inflammatory effect of poly-L-lysine in human and food tissues. The experiments were conducted on beamline intestinal epithelial cells mediated by calcium-sensing U2B at the National Synchrotron Light Source, Brookhaven receptor activation National Laboratory (NSLS-BNL, U.S. Department of Energy, New York, USA) and Saskatchewan Structural Sciences Center (SSSC, Saskatoon, Canada). The protein molecular images were system- Hua Zhang and Yoshinori Mine atically carried out from various structural layers under various chemical functional groups which were related to protein struc- Department of Food Science, University of Guelph, Guelph, ON, tures. The results showed that with the advanced SR-IMS N1G2W1, Canada plus multivariate molecular spectral analyses, unique cell architecture, the molecular spatial distribution, intensity and nutrient The calcium-sensing receptor (CaSR) is involved in maintaining make-up and conformation were revealed at an ultraspatial reso- cellular homeostasis and promoting recovery of damaged intestinal lution. This imaging technique provides a high potential that could epithelial cells (IECs). Poly-L-lysine is a basic polypeptide which has be used to develop a specific plant/feed/food with targeted nutrition been identified to activate CaSR through allosteric binding. The pri- qualify. mary goal of the current study was to identify anti-inflammatory Keywords: Synchrotron, SR-IMS, Protein image, Biological effect of poly-L-lysine on the human intestinal epithelial cell system, tissue and verified that these effects are mediated by CaSR activation.

123 S8 12th International Congress on Amino Acids, Peptides and Proteins

Human intestinal epithelial cell lines, Caco-2 and HT-29, were used been studied for many years. Recent research has shown that glycation as in vitro model to study intestinal inflammation. The CaSR acti- of plasma proteins by the physiological dicarbonyl compound, meth- vation induced by poly-L-lysine was determined by measuring ylglyoxal, forms quantitatively important advanced glycation intracellular calcium releasing. The Caco-2 or HT-29 was pre-incu- endproducts (AGEs). We investigated the impact of glycation by bated with poly-L-lysine for 2 h and the following inflammation was methylglyoxal on the structure and function of LDL and HDL—par- induced by recombinant human tumor necrosis factor (TNF)-a (2 ng/ ticularly in relation to the atherogenic transformation of LDL and the mL). The secretion level of interleukin (IL)-8 from IECs which is decreased stability in HDL associated with increased risk of cardio- recognized as a biomarker indicating the inflammation was measured vascular disease. We studied the effect of glycation of LDL, HDL2 and by sandwich enzyme linked immunosorbent assay (ELISA). The HDL3 by methylglyoxal to physiological extent on particle size, aortal poly-L-lysine reduced the IL-8 secretion from TNF-a-induced IECs. trapping and functional activities. LDL and HDL subfractions were The gene expressions of several pro-inflammatory cytokines, purified from venous plasma of healthy human volunteers by density including TNF-a, IL-6 and IL-1b, were also inhibited by poly-L-lysine gradient centrifugation and purity confirmed by SDS-PAGE. LDL and supplementation. Both anti-CaSR antibody and antagonist (NPS2143) HDL modified minimally by methylglyoxal were prepared. Choles- were used to block poly-L-lysine-mediated CaSR pathway and the terol and triglyceride contents and electron microscopy particle size suppression of IL-8 secretion in both IECs was abolished. Our current were analysed. Plasma clearance and aortal trapping of control and results demonstrated the anti-inflammatory activity of poly-L-lysine in modified LDL and HDL was studied in male rats. vitro in IECs, and indicated that the activity was primarily via CaSR- The particle sizes of both LDL and HDL were decreased by mediated activation. glycation with methylglyoxal. Glycation with methylglyoxal increased partitioning of LDL onto the aorta of rats. Cholesterol and triglyceride contents did not change by glycation with methylglyoxal. Arginine as a novel regulator of macroautophagy Glycation of HDL2 by methylglyoxal decreased particle size. Gly- in H4-II-E Cells cation by methylglyoxal leads to atherogenic transformation and aortal trapping of LDL, facilitating atherosclerosis in renal failure. Quantitation of methylglyoxal-modified LDL may improve epide- Motoni Kadowaki1,2, Kenji Kaneshiro1, Yasuhiro Daigaku1, 2 1,2 miological cardiovascular disease risk models. Glycation of HDL2 by Masatoshi Kubota , and Shinobu Fujimura methylglyoxal in may contribute to dyslipidemia by increasing HDL degradation. Small HDL2 is more readily hydrolysed by hepatic and 1Graduate School of Science and Technology, and 2 endothelial lipases with subsequent increased renal degradation of Center for Transdisciplinary Research, Niigata University, Japan apolipoprotein A1, decreasing HDL levels. Therapeutic agents to decrease methylglyoxal may improve current treatment to decrease Backgrounds and objectives: Macroautophagy is a major system of the risk of cardiovascular disease in high risk groups where protein intracellular bulk degradation and is regulated physiologically by a glycation by methylglyoxal is increased—particularly the elderly, number of amino acids. However, the signaling mechanism of amino patients with diabetes and patients with renal failure. acids is still elusive, which is quite complicated depending on cell types and amino acids. Arginine (Arg), which had been regarded as a non-regulatory amino acid, was recently shown to have a regulatory function of macroautophagy in rat hepatoma H4-II-E cells. The Biological characterization and structure based contribution of NO pathway as the signaling mechanism of Arg on prediction of IGFBP-5 macroautophagy was investigated. Materials and methods: H4-II-E cells were incubated in HBSS with Minkyung Sung, Eun Young Hwang, Mi Suk Jeong, complete amino acid mixture, regulatory amino acids, and Arg. Long- and Se Bok Jang* lived proteolysis was measured by 14C-Val release and calculated as %/h, and presented as % of HBSS (control) value. As an autophagy Department of Molecular Biology, College of Natural Sciences, marker, LC3 in the cytosol fraction was separated by western blotting Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan and detected by anti-LC3 antibody. The amount of LC3-I and LC3-IIs 609-735, Korea. were quantified by densitometric scanning and calculated as cytosolic *Corresponding author: E-mail: [email protected] LC3 ratio, a quantitative autophagy index. The NOS inhibitors, ami- noguanidine (AG, 1 mM), L-NIL (100 lM), L-NMMA (100 lM) were The insulin-like growth factor binding protein (IGFBP) family has employed for testing the contribution of NO pathway for the signaling. been shown to play a role in various functions such as cell growth, Results: Since Arg significantly suppressed proteolysis and cytosolic LC3 cell death, cell motility, and tissue remodeling. Among the 7 IGFBP ratio equally, the effect was autophagic. The suppressive effect of Arg was family members, IGFBP-5 was recently shown to play an important inhibited by these NOS inhibitors, so the signaling mechanism by Arg was role in breast cancer biology, especially in breast cancer metastasis. through NO pathway, independent from that by other amino acids. The three-dimensional structure of the mini IGFBP-5 domain (amino acids 40–92) is known, but structural information on the complete N, L, and C domains remains unknown. Due to difficulties associated Atherogenic transformation of LDL and impaired with expression and crystallization of full-length IGFBP-5, fragments have more frequently been studied. In this study, IGFBP-5 structures function of HDL by glycation containing N, L, and C domains were separately modeled from solved structures in protein data bank (PDB). In addition, the L domain of Naila Rabbani and Paul J Thornalley IGFBP-5 was expressed in Escherichia coli and purified for studying its structural characterization. Despite very low sequence homology, Warwick Medical School, Clinical Sciences Research Institute, the novel L domain structure of IGFBP-5 was unexpectedly similar to University of Warwick, University Hospital, Coventry CV2 2DX, U.K that of the corepressor of repressor element-1 silencing transcription factor (CoREST) linker in the lysine-specific demethylase 1 (LSD1)- Glycation by glucose of plasma proteins—including lipoproteins low CoREST complex. The purified L domain existed as a homogenous density lipoprotein (LDL) and high density lipoprotein (HDL)—has dimer in glutaraldehyde cross-linking and exhibited a typical a-helix

123 12th International Congress on Amino Acids, Peptides and Proteins S9 structure in the circular dichroism (CD) assay. This study has Potato scab disease is caused by several Streptomyces strains potential applications in medicine and other fields such as drug producing thaxtomin such as S. scabies, S. acidiscabies, and design, mutational study, and disease prediction. S. turgidiscabies. Thaxtomin is a plant toxin and a pathogenicity determinant that is conserved in scab-causing plant pathogenic streptomycetes. Also, AfsR acts as a transcriptional factor in both Citicoline influences kynurenic acid formation: the regulation of secondary metabolism and morphological differ- an in vitro study entiation in Streptomyces strains. In this study, PCR using the primers designed from the two highly conserved regions of the previously studied AfsR of Streptomyces strains gave 626-bp Halina Baran1 and Berthold Kepplinger1,2,3 products. The sequence of this product had a high similarity to the expected region of a afsR gene. An intact 3.0-kb afsR gene of S. 1Neurochemical Laboratory, Karl Landsteiner Research Institute acidiscabies was cloned by genomic Southern hybridization with for Pain Treatment and Neurorehabilitation, LKM Mauer-Amstetten, PCR product as a probe and then studied. To identify the function 2 Department of Neurology, Neuropsychiatric Hospital Mauer, of the cloned afsR gene, it was inserted into pSET152ET to con- Amstetten-Mauer, struct a high expression vector, and then was introduced into 3 Department of Neurology, General Hospital Amstetten, Amstetten, Streptomyces strains. The exconjugant including the high expres- Austria sion vector of afsR was constructed and its phenotype was Corresponding author: [email protected] compared with the control strain. This research was supported by Basic Science Research Program Citicoline (cytidine-50diphosphocholine) is a neuroprotectant and through the National Research Foundation of Korea (NRF) funded neurorestorative drug and is used in the treatment of acute stroke. by the Ministry of Education, Science and Technology (2010- Citicoline is involved in the formation of phosphatidylcholine, a 0008443). major brain phospholipid, and also serves as a choline donor in the biosynthesis of the neurotransmitter acetylcholine. Since an increased kynurenine metabolism has been documented in neonatal asphyxia and in acute stroke the aim of the present study was to investigate the biochemical properties of citicoline (solution of Compartmentalization of proteins in lipid droplet 125 mg citicholine/ml is called startonyl, Firma Torrex Chiesi biogenesis Pharma GmbH Austria) with respect to kynurenic acid formation in an in vitro study. Kynurenic acid is an endogenous metabolite of Horst Robenek, Insa Buers, Oliver Hofnagel, Mirko J. Robenek, tryptophan degradation along the kynurenine pathway and acts as David Troyer and Nicholas J. Severs an antagonist at the glutamate ionotropic excitatory amino acid and at the nicotine cholinergic receptors and exerts neuroprotective Leibniz-Institute for Arteriosclerosis Research, Department of Cell activity. The activities of the KYNA synthesising enzymes kynu- Biology and Ultrastructure Research, Domagkstr. 3, 48149 Mu¨nster, renine aminotransferase I, II and III (KAT I, KAT II and KAT III) Germany in rat liver homogenates were analysed in the presence 0, 15, 35 and 75 ll of startonyl, respectively. KAT I, II and III activities Our existing understanding of the protein organization and biogenesis were measured using a enzymatic method in the presence of 1 mM of the lipid droplet has relied heavily on microscopical techniques that pyruvate and 100 lM L-kynurenine. Citicoline, dose-dependently lack resolution and the ability to preserve protein composition. The and significantly increased KAT I activity of rat liver homogenate, electron microscopic technique of freeze-fracture replica immunola- whereas KAT II activity was dose-dependent and moderate beling (FRIL) overcomes these problems. Because of the property of reduced, while KAT III was not affected significantly. The present frozen lipids to deflect the fracture plane, en face views of the lipid study for the first time demonstrates the ability of citicoline to droplet and its component layers are revealed for high resolution influence kynurenic acid formation in rat liver homogenates. The visualization. By means of immunogold labeling, proteins involved in effect of citicoline varied notably between KAT I, II and III the accretion and mobilization of lipids, notably the PAT family activities. We suggest that the neuroprotective effect of citicoline proteins, can be localized at and in the droplet. This approach dem- could be in part due to influence of kynurenic acid formation onstrates that, contrary to prevailing wisdom, the PAT family proteins involving KAT I. are not invariably restricted to the surface of the lipid droplet but can occur throughout the core. The notion that lipid droplet biogenesis involves neutral lipid accumulation within the ER membrane bilayer followed by budding off, enclosed by a protein-containing phospho- Cloning and functional analysis of afsR, a global lipid monolayer, is not substantiated. Instead, lipid droplets appear to develop externally to both ER membranes at specialized sites in regulatory gene for secondary metabolism which the ER enwraps the droplet, and the facing leaflets of the ER in Streptomyces acidiscabies membrane and droplet surface are enriched in adipophilin. PAT family proteins are not, as often stated, specific to the lipid droplet, Min-Jeong Kim, Hye-Yun Park, Hyo-Ji Kim, Sun-Uk Choi, but are widely present in the plasma membrane where, after lipid and Yong-Il Hwang loading, they adopt a similar configuration to that of specialized sites in the ER. These examples highlight the contribution of the FRIL Department of Food Science and Biotechnology, Kyungnam technique to critical appraisal and development of concepts in the University, Changwon, 631-701, Republic of Korea lipid droplet field.

123 S10 12th International Congress on Amino Acids, Peptides and Proteins

Conformational stability of exchangeable The results of this study show that the photoproperties of apolipoproteins in solution and associated pheomelanin can be modulated by various experimental conditions, ranging from the photoprotection to the triggering of potentially with phospholipid damaging photochemical reactions. Noteworthy, the observed photosensitizing effect of pheomelanin on nitrative modiﬁcation of A. D. Dergunov tyrosine at pH 5.5 strengthens the hypothesis of a photodamaging role of this pigment in skin whose pH ranges from 5.0 to 6.0. National Research Centre for Preventive Medicine, 101990 Moscow, Russia

Guanidine-hydrochloride (GuHCl)-induced denaturation of a-helices Effects of T-2 toxin on low selenium state rats’ in human plasma apoA-I, apoA-IV, apoE3 and three recombinant chondrocyte expression of collagen II, matrix apoE isoforms in solution and discoidal complexes of apoA-I, apoA-II, metalloproteinase (MMP-1, MMP-3, and MMP-13) apoA-IV and apoE3 with phosphatidylcholine was studied. The pro- and TIMP1 tein conformational stability ðÞDGH2O and a slope of linear dependence of free energy of unfolding on GuHCl concentration (m-value) were estimated. The data for all proteins, except apoA-II, ﬁt with the three- XiaoRong Zhou, Jinghong Chen, and Haojie Yang state model, thus evidencing two-domain structure both in solution and in complexes. The predicted folding rate of the four apoE in solution The Second Hospital of Medical School of Xi’an Jiaotong University, correlated with their conformational stability, but the dependence Xi’an 710004, Shaanxi, People’s Republic of China disappeared at the inclusion of apoA-I and apoA-IV into analysis. In Corresponding Author: Zhilun Wang Institute of Endemic Diseases, addition, the m-values, adjusted for residue number in helices (mrh), Medical School of Xi’an Jiaotong University, Key Laboratory differed between those for apoE and apoA-I/apoA-IV. However, if of Environment and Genes Related to Diseases, Ministry analyzed together, the mrh-values for six proteins correlated positively of Education, Xi’an 710061, Shaanxi, People’s Republic of China with the fractional change in accessible surface area at unfolding for Phe, Lys and Asn, while negatively for Arg, Ala and Gly residues. The Kashin–Beck disease (KBD) is a chronic, endemic, deforming difference between the adjusted DGH2O values for apolipoproteins in osteoarticular disease with unclear etiology and severer damage to complexes and in solution decreased at the increase of reduced tem- sufferers’ working ability and quality of life. Low selenium and T-2 perature Tred, calculated as Tred = (Tobs - Tt)/Tt. Two-domain toxin are regarded as the most important etiological factors related to structure of apolipoproteins in complexes was much more uniﬁed. The the take place of the disease. But laboratory and epidemiologic induction of intrinsic disorder by arginine residues may be of primary experiments of either selenium or T-2 toxin separately cannot well importance in metabolism and function of exchangeable apolipopro- explained the endemically and wavy characteristic of KBD. So we teins, while their stability in nascent discoidal HDL is controlled by the established a new hypothesis that the KBD is due to an complicated physical state of phosphatidylcholine (RFBR grant 10-04-00270). causes of both factors mentioned above, shorting of selenium as an necessarily condition encountered with T-2 toxin leading to the happens of KBD. In this experiment we tried to explain the etiology Effect of pheomelanin on UVB radiation-induced of KBD by animal experiment to validate this hypothesis and tried to oxidation/nitration of tyrosine depict the molecular mechanism as well.

Mario Fontana, Alessia Baseggio Conrado, Simonetta Maina, Function comparisons between harpin and hpa Luciana Mosca and Laura Pecci Xoo Xm in the elicited ROS in tobacco against TMV Department of Biochemical Sciences, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy Lin Li, Wenbo Liu, Tingya Yang, Xiaoxi Lan, Xiang Li, Liang Sun, XiaoYan Wu, Weiguo Miao*, and Fucong Zheng* Pheomelanin is a natural yellow-reddish pigment derived from tyrosinase-catalyzed oxidation of 5-S-cysteinyldopa. Generally, the College of Environment and Plant Protection, Hainan University, formation of melanin pigments is a protective response against the Haikou 570228 China damaging effects of UV radiation in skin. However, pheomelanin *Corresponding author: Weiguo Miao increases UV-induced lipid peroxidation in liposomes, suggesting that E-mail: [email protected] Fucong Zheng this pigment could also act as pro-oxidant. E-mail: [email protected], [email protected] The photoproperties of this natural pigment have been studied by analyzing the effect of pheomelanin on photooxidation of tyrosine Harpin is a hypersensitive response elicitor. HarpinXoo and hpaXm -2 induced by UVB radiation (kmax 313 nm, total dose 4,800 J m ). encoded by hpa1/G from Xanthomonas oryzae pv. oryzae and X. citri 0 After exposure to UVB radiation, tyrosine is converted to 3,3 -dity- subsp. malvacearum, respectively. Although harpins (HarpinXoo and rosine and, in the presence of nitrite, also to 3-nitrotyrosine. It has hpaXm) are able to trigger the resistances of tobacco to Tobacco mosaic been observed that pheomelanin plays a protective role on the oxi- virus (TMV), it is unknown whether harpins induce the sequential dation/nitration of tyrosine at pH 7.4. The formation of 3,30-dityrosine generation of reactive oxygen intermediates (ROI) in tobacco. In this and 3-nitrotyrosine is completely inhibited at 4.2 lg/mL pheomelanin study, respective methods in detection of H2O2 and relative enzyme concentration. Conversely at pH 5.5, pheomelanin acts as photosen- activity, DAB (Diaminobenzidine) staining and quantitative real time sitizer and a 60% increase of the production of 3-nitrotyrosine is PCR were used for conﬁrming the ROI generation in tobacco com- observed with respect to control experiments without pheomelanin. pared with untreated tobacco. The results showed that after the leaves The photosensitizing action of pheomelanin on the production of were inoculated by TMV, compared with untreated tobacco leaves, 3-nitrotyrosine, is further increased in D2O, suggesting a predominant H2O2 content in harpins-treated leave tissue exhibited two peak values 1 role of singlet oxygen ( O2) in the reaction mechanism. during 0–8 h; PAL and POD activities were signiﬁcantly enhanced,

123 12th International Congress on Amino Acids, Peptides and Proteins S11

DAB staining revealed visual reddish-brown coloration spots, and amino acids (NAAs) are genetically encoded in vivo using a suitable host expressions of ghAOX1 and hsr203J genes were significantly induced. organism. The amino acids are encoded site-selectively in response to a However, the first peak of ROI induced by harpinXoo was detected recoded amber stop codon. The overall goals of our research are to ahead compared with that induced by hpaXm. There are no significant develop novel amino acids, but also to advance the understanding of the deference between harpinXoo and hpaXm in the expression level of ROI mechanism and function of bioorthogonal translation elements which is related genes. These evidences indicated that harpins possessed the described in a recent publication from my group. Using the fundamentals molecular basis for expression of some defense genes, and the of chemistry, NAAs may be chemically tailored to possess specific expression of harpinXoo and hpaXm was responsible to the generation properties for highly specialised applications, for example, in the charting of ROI and the expression of the related defense genes in tobacco of biological pathways or in unique post-translational modifications. In plant, and conferred the resistance of tobacco to TMV. these scenarios, there is a synergy between the chemistry of the NAA and Acknowledgments: This work was supported by grants from the the biochemistry of the host organism used to heterologously express the NKBRPC (2003CB114204 and 2006CB101902), NKPC (2004BA901 labeled protein. By increasing the sophistication of the amber suppression A36), RFDP (20104601110004), CARS-34-GW8 and KYQD1006 (China). method, a host of multidisciplinary applications related to drug delivery and biomaterials becomes possible, and preliminary work in these directions is presented. Functional genomics of amino acid transporters in the human pathogen Leishmania: the story of proline and alanine Glycation by methylglyoxal in physiological systems— the damage caused and strategies to prevent it Dan Zilberstein1, Ehud Inbar1, Doreen Schlisselberg 1, Marianne Suter Grotemeyer 2 and Doris Rentsch2 Paul J Thornalley and Naila Rabbani

1Faculty of Biology, Technion-Israel Institute of Technology, Clinical Sciences Research Institute, Warwick Medical School, Haifa 32000, Haifa, Israel and University of Warwick, University Hospital, Coventry CV2 2DX, U.K 2Institute of Plant Sciences, University of Bern, 3013 Bern, Switzerland Increased glycation of cellular and extracellular proteins by methyl- Protozoan parasites of the genus Leishmania are the causative agents glyoxal occurs physiological systems. Steady state levels of glycated of a wide range of visceral and cutaneous diseases in humans. Leish- proteins are generally low yet often has significant physiological mania species cause morbidity and mortality in 88 counties effect—particularly linked to ageing, diabetes development and pro- worldwide. Increasing cases of Leishmania-HIV co-infection is the gression of vascular disease, neurological disorders and arthritis. major reason for the recent emergence of leishmaniasis. Unlike all Identification of sites activated toward damaging modifications or other organisms, Leishmania (as well as all members of the Trypan- ‘‘hotspots’’ in functional domains within proteins appears key to osomatid family) maintain a large cellular pool of proline that, assessing targets of functional impairment. Disease progression is together with alanine, serves as an alternative carbon source and as a likely linked to instances where change in low level of hotspot damage reservoir of organic osmolytes. We cloned and characterized a new influences metabolic control or physiological function. Examples neutral amino acid transporter, LdAAP24 that translocates proline and discussed are: type IV collagen modification leading to endothelial cell alanine across the L. donovani plasma membrane. By knocking out the detachment and anoikis, mitochondrial protein modification leading to gene that encodes for LdAAP24 we showed that this transporter fulfills oxidative stress, and apolipoprotein B100 modification in low density multiple functions: it is the sole supplier for the intracellular pool of lipoprotein leading to vascular retention and atherosclerosis. The role proline and contributes to the alanine pool; it is essential for cell of mathematical systems biology, bioinformatics and proteome volume regulation after osmotic stress; and it regulates transport and dynamics in future investigations is discussed. Therapeutic interven- homeostasis of glutamate, arginine and glycine, none of which are its tions to decrease glycation by methylglyoxal may be achieved by substrates. Interestingly, loss of cellular proline signal for parasites in agents that decrease triosephosphate precursors—such as high dose the insect to develop virulence, a process called metacyclogenesis; thiamine therapy in diabetes, scavenging agents and inducers of Dldaap24 mutants transiently express gene markers of metacyclo- enzymes that metabolize methylglyoxal such as glyoxalase 1. genesis, at late log rather than late stationary phase. In addition, they change to metacyclic-like morphology at mid log, but resume oval shape at stationary phase. Finally, Dldaap24 are significantly more High pH solubilization and chromatography based susceptible to oxidative stress than wild type. We conclude that by renaturation and purification of recombinant human controlling the cellular pool of proline LdAAP24 plays a key role in virulence development of Leishmania parasites inside the vector. granulocyte colony-stimulating factor from inclusion bodies**

Genetically encoded nonnatural amino acids: novel Ming Li, Jiahua Liu, Lili Wang, and Chaozhan Wang* methods for bioengineering structure–function Key Laboratory of Synthetic and Natural Functional Molecule into biomaterials Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, China Eric M. Tippmann *Corresponding author: Dr. Chaozhan Wang, College of Chemistry and Materials Science, Northwest University, 229 Tai Bai North Cardiff University School of Chemistry, Cardiff United Kingdom Road, Xi’an 710069, People’s Republic of China. CF10 3AT Tel: +86-29-88302604, Email: [email protected]

New approaches for the optimization of translational and post-transla- Recombinant human granulocyte colony-stimulating factor (rhG- tional modiﬁcation of proteins are presented. We show that nonnatural CSF) is a very efﬁcient therapeutic protein drug which has been

123 S12 12th International Congress on Amino Acids, Peptides and Proteins widely used in human clinics to treat cancer patients suffering from Illuminating the interactions of the C-terminal region chemotherapy-induced neutropenia. In this study, rhG-CSF was of TRPV1 channel with membrane phospholipids solubilized by using high pH solution containing low concentration of urea from inclusion bodies. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the pH of Lenka Grycova, Blanka Holakovska, and Jan Teisinger employed buffer, alkaline pH drastically favored the solubilization. In addition, when small amount of urea was added to the solution Institute of Physiology, Academy of Sciences of the Czech Republic at high pH, the solubilization was further enhanced. After solubi- 2+ lization, the rhG-CSF was renatured by using weak anion TRPV1 channel is non-selective Ca -permeable cation channels activated by a wide range of stimuli. As a result of its activation, the exchange, strong anion exchange and hydrophobic interaction 2+ chromatography separately, with simultaneous purification. The cellular level of Ca increases, which initiates a wide variety of results indicated that the rhG-CSF solubilized by high pH com- cellular responses. It is assumed that TRPV1 channel has six trans- bined with low concentration of urea had much higher mass membrane domains with a pore domain between the fifth and the sixth recoveries than the one solubilized by 8 M urea in all three segments. Both C- and N-termini are located intracellularly and have employed refolding methods. In the case of weak anion exchange, been shown to be involved in the regulation of the channel activity. the high pH solubilized rhG-CSF could get a mass recovery of Both tails recruits agonist and regulatory molecules such as ATP, 73%. A strategy combining solubilization of inclusion bodies at calmodulin (CaM), phosphatidyl inositol 4, 5 bisphosphate (PIP2). high pH and refolding of protein using liquid chromatography may In this study using the combination of biochemical and biophysical become a routinely method for protein production from inclusion approaches the highly purified C-terminal fusion protein TRPV1 bodies. (TRPV1-CT) was shown to interact directly with PIP2 and this interaction ** This work was supported by the National Natural Science has been characterized in detail. As this sequence contains several basic Foundation in China (No. 20705028). amino acids which may interact with anionic lipids. We tested influence of liposome composition on PIP2—fusion protein TRPV1 interaction. Moreover, we have provided the structural insight to the TRPV1-CT/PIP2 interaction using homology modelling and computer ligand docking. Hydroquinone regulates tumorigenic responses through This work was supported by Grant GAAV IAA600110701, GACR Src activation by binding to cysteine residues P205/10/P308, GACR P301/10/1159, Centre of Neurosciences No. LC554 MSMT CR3 Se Eun Byeon1, Ji Hye Kim1, To Thi Mai Dung1, Byung Chul Kim2, Byoung Chul Yoo3, Won-Jea Cho4, Jae Youl Cho1, and Sungyoul Hong1 Induction of actinorhodin production from Streptomyces lividans TK24 by high expression 1 Department of Genetic Engineering, Sungkyunkwan University, of the two-component system derived Suwon 446-746, Korea; 2College of Natural Science, Kangwon National University, from Streptomyces acidiscabies Chuncheon 200-701, Korea; 3Research Institute and Hospital, National Cancer Center, Hye-Yun Park, Min-Jeong Kim, Hyo-Ji Kim, Sun-Uk Choi, Goyang 410-769, Korea; and Yong-Il Hwang 4College of Pharmacy, Chonnam National University, Kwangju 500-757, Korea Department of Food Science and Biotechnology, Kyungnam University, Changwon, 631-701, Republic of Korea The hydroxylated benzene metabolite hydroquinone (HQ) is mainly generated from benzene, an important industrial chemical, and is also Streptomyces acidiscabies produces thaxtomins that cause a scab a common dietary component. Although few reports have addressed disease of potatoes and have structure of cyclic dipeptide consisting the immunosuppressive effects of hydroquinone, numerous papers of L-4-nitrotryptophyl-L-phenylalanyl. In this study, the two-compo- have explored its roles in tumorigenic responses. In this study, we nent system, which performs an important role in secondary characterized Src-targeted tumorigenic responses by hydroxylated metabolite production of actinomycetes, cloned from Streptomyces benzene metabolites, hydroquinone and its derivatives, in activating acidiscabies, and its antibiotic production-increasing activity was various cancerous responses using cellular, molecular, biochemical confirmed. Genes (tcsK and tcsR) encoding the sensor kinase (TCSK) and immunopharmacological approaches. HQ up-regulated tran- and the response regulator (TCSR) were acquired via PCR. PCR was scriptional activation of NF-B and ARE responses so that carried out using the primers designed from the two highly conserved hemeoxygenase-1 and pro-inflammatory gene expressions were regions of previously studied two-component system of Streptomyces increased. Several of evidence have raised a possibility that HQ strains, and gave a 300-bp and 498-bp products for tcsK and tcsR. The directly activates Src kinase activity. Thus, Src knockdown or defi- obtained PCR products showed a high homology to the two-compo- ciency by siRNA treatment and infection with retrovirus expressing nent systems of Streptomyces strains. By Southern hybridization using shRNA to Src as well as exposure of PP2, a Src kinase inhibitor, PCR products as a probe, intact 1.6-kb tcsK gene and 0.7-kb tcsR gene strongly abrogated HO-1 expression. Interestingly, HQ directly tar- were obtained from genomic DNA of S. acidiscabies. In order to geted and bound to the sulfhydryl group of Cys-483 and Cys-400 of identify the effects of the cloned genes, the high expression vectors Src, potentially leading to breaking intracellular disulfide bonds. were constructed by using pSET152ET and introduced into S. lividans Indeed, Src kinase activity was dramatically enhanced by the muta- TK24, which was employed as a host for the confirmation of inducer tion of these Cys sites, implying that these sites may play an gene activity for secondary metabolite production. And then the important role in Src activity regulation. Therefore, our data suggest actinorhodin production of the exconjugants was assessed. As a result, that Src and its target site Cys-483 can be considered as a prime it was determined that the expression of tcsK and tcsR induced molecular target of HQ-mediated tumorigenic responses and for 5.4-fold and 5.2-fold increases in actinorhodin production from strong Src kinase regulatory loop. S. lividans TK24, respectively, compared to the control strains.

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This research was supported by Basic Science Research Program which is immunologically identical to PIF. The WF has no cytotoxic through the National Research Foundation of Korea (NRF) funded by effect in C2C12 cells after 24 h incubation. Higher WF concentra- the Ministry of Education, Science and Technology (2010-0008443). tions increased the chymotrypsin-like and proteasome pathway activities in myotubes cells. However, adding leucine previously to WF-treated muscle cells, the cell growth was increased associated to enhance on phosphorylated STAT3 and Akt and reduction on Interaction of microcystin-LR with human serum albumin phosphorylated Erk in parallel to increased protein synthesis. Taken together, these results strongly suggest an important modulatory Chao Song and Hong-Wen Gao1 effect of leucine when acting previously under the WF effects on C2C12 muscle cells. State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, China 1To whom correspondence should be addressed, Mimicking a biological cellular structure Fax: (86) 21-6598-8598. E-mail: [email protected] with incorporation of interior organelles and protein cytoskeletal network in the cytoplasm The binding of microcystin-LR (MC-LR) onto human serum albumin (HSA) was investigated by fluorescence and UV–visible spectrome- Dooho Lee1, Sujin Park1, and Kwanwoo Shin1,2,3* try. The results showed that MC-LR can quench strongly the intrinsic fluorescence of HSA. The binding number of MC-LR was determined Department of 1Chemistry and to be 1 per HSA and the thermodynamic constants (DH, DS and DG) 2Interdisciplinary program of integrated Biotechnology, Sogang calculated. The electrostatic attraction and hydrophobic stack are the University, Shinsu dong 1, Mapo gu, Seoul 121-742, South Korea main binding forces in the MC–LR/HSA interaction. From the syn- 3Sogang-HANARO Joint Center for Biological Interfaces, KAERI, chronous and three-dimensional fluorescence spectra, the different Daijeon, Korea *Email: [email protected] concentrations of MC-LR exposed in HSA solution can cause the different quenching peak. For example, the low concentration of MC- A giant unilamellar vesicle (GUV) have received much attention LR mainly caused the quenching of peak 3 (k 230.0 nm, k ex em recently because they, as a biological cell mimic in size and shape, 330.0 nm), indicating a change of the peptide strands structure. The can be utilized as a biological mimics. Up to now, many researchers high concentration of MC-LR caused the quenching of peak 4 (k ex have reported the dynamics and physical properties of GUV, for 280.0 nm, k 330.0 nm) and MC-LR interacted with the Trp and Tyr em examples, dynamics of GUV with shear elasticity, or lipid phase residues by hydrophobic stack. Therefore, the interaction of MC-LR behaviors of GUV, etc. However, most biological cells contain with HSA exhibited a two-step reaction: first to change the poly- organelles, and cytoskeletal network in their cytoplasm, and it is not peptide backbone structure, and then to combine with the hard to expect that the physical properties of GUV will be greatly hydrophobic amino acids. affected when a cellular vesicles contains complex sub-cellular structures (organelles and protein networks) in the cytoplasm. Ini- tially, we mimicked various non-spherical structures of cellular Leucine modulated the effects of Walker factor organelle mimics (tadpole, snowman, dumbbell, etc.) by varying major membrane components. We could successfully injected the in C2C12 myotubes organelle mimics into a GUV. Furthermore, we introduced the collagen-mediated fibers into the cytoplasm, resulting in the fiber E. M. Gonçalves and M. C. C. Gomes-Marcondes network in the inner volume of GUVs. We, then could study how the mechanical/physical properties of GUV membranes were affected Depart Physiology and Biophysics, Biology Institute, State University with the incorporation of interior-organelles and protein-cytoskeletal of Campinas, Saõ Paulo, Brazil. Financial support: Fapesp, CNPq. network. Statistical support: Dr. J Marcondes Neto

Factors produced by tumors or host tissues have been signed in tissue wasting in cancer cachexia. Protein degradation in skeletal Molecular imprinting of thymopentin in aqueous media muscle during cancer cachexia is also mediated by a proteolysis- inducing factor (PIF). The three main proteolytic systems respon- Liang Zhao, Yan-Ping Huang*, and Zhao-Sheng Liu* sible for proteolysis in skeletal muscle, especially during cachexia, are the lysosomal, the calcium-dependent, and the ubiquitin–pro- College of Pharmacy, Tianjin Medical University, Tianjin 300070, teasome pathways. In skeletal muscle, the effect of PIF on protein China Correspondence: Dr. Yan-Ping Huang, College of Pharmacy, degradation is mediated through an increased expression and activity Tianjin Medical University, Tianjin 300070, China, of the ubiquitin–proteasome proteolytic pathway. The branched- E-mail: [email protected] chain amino acid leucine has been investigated as an important cellular signalling and studies have shown that this amino acid can Molecular imprinting of peptides in aqueous media is always a inhibit the activity of the ubiquitin–proteasome pathway in muscle challenge in the synthesis of artificial receptors. In this work, a new of cachectic rats and can improve protein synthesis of muscle in molecularly imprinted polymer (MIP) for thymopentin (TP-5) was tumour-bearing rats. In this study were investigated the effects of prepared by the combined use of N-isopropylacrylamide (NIPAm) leucine in C2C12 skeletal muscle cells exposed to the proteolysis- and N-t-butylacrylamide (TBAm) as functional monomers in the inducing factor (PIF)-like protein, purified from the ascitic fluid of presence of N, N0-methylenebisacrylamide (BIS) as cross-linker, and the Walker 256 tumour-bearing rat, called Walker Factor (WF), the imprinted nanoparticles can be synthesized using a simple

123 S14 12th International Congress on Amino Acids, Peptides and Proteins precipitation polymerization method. The preparation conditions Mutation of amino acid within the n terminus of human were optimized by investigating the effect of the concentrations of O-GlcNAcase results in impaired function the monomer and cross-linker on the adsorption properties. Dynamic light scattering (DLS) was used to investigate the dynamic process 1 1 1 1,2, of particle growth. The resulting MIPs nanopartile were average Lin Lin , Zhonghua Li , Jing Li *, and Peng George Wang * diameter of 300 nm with the polydispersity (PDI) of 0.217. The 1 adsorption properties of resultant imprinted polymers were evaluated College of Pharmacy and State Key Laboratory of Element-Organic Chemistry, Nankai University, Tianjin 300071, China by equilibrium rebinding experiments in an aqueous media. The 2 highest binding capacity of TP-5 achieved from the optimized Departments of Biochemistry and Chemistry, the Ohio State imprinted polymer was 194.72 lmol/g with an imprinting factor of University, Columbus, OH 43210 2.04. The results showed that the method established owns potential *Corresponding author. Tel./Fax: +86 22 23507760 promises. E-mail Addresses: [email protected] (J. Li), This work was supported by the National Natural Science Foun- [email protected] (P. G. Wang) dation of China (Grant No. 21075090). O-GlcNAcylation is a newly discovered post-translational modification of cytosolic proteins in all metazoans. Analogous to phosphorylation, O-GlcNAcylation plays critical roles in cellular Monitoring of NTH1 phosphorylation sites important events, including cell-cycle progression, cellular development, cellular stress and extracellular stimuli, but its faulty regulation is for BMH binding involved in the etiology of type II diabetes, cancer, and neurological disorders. O-G1cNAcase (OGA), belongs to GH84 family (http//: D. Veisova1, E. Macakova1, L. Rezabkova1,2, and V. Obsilova1 www.cazy.org), is the only enzyme responsible for removal of O-GlcNAc from protein and thus plays a key role in O-GlcNAc 1Institute of Physiology Academy of Sciences of the Czech Republic, metabolism. Due to unstable expression and poor purification, until 14220 Prague, Czech Republic recently the structure and reaction/substrate recognition mechanisms 2Department of Physical and Macromolecular Chemistry, Faculty of human OGA have not yet been understood. of Science, Charles University, 12843 Prague, Czech Republic Using deletion mutation, our previous study revealed that the [email protected] N-terminal region (a.a. 1-350) of OGA (sOGA, the smallest OGA) contains the catalytic site for glycoside hydrolase. Here using Geno Trehalase (EC 3.2.1.28) is an intrinsic glycoprotein of the small 3D and the crystal structure of human OGA orthologue in GH84 intestine and renal brush-border membranes that hydrolyzes from C. perfringens (CpOGA), we modeled sOGA and identified a,a- trehalose (1-a-D-glucopyranosyl a-D-glucopyranoside) to two key amino acids involved in glycosidase hydrolysis. Key amino glucose molecules in animals. Three trehalases have been identified acids were subjected to site-directed mutagenesis, and the mutated in Saccharomyces cerevisiae so far: neutral trehalase 1 (NTH1), enzymes were expressed in E. coli BL21 cells and assayed with neutral trehalase 2 (NTH2) and acidic trehalase 1 (ATH1). NTH1 is 4-MU-GlcNAc (4-methylumbelliferyl-2-acetamido-2-deoxy-b-D- responsible for trehalose degradation, which is accumulated after glucopyranoside) as substrate. D174N showed a severely impaired stress. The activity of the NTH1 enzyme was just recently found to catalytic activity (about 2,000-fold reduction in Kcat), consistent be mediated by BMH1 and BMH2 binding in yeast. Yeast BMH1 with its role in polarized and oriented the 2-acetamido group for a and BMH2 proteins (yeast 14-3-3 isoforms) form a complex with nucleophilic attack. D175N showed only modest 26-fold decrease neutral trehalase after its phosphorylation by PKA. Either one of in Kcat, in good agreement with its role in general acid/base cat- the two 14-3-3 yeast isoforms are required for complete activation alyst. D285 interacts with the O4/O6 hydroxyls through tight of neutral trehalase (NTH1). However, details concerning the hydrogen bonds, which aid in formation of the 4E envelope con- mechanism of BMH-dependent activation of NTH1 remain still formation of the pyranose ring in the transition state, providing a unknown. possible explanation for the large mutational effect on Km (about We showed that PKA phosphorylates NTH1 in vitro on four Ser twofold increase) and Kcat (about 120-fold reduction). N280 is residues: 20, 21, 60 and 83. To find out which site or sites are involved in stabilizing the conformation of the acetamido group essential for the 14-3-3 binding we produced NTH1 WT (both and the oxazolinium intermediate, compatible with its 300-fold phosphorylated and non-phosphorylated), four NTH1 mutants con- reduction in Kcat. C215 and Y219 are hydrogen bonded to the taining single phosphorylation site, one double phosphorylated oxime nitrogen (approximately equivalent to the position of the NTH1 mutant (at Ser20 and 21) and a mutant containing none of substrate glydosidic oxygen), in agreement with this, mutations of these studied phosphorylation sites as well. The interaction between C215A and Y219F show modest increase in Km. In contrast, the BMH1 and BMH2 protein with enzyme NTH1 was monitored using Y69F mutant enzyme displayed comparable level of O-GlcNAcase native electrophoresis and sedimentation velocity measurements. activity as that of the wild type enzyme-sOGA, indicating Y69 may The sedimentation equilibrium analysis was used to define the not be an essential residue in substrate binding and catalysis. This stoichiometry of NTH1/BMH complexes. Finally, we used enzyme study provides, to our knowledge, the fist data on the three- kinetic measurements to monitor the BMH-dependent activation of dimensional homology modeling to analyze the essential role of six NTH1. essential residues in human OGA catalysis. Acknowledgments: This work was funded by Grant P207/11/0455 of Keywords: O-GlcNAcylation, OGA, Site-directed mutation the Grant Agency of the Czech Republic and Centre of Neurosciences This research was supported by the National Basic Research LC554 of the Ministry of Education, Youth, and Sports of the Czech Program of China (973 Program, grant No.2007CB 914403) the Republic, by Research Project AV0Z50110509 of the Academy of National Natural Science Foundation of China (31000371) and Sciences of the Czech Republic and by Grant 350111 of the Grant the Fundamental Research Funds for the Central University Agency of the Charles University. (65011091).

123 12th International Congress on Amino Acids, Peptides and Proteins S15

Preparation and characterization of the yeast enzyme cluster of calcium-elicited kinases was suppressed by nicotine. These Neutral trehalase 1 and its complex with BMH proteins ﬁndings were further supported by Western blotting and calcium imaging experiments. Taken together, our results indicate that nico-

1 1 1,2 1 tine has significant down-regulatory effects on Poly (I:C)-induced E. Macakova , D. Veisova , L. Rezabkova , and V. Obsilova pathways, and that calcium signaling is actively involved in nicotine’s immunomodulatory effects during viral-induced inflammation. 1Institute of Physiology Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic, 2 Department of Physical and Macromolecular Chemistry, Faculty Replication study of prior GWAS candidate genes/loci of Science, Charles University, 12843 Prague, Czech Republic for coronary artery diseases in the genetic isolated Neutral trehalase (EC 3.2.1.28) is a trehalose hydrolyzing enzyme, Newfoundland and Labrador population found in the yeast Saccharomyces Cerevisiae. It hydrolyzes a,a-trehalose (1-a-D-glucopyranosyl a-D-glucopyranoside,) which plays an Y.-G. Xie1, J. Cui1, E. Randell1, J. Renouf5,S.Li1, A. Pope4, G. Sun2, important role as a reserve and stress metabolite in yeast cells, to two W. Gulliver2, B. Sussex2 and F.-Y. Han1 glucose molecules. According to former results, the activity of NTH1 is mediated by BMH1 and BMH2-yeast isoforms of 14-3-3 proteins. Disciplines of 1Laboratory Medicine, When the NTH1 is phosphorylated by PKA, it forms a complex with 2Medicine, Memorial University, St. John’s, NL, Canada one of the 14-3-3 isoforms, which is required for activation of NTH1. 3Laboratory Medicine Program, Eastern Health, St. John’s, NL, Canada However, the exact mechanism of BMH controlling the NTH1 acti- 4Dept. of Molecular Genetics, Newfound Genomics, St. John’s, NL, vation remains still elusive. Canada BMH proteins were expressed as described previously. All mutants of NTH1 were expressed as 6xHis tag fusion proteins and Genome-wide association studies (GWAS), have provided some were purified from Escherichia coli Rosetta cells using Chelating- promising achievements for a number of multifactor diseases includ- Sepharose Fast Flow, cation-exchange chromatography on HiTrap SP ing coronary artery disease (CAD). However, many of these GWAS column and using gel filtration on Superdex 200 column afterwards. candidate genes failed to been replicated in studies using different Purified NTH1 mutants were phosphorylated by cyclic AMP-depen- ethnic populations which indicates the variety of genetic modifiers dent protein kinase (PKA). The completeness of the phosphorylation among different ethnic populations. Additionally, the genetic hetero- reaction was checked using MALDI-TOF mass spectrometry. geneity in the studied population can reduce the sensitivity in detection The interaction between BMH1 and BMH2 protein with enzyme of weak genetic effects and leads to false negative results in replication NTH1 was monitored using native electrophoresis and sedimentation studies. The population of Newfoundland and Labrador (NL) is a well velocity measurements. Limited proteolysis was used to show how is known genetic isolated population, and this population has a high NTH1 in complex with BMH protected from cleavage. Finally, we used prevalence of CAD in Canada. As a part of our ongoing study, 15 enzyme kinetic measurements to monitor the BMH-dependent acti- genetic variants from 12 selected prior GWAS candidate genes/loci vation of NTH1. have been genotyped in 500 patients with myocardial infarction (MI) and 500 age and sex matched controls from the NL population to determine the disease risk impact in the studied population. Geno- TM Regulatory effect of nicotine on Poly (I:C)-induced typing was carried out by using the Sequenom’s MassARRAY signaling using PCR and antibody arrays: system. Among the 12 studied genes/loci, only the 9p21 locus (rs 133049, rs 10757274, rs238306 and rs238307) was successfully from expression to activation associated to the patients (P \ 0.000 in all SNPs). This association was further confirmed in another study with enlarged sample size (1,000 Wen-Yan Cui MI patients and 1,000 controls) from the same population (P \ 0.000 in all SNPs). We, therefore, conclude that the 9p21 locus is a genetic State Key Laboratory for Diagnosis and Treatment of Infectious susceptibility for CAD in the NL population. The failure of replicating Diseases, First Affiliated Hospital, College of Medicine, other 11 GWAS candidate genes for CAD in NL patients strongly Zhejiang University suggests the diversity of genetic modifiers for CAD in NL population.

Nicotine, the primary psychological stimulant in tobacco, exerts Structural insight into interactions of calmodulin broad physiological and pharmacological effects on both neuronal and peripheral systems. In recent years, the anti-inflammatory func- with TRPV2 and TRPV5 C-termini tion of nicotine has drawn particular attention. However, most of the previous studies on the immunomodulatory effect of nicotine are Blanka Holakovska, Lenka Grycova, and Jan Teisinger limited to single gene level. In this study, we used multiple high throughput molecular techniques to characterize nicotine’s anti- Institute of Physiology, Academy of Sciences of the Czech Republic inflammatory actions during viral infections at both gene expression and pathway activation level. We examined RNA expression of 51 TRPV2 and TRPV5 ion channels belong to vanilloid subfamily of key genes in TLR (Toll-Like Receptor) pathways using quantitative transient receptor potential channels (TRPs). These channels are RT-PCR array, and determined the protein levels for TNF-a and IL-6 ubiquitously expressed in all eukaryotic cells and are involved in as indicators of the pathway using ELISA assay, in Poly (I:C)-induced many cellular processes like transduction of sensory signals and macrophages. We found that Poly (I:C)-induced innate immune sig- regulation of Ca2+ and Mg2+ homeostasis. naling was significantly modulated by nicotine. We further TRPV2 falls within the so called thermoTRPs and has been pro- demonstrated that such modulation was mediated by the a7 nicotine posed to mediate high-threshold noxious heat sensation. TRPV5 is a acetylcholine receptor (nAChR). We also employed an antibody array rather distinct member of TRPV subfamily and features quite dif- containing 1,318 antibodies to determine nicotine’s effects on Poly ferent properties then the rest of this subfamily. It is strictly Ca2+ (I:C)-triggered kinases at the phosphorylation level, and found a selective and involved in renal Ca2+ absorption/reabsorption.

123 S16 12th International Congress on Amino Acids, Peptides and Proteins

It is known that calmodulin (CaM) serves as an important TRP Pesticides have become an integral part of ecosystems, although channels regulator via binding to their intracellular termini in a cal- many of them are extremely toxic, even to non-target species. cium dependent manner. Hence, it is crucial to detect pesticide-induced molecular alterations We identified CaM binding sites on the C-termini of TRPV2 (654- in exposed species. In this study, Attenuated Total Reflectance 683) and TRPV5 (587-616) corresponding to the consensus CaM Fourier Transform Infrared (ATR–FTIR) spectroscopy was utilized binding motif 1-5-10 using steady-state anisotropy measurement, CD as a novel method to better understand the impact of cypermethrin spectroscopy and computer homology modeling. Moreover, it is the toxicity (0.04–3.60 lg/L) on the protein profile of Daphnia pulex— first time that such sequence has been recognized on the intracellular key-stone species in lake ecosystems. Spectral analysis revealed a parts of TRPV2. We also investigated the role of basic residues decrease in protein content at all cypermethrin concentrations from present in the region. The exchange of positively charged residues to the peak area values of both the amide I and II bands. In order to neutral alanine resulted in some cases in total loss of binding ability to determine protein secondary structures, Neural Network (NN) CaM. Also the data from the CD spectroscopy experiment showed method using the 1,700–1,600 cm-1 spectral region of the FTIR changes in the distribution of the secondary structure elements in the spectra put forward a cypermethrin-induced protein denaturation at mutants compared to the wild type. high cypermethrin doses (0.90–3.60 lg/L) through a decrease in a- Based on these results we concluded that basic residues play helix and turn content and an increase in total b-sheet and random crucial role in TRP channels binding to CaM. coil content. Increased random coil content in high-dose groups was This project was supported by Grant GAAV IAA600110701, observed also with vector normalization in the same spectral region. GACR 301/10/1159, GACR P205/10/P308, project No. 305/08/H037, Vector normalization method further suggested that the increase in Centre of Neurosciences No. LC554 MSMT CR. total b-sheet content observed at high doses by NN studies was mainly due to an increase in antiparallel and aggregated b-sheet elements, regardless of the decrease in native b-sheet structures. The binding of hepatitis B virus X protein to GLI1 Supporting the findings on the different attitude of high cyper- and its biological characterization in vitro methrin doses, hierarchical cluster analysis successfully differentiated the highdose groups from the control and low-dose groups. Therefore, the ATR–FTIR spectroscopic results put forward So Young Park, Young-hoon Park, Se Bok Jang and Mi Suk Jeong that the protein profile of Daphnia pulex are adversely affected by cypermethrin. Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Korea Two-dimensional glycoproteome maps: a new insight Hepatitis B virus (HBV) X protein (HBx) is a 17-kDa transcriptional into protein glycosylation patterns during larval coactivator that plays a significant role in the regulation of genes development and metamorphosis in marine involved in inflammation and cell survival. It has been known to be involved in the development of liver cancer and alteration of the invertebrates cellular HBx level may influence the pathogenesis of HBV-induced liver diseases. The transcription factor GLI1, a member of the glioma- K. H. Chandramouli, Y. Zhang, Y. H. Wong, S.Y. Mok, and P.-Y. Qian associated oncogene homologue (GLI) subfamily of Kru¨ppel-like zinc finger proteins is involved in signal transduction within the hedgehog Section of Marine Ecology and Biotechnology, Division of Life (Hh) signaling pathway, which is involved in the development of Science, The Hong Kong University of Science and Technology, many human malignancies. GLI activation is important for cell pro- Hong Kong SAR. liferation and anti-apoptosis in various cancers. To investigate *Presenting author Email: [email protected] whether the transcriptional coactivator HBx binds to the zinc finger transcription factor GLI1, recombinant HBx and GLI1 were isolated. The life cycle of most marine invertebrates has two distinct stages: Expression and purification of the HBx and GLI1 proteins were the pelagic larvae and the adults attached to marine surface. The successfully performed in Escherichia coli. The binding of HBx to transition between these two stages (called ‘‘larval settlement and GLI1 was detected by surface plasmon resonance spectroscopy metamorphosis’’) is often abrupt and may involve differential (BIAcore), fluorescence measurement, and a His-tagged pull-down protein modifications. Glycosylation, a very important post-trans- experiment. After measuring the fluorescence emission spectra of lational modification of proteins plays fundamental roles in purified HBx and GLI1, it was found that the interaction of these controlling various biological processes. However, to date, no study proteins is accompanied by significant conformational changes in one has addressed glycosylation patterns of proteins during larval set- or both. This study provides important clues for the structural iden- tlement and metamorphosis in marine invertebrates. To understand tification of signal transduction pathways involving the HBx and the common molecular patterns of protein glycosylation associated GLI1 proteins. with larval metamorphosis, we used comparative approach to investigate glycosylation patterns in barnacles, bryozoan, and Toward a protein profile of the zooplankton polychaete species. We applied a fluorescence-based multiplexed proteomics technology which included two-dimensional gel elec- Daphnia pulex exposed to the pesticide cypermethrin, trophoresis (2-DE) followed by the fluorescence staining of using ATR–FTIR spectroscopy as a novel method glycoprotein and mass spectrometry identification of proteins. Two- dimensional proteome maps showed that protein glycosylation S. B. Akkasa, M. Severcanb, M. Beklioglua, and F. Severcana pattern changed markedly from larval to juvenile stages of selected marine species. We identified twenty abundant and differentially aDepartment of Biological Sciences, expressed glycoproteins, of these cytoskeleton proteins showed bDepartment of Electrical and Electronic Engineering Middle East distinct glycosylation in all three species. Oxidative stress proteins Technical University, 06800 Ankara, Turkey revealed distinct glycosylation in B. amphitrite and energy

123 12th International Congress on Amino Acids, Peptides and Proteins S17 metabolism proteins were glycosylated in B. neritina. Expression Computational studies of protein phosphorylation: from patterns of selected proteins were confirmed at the protein level by site-specific prediction to systematic network analysis Western blot analysis while some proteins were further studied at the mRNA level by real-time PCR. The identified glycoprotein Zexian Liu1, Xinjiao Gao1, Jun Cao1, Qian Ma1, Jian Ren2, proteins were shown to be involved in development, cell differ- 1, entiation and integrity, apoptosis, oxidative stress and energy and Yu Xue * metabolism. The protein glycosylation alteration provide essential 1 basis to understand molecular processes and pathways involved Hefei National Laboratory for Physical Sciences at Microscale in larval development and metamorphosis of multiple marine and School of Life Sciences, University of Science & Technology species. of China, Hefei, Anhui 230027, China 2Life Sciences School, Sun Yat-sen University (SYSU), Guangzhou, 510275, China 3Hubei Bioinformatics and Molecular Imaging Key Laboratory, Bioinformatics Department of Systems Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China *Correspondence: [email protected] Application of fluorogenic substrate libraries containing natural and unnatural amino acids for profiling Phosphorylation catalyzed by protein kinases precisely regulates cell of proteolytic enzymes signaling pathways. Comprehensive elucidation of kinase-specific phosphorylation sites will reveal a complex protein phosphorylation network (PPN), which provides a systematic insight for understanding Marcin Drag1,2, Anna Gajda1, Paulina Kasperkiewicz1, cell dynamics and plasticity. Recent progresses on phosphoproteomics Marcin Poreba1, Rafal Latajka1, Malgorzata Pawelczak3, with high-throughput mass spectrometry (MS) produce hundreds of Aline Marschner4, Christian Klein4, Matthew Bogyo5, thousands of phosphorylation sites. However, the upstream kinases are Jonathan A. Ellman6, and Guy S. Salvesen2 difficult to be determined, while further in vitro and in vivo studies were greatly hampered by lack of accurate and efficient computational tech- 1Division of Medicinal Chemistry and Microbiology, Faculty niques. Here we show that kinases and substrates form highly complex of Chemistry, Wroclaw University of Technology, Poland PPNs in eukaryotes. Based on a general model, we accurately predict 2Apoptosis and Cell Death Research Program, Sanford Burnham 188,288 site-specific kinase-substrate relationships between 9,247 tar- Medical Research Institute, La Jolla, CA, USA gets and 1,079 kinases for 44,290 phosphorylation sites. An integrated 3Institute of Chemistry, University of Opole, Poland and automated computational platform was developed for analyzing 4Department of Medicinal Chemistry, Universita¨t Heidelberg, various organism- or tissue-specific PPNs. Particularly, we discovered a Germany small potential core PPN specifically in liver conserved between human 5Department of Pathology, Stanford School of Medicine, Stanford, and mouse. Furthermore, we found that PPNs evolved rapidly even CA, USA among near species, and consistent with other recent observations. Our 6Department of Chemistry, Yale University, New Haven, USA results demonstrate that MS-based large-scale phosphoproteomic analysis with subsequently computational prediction could generate flood of Proteases are known to participate in many cellular processes due useful information, which might be helpful for further experimental to their ability to process the peptide bond. They are key players in manipulation. We anticipate our study to be a starting point for more several cascades taking place in the cell with apoptosis, fibrinolysis, sophisticated analyses of PPNs. For example, human PPNs influenced by blood clotting, complement fixation, gastrulation or general cell the individual polymorphisms could be tested. Furthermore, tissues cycle being only a few examples. However, proteases can be also under different developmental stages might also exhibit distinct PPNs, bad guys and participate in the cellular events, which leads to while some key pathways will be rewired in diseases and cancer. severe diseases such as cancer, diabetes, pathogens infections or hypertension. Each protease recognise only substrates, which can fit into the New alignment and software package for sequence analysis recognition pockets. There are several methods of determination of substrate specificity. One of the most versatile is Positional Scan- ning Substrate Combinatorial Library approach, which allows fast *Toshihide Hara, Keiko Sato, and Masanori Ohya and reliable determination of substrate preferences for most of the proteases. To date used libraries contained only natural amino acids We develop a new alignment for bio-sequences called MTRAP by and only very occasionally unnatural amino acids were incorporated introducing a measure based on entangled correlation in two consecutive into the structure of synthesized substrates. To get better insight into residues. We show that the alignment accuracy could be improved by our substrate specificity, we have applied several different tailored to approach not only for pairwise sequences but also multiple sequences. certain type of protease approaches using broad range of unnatural amino acids. This allowed us to obtain much better substrates The code structure of the receptor binding site comparing to natural amino acids derivatives for several different aminopeptidases, dipeptidylpeptidases and endopeptidases. Here we of influenza A virus hemagglutinin will present the latest findings for each respective group of proteases. *Keiko Sato, and Masanori Ohya Acknowledgments: The Drag laboratory is supported by the Foun- dation for Polish Science and the State for Scientific Research Grant Information of life is stored as a sequence of nucleotides, and the N N401 042838 in Poland. sequence composed of four bases seems to be a sort of code. The

123 S18 12th International Congress on Amino Acids, Peptides and Proteins hemagglutinin (HA) of influenza A viruses is responsible for binding perspective, we proposed a model to explain the DAAO/pLG72/D- the virus to host receptors. In the coding theory, information is Ser association with the onset of the pathology: an anomalous properly coded for its transmission. The artificial codes in information hypoexpression of pLG72 yields to an increase of DAAO activity, transmission were applied to find the code structure of the left and then to a decrease of D-Ser released at the synapse, finally leading right edge of the receptor binding site in HA. Various influenza A to hypoactivation of NMDA receptors. This intriguing mechanism viruses are classified according to the method above. of D-Ser regulation does not match with the proposed subcellular localizations for DAAO (peroxisomes) and pLG72 (mitochondria). By using U87 human glioblastoma cells expressing EYFP-DAAO and/or pLG72-ECFP fusion proteins, we provide evidence that Walking on the thin line between order and randomness newly synthesized DAAO is located in the cytosol where it can in conformational transitions in polypeptides: chiral interact with pLG72, placed on the external mitochondrial mem- bifurcation brane. Subcellular immunolocalization and FRET analyses strongly support this proposal. Furthermore, in cotransfected cells pLG72 overexpression decreases the half life of DAAO. Since neosyn- Wojciech Dzwolak thesized cytosolic DAAO was demonstrated to be catalytically active, we suggest that pLG72 binding to DAAO (and ensuing Department of Chemistry, University of Warsaw, Pasteur 1, 02-093 inactivation and degradation) might play a protective role against Warsaw, Poland excessive D-Ser depletion. Our results uncover basic molecular aspects of the D-Ser deg- Recently, we have described an unexpected phenomenon consist- radation pathway and open novel perspective for therapeutic ing in vortex-induced formation of two conformational variants strategies of schizophrenia by targeting DAAO and the metabolism of insulin amyloid fibrils with opposite chiral biases, as revealed of D-Ser. by CD spectroscopy. Under certain conditions of the aggregation process, the sign of the CD spectrum cannot be predicted, suggesting thereby that stochastic fluctuations determine outcome of the experiment. This is the first case of chiral bifurcation effect in Levels of amino acids in the brain and peripheral biophysics, although similar phenomena have been reported in con- organs of serine racemase knock-out mice densed matter physics—e.g. upon crystallization of NaClO3. Our findings point to a new aspect of topological complexity of 1 1 1 1 protein fibrils: a chiral feature of hierarchically assembled poly- Mao Horio , Tamaki Ishima , Mami Kohno , Yuko Fujita , 2 2 1 peptide chains, which is not determined by the innate left- Ran Inoue , Hisashi Mori , and Kenji Hashimoto handedness of amino acids. Because altering chirality of a molecule 1 changes dramatically its biological activity, these findings may Division of Clinical Neuroscience, Chiba University Center have important ramifications in the context of structural basis of for Forensic Mental Health, Chiba, Japan 2 ‘amyloid strains’. Hydrational forces may play an important role in Department of Molecular Neuroscience, Graduate School defining pathways of conformational transitions of misfolded proof Medicine and Pharmaceutical Sciences, University of Toyama, teins both in vitro and in vivo. Toyama, Japan

D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA) receptor, plays an important role in mammalian brain neurotransmission, via the NMDA receptor. D-Serine is synthesized D-Amino acids from L-serine by serine racemase (SRR), and D-serine is metabolized by D-amino acid oxidase (DAAO). In this study, we measured levels of the neurotransmission related amino acids, D-serine, L-serine, D-Serine level in glial cells: spotlight on the role glycine, glutamine and glutamate in the frontal cortex, hippocampus, striatum and cerebellum as well as in peripheral tissues of blood, of D-amino acid oxidase heart, pancreas, spleen, liver, kidney, testis, epididymis, heart, lung, muscle and eyeball, in wild-type (WT) and Srr-knockout (Srr-KO) S. Sacchi, P. Cappelletti and L. Pollegioni mice. Levels of D-serine in the frontal cortex, hippocampus, and striatum of Srr-KO mice were significantly lower than in WT mice, ‘‘The Protein Factory’’ Research Center, Universita` degli Studi while levels in the cerebellum stayed the same. In contrast, levels of dell’Insubria (Dip. Biotecnologie e Scienze Molecolari) L-serine, glycine, glutamine and glutamate remained the same in all and Politecnico di Milano, via Dunant 3, 21100 Varese, Italy; tested brain regions. In vivo microdialysis using free-moving mice [email protected] showed that extracellular levels of D-serine in the hippocampus of Srr-KO mice were significantly lower than in WT mice while the D-Serine is a brain enriched transmitter-like molecule that physio- other amino acid levels remained the same between mice. In logically activates NMDA receptors, playing a main role in neuron- peripheral organs, levels of D-serine in the liver, kidney, testis, epi- glia communication and excitatory neurotransmission. D-Ser is didymis and eyeball of Srr-KO mice were significantly lower than in synthesized by serine racemase and degraded by D-amino acid WT mice. Tissue levels of the other tested amino acids in peripheral oxidase (DAAO). We demonstrated that D-Ser cellular concentra- organs were not altered. These results suggest that SRR is the major tion depends on the expression of active DAAO and of its negative enzyme responsible for D-serine production in the mouse forebrain, regulator pLG72. Genetic evidences indicated that both pLG72 and that other pathways of D-serine production may exist in the brain and DAAO are related to schizophrenia susceptibility. In this and peripheral organs.

123 12th International Congress on Amino Acids, Peptides and Proteins S19

Pain biology of spinal D-amino acid oxidase (DAAO) the homozygous mouse. Methionine oxidation and protein disulfide formation in crystallins are highly increased at 6 months of age. Mice Yong-Xiang Wang, Nian Gong, Xiao-Lin Chen, Jin-Lu Huang, develop nuclear opacities as in human lens. In the NEGSKO mouse, Yan-Chao Wang, Xin-Yan Li and Ai-Niu Ma molecular, cellular and behavioral phenotypes resembling amyotrophic lateral sclerosis (ALS) develop at 1 month of age, which include low King’s Lab, Shanghai Jiao Tong University School of Pharmacy, 800 pan-neuronal levels of GSH, increased apoptosis, diminished neuronal Dongchuan Road, Shanghai 200240, China. Tel.: 86-21-3420-4763; cell count, increased tau phosphorylation, and cleavage of the ALS Email: [email protected] marker TDP-43. The availability of these novel disease models is expected to be fruitful for the development of drugs against age-related cataract and ALS. D-Amino acid oxidase (DAAO) is a peroxisomal flavoprotein that catalyzes the oxidative deamination of neutral and polar D-amino acids to a-keto acids, NH3 and hydrogen peroxide with strict ste- Protein glycation, aggregation and cognitive reospecificity. DAAO is restrictedly distributed in the kidneys, liver impairments and central nervous system including the spinal cord exclusively located within astrocytes. The role of spinal DAAO in pain has been recently discovered and validated. Yan Wei, Chanshuai Han and Rongqiao He* Systemic and intrathecal injections of a series of DAAO inhibitors, blocked formalin-induced tonic pain and bone cancer-induced State Key Laboratory of Brain and Cognitive Sciences, Institute mechanical allodynia, with maximum inhibition of 50–60%, in rats of Biophysics, Chinese Academy of Sciences, Beijing, China and mice, correlated to their inhibition of spinal DAAO activity. Intrathecal administration of siRNA/DAAO delivered by PEI com- D-Ribose, one of the most important reducing monosaccharides, is plexation or adenovirus vector prevented tonic pain in rats. DAAO present in all living cells and also in blood, cerebrospinal fluid as well as gene mutation also caused similar inhibition of tonic pain in ddy/- the human brain. Recently, D-ribose has been found to be very active in DAAO-/- mice. DAAO inhibitors produced analgesia via the glycation of proteins such as bovine serum albumin, a-Synuclein and blockade of raised spinal hydrogen peroxide. Tau protein, resulting in advanced glycation end products (AGEs). The effects of DAAO inhibitors in chronic pain were specific as they Ribosylation occurs much more rapidly than glucosylation in vitro. That were not effective in acute pain such as escape responses in the tail-flick is, in the presence of D-ribose, serum albumin, a-Synuclein and Tau and hot-plate tests, and thermal hyperalgesia in the carrageenan model. In protein are rapidly glycated, generating globule-like aggregations which addition, DAAO inhibitors interacted with morphine in an additive manner can induce apoptosis and oxidative stress in several neuronal cell lines. in their analgesia, and did not produce either self-tolerance to analgesia or However, we did not observe that the globule-like aggregations could cross-tolerance to morphine after 7 days exposure, in contrast to morphine. advance into fibrils deposits after incubation with D-ribose over Thus, spinal DAAO is a potential new target molecule for dis- 2 weeks. We also find that D-ribose, but not D-glucose under our covery and development of novel medicines for chronic pain experimental conditions, can decrease cell viability and accelerate including cancer pain, with high efficacy, pain signal transduction and protein glycation and AGEs accumulation in SHSY5Y cells, HEK-293 tissue expression specificity, and without tolerance to algesia. cells and primary cultured hippocampal neurons. To investigate the The study was supported by NSFC grants: No 81072623 and effect of D-ribose and ribosylation in vivo, C57BL/6J mice were intra- 30973581, China. peritoneally injected with D-ribose for 30 days. After injection, these mice had got remarkably high levels of glycated proteins and AGEs in their blood. AGEs were highly accumulated in mice brains and localized in the hippocampus and cortex, but not significantly observed with the Glycation mice administrated with D-glucose or saline control. More interestingly, the ability of spatial learning and memory of D-ribose-treated mice Mouse models of accelerated aging by carbonyl declined in the Morris water maze test. These data demonstrated that D- ribose, as an efficient glycation agent, could rapidly glycate proteins and and oxidant stress produce AGEs both in vitro and in vivo. The accumulated AGEs in brains by ribosylation could impair spatial cognition of mice. V. M. Monnier, and X. Fan Dept. of Pathology and Biochemistry, Case Western Reserve Medicine University, Cleveland, OH 44106, USA

Aging is associated with progressive protein damage in lens, collagen- rich tissues, neurodegenerative diseases and conditions with weakened Abundances of intestinal apical neutral amino acid antioxidant defenses. While the nature of the chemical protein changes transporter B0AT1 and exchanger ASCT2 proteins are is getting better understood, it has been difficult to directly implicate reduced in pigs with dextran sodium sulfate-induced colitis damage in the disease process itself. To close this gap, we developed novel mouse models of accelerated tissue aging, by selectively over- 1,2 1 1 expressing or knocking out genes controlling carbonyl and oxidant Chengbo Yang, Dale Lackeyram, Tania Archbold, 2 1 stress. The first model was the hSVCT2 mouse in which we showed that Yoshinori Mine, and Ming Z. Fan expression of the human vitamin C transporter 2 in the lens accelerated 1 the protein modifications observed during lenticular aging. Two new Center for Nutrition Modeling, Department of Animal and Poultry Science, 2 models of accelerated protein aging by oxidation have now have been Department of Food Science, University of Guelph, Guelph, Ontario N1G generated by conditionally targeting glutathione synthesis in lens 2W1 (LEGSKO mouse) (to mimic the low GSH levels in the core of the 0 human lens) and nerve (NEGSKO mouse). c-Glutamyl cysteine ligase We investigated changes in the expression of B AT1 and ASCT2 mRNA, activity and glutathione (GSH) levels are severely depressed in genes as affected by inflammation in a porcine model of colitis

123 S20 12th International Congress on Amino Acids, Peptides and Proteins induced with dextran sodium sulfate (DSS). Real time RT-PCR reduced the BSCB breakdown, edema formation and cell injury. analyses showed no difference (P [ 0.05) in the relative abundance Functional recovery was also markedly improved by treatment with this of the B0AT1 mRNA between the DSS and the control groups in the combination of neurotrophins. Expression of HO-2 in the spinal cord colon. However, lower jejunal B0AT1 mRNA abundance was was significantly reduced. However, co-application of CNTF with either observed (P \ 0.05) in the DSS group. There were no differences BDNF or GDNF was not that effective in inducing neuroprotection or (P [ 0.05) in the relative abundances of the ASCT2 mRNA in both downregulation of HO-2 expression in SCI. These observations suggest jejunum and the colon between the DSS and the control groups. Both that (i) a combination of CNTF with BDNF and GDNF is necessary to B0AT1 and ATB0 protein abundances were lower (P \ 0.05) in the induce effective neuroprotection even during the later phase of SCI (i.e., jejunal homogenate and cytosol, and on the apical membrane in the 120 min after primary insult), and (ii) the neuroprotective effects of DSS group compared with the control. Abundances of the B0AT1 and neurotrophins in SCI are some how interrelated with increased CO ATB0 proteins on the colonic apical membrane was lower (P \ 0.05) production. Taken together our novel observations suggest an active in the DSS group than in the control, whereas there were no differ- interaction of BDNF, CNTF and GDNF with CO system for inducing ences (P [ 0.05) in abundances of the B0AT1 and ATB0 proteins in neuroprotection in the spinal cord after trauma. the colonic tissue homogenate and the cytosolic fraction between the two groups. These results collectively suggest that abundances of neutral amino acid transporter B0AT1 and exchanger ASCT2 are Affibody molecules. New targeting scaffold proteins decreased in the small intestine and colon in pigs with colitis induced for radionuclide molecular imaging and therapy 0 0 by DSS. Considering the reduced B AT1 and ATB protein of cancer abundances on the apical membrane, combination of dietary supplementation of neutral amino acids mixture and other strategies to enhance the B0AT1 and ATB0 protein expression on the apical Anna Orlova membrane may have the potential for the treatment of colitis. Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Sweden; Email: [email protected] A combination of CNTF, GDNF and BDNF applied topically over the injured spinal cord improves functional Specific recognition of cancer-associated molecular abnormalities, i.e. recovery and reduces hemeoxygenase-2 (HO-2) targeting, is a promising way to improve diagnostics and treatment of malignant tumors. Affibody molecules represent a new class of affinity expression, blood-spinal cord barrier permeability, proteins based on a scaffold of B-domain of staphylococcal protein A. edema formation and cellular damage in the rat Affibody molecules combine small size (*6.5 kDa) with high affinity and specificity. Selection of high-affinity (low nanomolar or subnanom- 1Hongyun Huang, 2Aruna Sharma, and 2Hari Shanker Sharma olar) Affibody molecules binding to cancer-associated molecular targets, such as HER2, EGFR, IGF-1R, HER3 and PDGFbR, has been reported. 1Neuroscience Institute of Taishan Medical University, China; Email: The small size and simple structure of Affibody molecules allow [email protected], their production by chemical synthesis. This permits the use of 2Cerebrovascular Research Laboratory, Department of Surgical unnatural amino-acids, site-specific incorporation of chelators for Sciences, Anesthesiology & Intensive Care Medicine, University labeling with variety of nuclides, or toxins and drugs for therapy. As Hospital, Uppsala University, SE-75185 Uppsala, Sweden, Affibody scaffold does not contain cysteine, an introduction of a Email: [email protected] single cysteine by gene-engineering enables site-specific conjugation of linkers and chelators to recombinantly produced Affibody mole- Previous reports from our laboratory show that neurotrophins when cules using thiol-directed chemistry. Fusion of Affibody molecules applied topically over the injured spinal cord in combination is able to with streptococcal albumin-binding domain (ABD) permits modula- reduce cord pathology and improves functional recovery. In this tion of their residence time in the blood circulation. regard, a combination of BDNF and GDNF showed remarkable Pre-clinical studies have demonstrated that Affibody molecules neuroprotection even when applied 90 min after spinal cord injury can be labeled with different nuclides for imaging (PET and SPECT) (SCI). However, a combination of BDNF or GDNF with NGF was not and for radionuclide therapy. In vivo, Affibody molecules provide so effective. In present investigation, we wanted to know the role of rapid and specific binding to tumors and rapid renal clearance for non- CNTF in enhancing neuroprotection in SCI in combination with bound tracer, permitting high contrast in vivo images already a few 111 BDNF or GDNF treatment. Previously, we have shown that BDNF is hours after injection. A pilot clinical study confirmed that [ In]- and 68 able to attenuate nitric oxide (NO) production in the spinal cord after [ Ga]-labeled Affibody molecules can visualize HER2-expressing injury that correlates well with its neuroprotective ability. Since metastases. In conclusion, Affibody molecules constitute a promising carbon monoxide (CO) is also a free radical gas and has many sim- class of targeting proteins for tumor targeting. ilarities with NO in inducing cell damage, in present investigation, we explored the role of neurotrophins in modulating CO production in Altitude hypoxia on the ultrastructure of rat brain the spinal cord in relation to neuroprotection. For this purpose, we used immunohistochemistry of the constitutive isoform of CO syn- and Hsp70 expression thesizing enzyme, hemeoxygenase-2 (HO-2) to understand the functions of CO in SCI in relation to neurotrophins treatment. Li Wenhua, Yuan Dongya, Zhang Min, Li Yang, Sun Zhenqi, A focal SCI on the right dorsal horn on the T10–11 segment Zhao Fengcang markedly increased the HO-2 immunostaining in the T9 and T12 segments at 5 h. At this time, breakdown of the blood-spinal cord barrier Medical School, Tibet Institute of Nationalities, Xianyang, (BSCB), edema formation and cell changes were seen in several spinal Shanxi 712082, China; Email: [email protected] cord segments adjacent to the lesion site. Topical application of BDNF, CNTF and GDNF in combination (10 ng each in 10 ll, Total 30 ll Objective means of cell biology and molecular biology of high alti- from a solution of 1 lg/ml BDNF, GDNF or CNTF solution) 60, 90 and tude adaptation in rat, the rat heat shock response, heat shock protein 120 min after injury over the exposed surface of the cord significantly in the organism to understand high altitude adaptation in biological

123 12th International Congress on Amino Acids, Peptides and Proteins S21 significance. Methods 90 male SD rats were randomly divided into 10 induce marked neuroprotection in TBI and these effects are further groups, three experimental animals brought by the Golmud, Qinghai, potentiated by nanowiring of the HO-2 antibodies. Xi’an time-consuming and 1d (elevation 2,700 m), 2d to the Tibetan Tanggula (elevation 5,000 m), 3d to the Naqu (elevation 4,500 m), two experimental groups 1d directly to Lhasa, Tibet (altitude 3,658 m) and Naqu, feeding more than five groups to plateau after Arginine transport in the human pathogen Leishmania 24 h of slaughter; three different experimental groups 1d directly to and its possible role in parasite-host interactions high altitude (above sea level in Qinghai Golmud 2,700 m, Lhasa, Tibet, 3,658 m, Naqu 4,500 m) after 4 weeks of feeding killed, two 1 1 2 group (in Xi’an, elevation 5 m), with the Western blot and conven- Adele Goldman-Pinkovich , Ilona Darlyuk , Doris Rentsch 1 tional RT-PCR and real-time fluorescence quantitative PCR (real- and Dan Zilberstein time PCR detection) measured at different altitudes SD Heat shock 1 protein in rat brain 70 (Hsp70) expression and brain differences in the Faculty of Biology, Technion-Israel Institute of Technology, natural expression of Hsp70 gene, light microscopy and transmission Haifa 32000, Israel, 2 electron microscopy observation of animals in each group structural Institute of Plant Sciences, University of Bern, 3013 Bern, changes in brain tissue. Results Mammals have a different elevation Switzerland of heat shock response genes, stress, high altitude when the rapid increase in the expression of mammalian Hsp70; Hsp70 can be high Arginine is an essential amino acid for the intracellular parasitic pro- altitude (high altitude) induced. Conclusion: Hsp70 heat shock tozoan Leishmania but not for its host. Thus, maintaining cellular response in the rapid synthesis of high altitude hypoxic stress is homeostasis of arginine is critical for parasite survival and virulence. conducive to maintaining the normal physiological function when the Previously, we cloned and functionally characterized a high affinity cells, Hsp70 and cell formation is proportional to hypoxia tolerance. arginine-specific transporter, LdAAP3, from Leishmania donovani. Keywords: High altitude, SD rats, Heat shock protein 70 (Hsp70), Here we characterized the relationship between arginine transport via Western blot, RT-PCR real time quantitative PCR LdAAP3 and amino acid availability. Exposing parasites to amino acids Project: Tibet, the Office of Science and Technology Research starvation decreased the cellular level of most amino acids including Fund (2010), the State Ethnic Affairs Commission research project arginine, while the abundance of LdAAP3 mRNA and protein greatly (2011), initial results. increased. Consequently, arginine transport activity was up-regulated *fivefold. We also found that genetic obliteration of the polyamine biosynthesis pathway, for which arginine is the sole precursor, caused a significant decrease in the rate of arginine transport. Cumulatively, we Antibodies to hemeoxygenase-2 tagged with nanowires established that LdAAP3 expression and activity changed whenever the cellular level of arginine changed, and thus hypothesized that L. enhances neuroprotection in traumatic brain injuries donovani promastigotes have a signaling pathway that senses cellular concentrations of arginine and subsequently activates a mechanism that Hari S Sharma, and Aruna Sharma regulates LdAAP3 expression and activity. Although starvation for amino acids is an artificial condition Department of Surgical Sciences, Anesthesiology & intensive Care examined in an in vitro system, it is likely that Leishmna- Medicine, University Hospital, Uppsala University, SE-75185 nia also experience starvation in vivo. Inside macrophage Uppsala, Sweden. Email: [email protected] phagolysosomes the parasites proliferate as non-motile amastigotes that need to compete for host arginine, possibly by employing their Previous reports from our laboratory showed that traumatic brain or starvation response mechanism. Therefore, we hypothesize that spinal cord injuries upregulates hemeoxygenase-2 (HO-2) proteins in LdAAP3 plays a vital role in the parasites survival within its host. various regions in the CNS. Drugs downregulating HO-2 protein expression resulted in neuroprotection. Thus, in present investigation the neuroprotective role of HO-2 antibodies in brain injuries was investigated in a rat model. Since nanowiring of drugs enhances then Association study of hypoxic gene single nucleotide neuroprotective capabilities, influence of nanowired HO-2 antibodies polymorphism with the susceptibility of acute mountain was also examined in present investigation. sickness Traumatic brain injury (TBI) was produced by making a longitudinal incision into the right parietal cortex under Equithesin anesthesia and the rats were allowed to survive 5 h after the lesion. In Li Wenhua, Yuan Dongya, Zhao Fengcang, Sun Fangyun separate group of rats HO-2 antibodies (1:20, monoclonal) were applied 10, 30 or 60 min after TBI. Another group of rats received Medical School, Tibet Institute of Nationalities, Xianyang, Shanxi identical HO-2 antibodies but tagged with TiO2 nanowires applied 712082, China. Email: [email protected] over the lesion at the same time intervals after trauma. Topical application of HO-2 antibodies given 10 or 30 min after TBI resulted Background and objective: Acute mountain sickness (AMS) is in marked neuroprotection in terms of reduction in the blood–brain apotentially serious affliction to health in immigrants to Tibetan Pla- barrier (BBB) breakdown to Evans blue and radioiodine traces, edema teau above 3,000 m. There are no effective treatment and prevention formation and neuronal injuries. However, normal HO-2 antibodies strategy of AMS now. Interindividual variation in acclimatization and did not give any effects when they are applied 60 min after TBI. On adaptation to high altitude suggest that the probability of developing the other hand nanowired HO-2 was able to significantly reduce AMS depends on genetic and environmental factors. So we speculated neuronal injuries and BBB disruption or brain edema even applied that genetic factors may be associated with AMS susceptivity. The after 60 min TBI. The neuronal damages were tightly correlated with research aimed to explore the association between single nucleotide upregulation of HO-2 expression in both untreated and following HO- polymorphism (SNP) of hypoxic gene and AMS by comparing the 2 antibodies treatments with or without nanowiring in TBI. Taken difference of hypoxic gene SNP between AMS-susceptible and together our results for the first time show that HO-2 antibodies could acclimatized individuals.

123 S22 12th International Congress on Amino Acids, Peptides and Proteins

Methods: The study enrolled Han Chinese students into Tibet in our rs8005745, rs2301108 and 10873142 of HIF 1 A between the AMS hospital for the first time flew to high altitude (3,658 m, Lhasa) from group and control group at high altitude (P [ 0.05). lowland (505 m). AMS was diagnosed on the basis of the AMS Conclusions: 1, Morbidity of AMS in Han Chinese students into Tibet Questionnaire. Venous blood was collected and then analyzed by in our hospital for the first time flew to high altitude was 35.68%; 2, ELISA for VEGF and for extraction of genomic DNA and peripheral SPOZ and VEGF is down-regulated significantly in AMS group and arterial oxygen saturation (SP02) was recorded at low altitude and control at athigh altitude but VEGF level of AMS group still higher after 24–48 h at high altitude. By case–control study method, we significantly than control not only at high altitude but also at sea level; selected 200 cases of AMS as AMS group and 200 individuals which 3, Polymorphism of rs3025039 and rs3025030 in VEGF gene were not developed AMS as control group randomly from these soldiers significantly associated with AMS. Individuals carrying the allele T of respectively. The selected subjects were genotyped for the fourteen rs3025039 and C of rs3025030 significantly decrease the risk of AMS polymorphisms of the hypoxic genes by primer extension of multiplex in a Chinese population. 4, Polymorphic loci of rs1137933 in NOS2 products with detection by matrix-assisted laser desorption ionization- and rs4309 in ACE were significantly associated with AMS. Individ- time of flight (MALDI-TOF) mass spectroscopy. These hypoxic uals carrying the genotypic TT of rs1137933and genotypic CC rs4309 genes include vascular endothelial growth factor (VEGF), hypoxia significantly increase the risk of AMS. 5. This study did not provide inducible factor I alpha subunit (HIF I A), Glutathione S-transferase evidence that SNPs of the rs2297518 of NOS2, rsl799983,rs3918188, mu 3(GSTM3), Glutathione S-transferase pi 1(GSTPI), Nitric oxide rs7830 of NOS3, rs1695 of GSTP1, rs7483 of GSTM3, rs8005745, synthase 2(NOS 2), nitric oxide synthase 3 (NOS 3), Angiotensin I rs2301108, rs10873142 of HIFIA and rs35853823 of HSPA4 gene are converting enzyme (ACE) and heat shock 70 kDa protein 4 (HSPA4). associated with susceptibility to AMS in a Chinese population. The association between SNP and AMS was analysed by genetic Keywords: VEGF, Hypoxic gene, Single nucleotide polymorphism, statistical software. AMS Results: Morbidity of AMS in Han Chinese students into Tibet in our Project: Tibet, the Office of Science and Technology Research hospital for the first time flew to high altitude was 35.68%. SP02 fell Fund (2010), the State Ethnic Affairs Commission research project from 98.0211.69% at 505 m to 85.16 ± 5.42% at 3,658 m in subjects (2011), initial results. with AMS (P \ 0.01) and from 98.02 ± 1.40% at 505 m to 86.3014.63% at 3,658 m in subjects without AMS (P \ 0.01). SP02 in subjects with AMS fell significantly compared with those without AMS Autoantibodies against heat shock proteins are at a height of 3,658 m (P \ 0.05). Plasma VEGF concentration decreased at an altitude of 3,658 m (16.98 ± 11.61 vs. increased in patients with amyotrophic lateral sclerosis 41.03 ± 37.37 pg/m1, P \ 0.01, in group without AMS; 55.58120.19 vs. 79.27127.48 pg/ml, P \ 0.01, in group with AMS) compared with I-Lin Liao1,2#, Chi-Shin Hwang3,4#, Guan-Ting Liu1,5, the baseline level. The plasma VEGF concentration were significantly Yi-Chen Wu1,6, Margaret Dah-Tsyr Chang3*, and Hao-Teng Chang1,5,7* higher in group with AMS than in group without AMS at baseline level and at altitude of 3,658 m (79.27 ± 27.48 vs. 41.03 ± 37.37 pg/ml, 1Graduate Institute of Molecular Systems Biomedicine, 55.58 ± 20.19 vs. 16.98 ± 11.61 pg/ml, respectively, P \ 0.01). All 2Department of Medical Laboratory Science and Biotechnology, 13 gene locuses exist polymorphisms and were in Hardy–Weinberg 3Institute of Molecular and Cellular Biology & Department equilibrium in both AMS group and controls except for HSPA4 which of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, only have a allele T. The T allele of rs3025039 of VEGF was signifi- Republic of China., cantly associated with AMS (P = 0.012, OR = 0.62, 95% CI 4Department of Neurology, Taipei City Hospital–Zhong Xiao Branch, 0.43–0.90). The rs3025039 of VEGF genotypic frequency distribution Taipei, Taiwan, Republic of China. differed significantly between AMS and control group (P = 0.0297). E-mail: [email protected]; [email protected] CT genotype of rs3025039 was significantly associated with AMS 5Graduate Institute of Basic Medical Science & Ph.D. Program (P = 0.010, OR = 0.56, 95% CI 0.36–0.87) assuming a additive effect for Aging, of the T allele; CT + TT genotype of rs3025039 was significantly 6Department of Biological Science and Technology, associated with AMS (P = 0.008, OR = 0.56, 95% CI 0.37–0.86) 7Graduate Institute of Clinical Medical Science, China Medical assuming a dominant effect of the T allele. The C allele of rs3025030 of University, Taichung, Taiwan, Republic of China, VEGF was significantly associated with AMS (P = 0.019, OR = 0.64, 95% CI 0.44–0.93). CC + CG genotype of rs3025030 was significantly Amyotrophic lateral sclerosis (ALS) is one of severe neurodegener- associated with AMS (P = 0.018, OR = 1.66, 95% CI 1.10–2.45) ative disorders. With the loss of motor neurons in the brain and spinal assuming a dominant effect of the T allele. In terms of NOS2, TT cord, it causes progressive muscular atrophy. Therefore, ALS patients genotype of rsl137933 was significantly associated with AMS die due to respiratory failure caused by bronchial muscular dystrophy (P = 0.049, OR = 3.045, 95% CI 0.96–9.68) assuming a additive within 2–5 years postdiagnosis. The exact pathogenesis of ALS effect of the T allele. TT genotype of rs1137933 was significantly remains unclear to date. Though there are ways to slow down the associated with AMS (P = 0.04, OR = 3.13, 95% CI 0.99–9.87) progression of the disease, no absolute treatment is effective. In assuming a recessive effect of the T allele. For ACE, CC genotype of addition, discovery of ALS biomarkers is needed both for early rs4309 was significantly associated with AMS (P = 0.049, diagnosis and to monitor disease progression. OR = 1.83, 95% CI 1.00–3.34) assuming a recessive effect of the C Previous studies showed that the increasing of intracellular oxi- allele. Genotypic and allelic frequencies of the rs2297518 of NOS2; dative stresses would raise the rate of suffering ALS. Similar with rs8005745, rs2301108, rs 10873142 of HIF 1 A; rs 1695 of GSTP those studies, Western blotting showed heat shock protein 60 (HSP60) 1;rs7483 of GSTM3; rsl799983, rs3918188 and rs7830 of NOS3 and and HSP70, which belong to the damage-associated molecular pat- rs35853823 of HSPA4 were not significantly different between AMS terns (DAMPs), were elevated in sera of patients with ALS. However, and control group (P [ 0.05). At the haplotype level, a haplotype GC/ in our cases it was fail to distinguish the difference between patients CT frequencies consisting of rs3025030 (G/C) and rs3025039 (C/T) of and control using enzyme-linked immunosorbent assay (ELISA) to VEGF was significantly differences between the AMS group and detect HSP60. We proposed that HSP60 might be neutralized by the control group (P = 0.029). There were no statistically significant dif- autoantibody against itself. Thus, the levels of autoantibodies against ferences in the haplotype AGT/TAC/AGC frequencies consisting of HSP60 and against HSP70 would be measured to distinguish the

123 12th International Congress on Amino Acids, Peptides and Proteins S23 patients with ALS from the controls. Compared with 40 subjects with and screened for inhibitory activity against human O-GlcNAcase and ALS and 40 age-matched controls, serum levels of the autoantibodies human lysosomal b-hexosaminidase (Hex A & B). As a result, 4-pyridyl- against HSP60 and HSP70 were 1.64 and 1.37 folds higher in patients 1-(20-deoxy-20-acetamido-b-D-glucopyranosyl)-1,2,3-triazole, displayed with ALS than in controls. The ROC curve also revealed that the specific inhibitory activity against human O-GlcNAcase, with Ki = AUC of 0.7651 and 0.6179 for autoantibodies against HSP60 and 48 lM and 200-fold selectivity against Hex A & B. HSP70, respectively. According to this study, we firstly identified the autoantibodies against HSP60 and HSP70 may serve as biomarkers of detecting ALS, and in both HSP60 autoantibody is more potent. Diabetes-induced structural alterations in rat liver proteins and the recovery effect of selenium: an FTIR Clotting activity changes of fibrinogen induced microspectroscopy and neural network study by peroxynitrite Ozlem Bozkurt1, Sevgi Haman Bayari2*, Mete Severcan3, 4 4,5 1 Yunjing Luo, Zhiguo Han, Qi Zhu, Shuang Cui, and Rugang Zhong Christoph Krafft ,Ju¨rgen Popp , and Feride Severcan

1 College of Life Science and Bioengineering, Beijing University Department of Biological Sciences, Middle East Technical of Technology, 100124 People’s Republic of China. University, 06531 Ankara, Turkey 2 E-mail address: [email protected] Department of Physics, Hacettepe University, 06800 Ankara, Turkey 3Department of Electrical Engineering, Middle East Technical Fibrinogen plays an important role in blood clot formation. The injury University, 06531 Ankara, Turkey 4 of fibrinogen induced by perxoynitrite is related to thrombotic and Institute of Photonic Technology, 07745 Jena, Germany 5 cardiovascular disease. In this paper, we investigated the changes on Institute of Physical Chemistry, University Jena, 07743 Jena, the secondary structure and clotting activity of fibrinogen treated by Germany peroxynitrite with FTIR spectra and Von Clauss method. With the increase of peroxynitrite, the content of a-helix in fibrinogen reduced Diabetes mellitus is a major endocrine disorder and a growing health from the original 38.03 to 29.69% whereas b-sheet increased from problem in the world, characterised by hyperglycemia resulting from 29.89 to 43.66%. The increase of b-sheet structure makes folded defects in insulin secretion, insulin action or both of them. In the fibrinogen looser, consequently, the ability of fibrinogen to release current study, the changes in Streptozotocin (STZ)-induced diabetes fibrin clot is changed. Although the clotting activity of fibrinogen in content and structure of proteins in rat livers were studied by decreased with the increase of peroxynitrite in overall trend, trace of Fourier transform infrared microspectroscopy (FTIRM). The potential peroxynitrite could enhance the clotting activity. We speculated slight role of selenium in recovery of diabetes induced alterations was also nitration could make fibrinogen release fibrin monomers easier in the investigated. FTIRM results demonstrated an increase in protein presence of thrombin. When fibrinogen treated with hydrogen per- content of diabetic group. In order to monitor the changes in other oxide, the clotting activity of fibrinogen decreased sharply, which macromolecules with respect to protein, different ratios, such as lipid suggested fibrinogen is more susceptible to oxidation than nitration. to protein and glycogen to protein ratio were calculated. The alterations in protein secondary structures were further determined using neural network (NN) analysis based on amide I band. According to Development of O-GlcNAcase inhibitors NN results, the content of a-helical structures was decreased, while for Alzheimer’s disease therapy the b-sheet content was increased in diabetic group with respect to the control. Restoring effect of selenium was observed in all of the

1 1 1 1 1 spectral parameters investigated. Furthermore, the hierarchical cluster Zhonghua Li , Tiehai Li , Lin Lin , Jing Li *, Wei Zhao * -1 1,2,3* analysis performed in fingerprint region (1800–900 cm ) resulted in and Peng George Wang the successful differentiation between control, diabetic and selenium treated diabetic groups. The sensitivity and specificity values calcu- 1College of Pharmacy and State Key Laboratory of Element-Organic 2 lated from the cluster revealed the power of FTIRM in characterizing Chemistry, Nankai University, Tianjin 300071, China Departments diabetic liver tissues. The results of this study established that FTIRM of Biochemistry and Chemistry, the Ohio State University, Columbus, together with chemometric methods can be used as a powerful tool in OH 43210 determination of diabetes-induced alterations at molecular level. O-GlcNAcylation is an abundant, essential and dynamic protein post- translational modification of cytosoic proteins in metazoans and can compete with phosphorylation at similar Ser/Thr sites of target pro- Dialyl trisulfide affects sulfurtransferases and Bcl-2 teins. The enzyme of O-GlcNAc metabolism (OGT/OGA) are expression in human cancer cells SH-SY5Y enriched in the brain and many proteins important for neuronal function are modified by O-GlcNAc including microtubule-associated and U-87 MG protein tau, b-amyloid precursor protein (AP), clathrin-assembly proteins and neurofilaments. Pathological hyperphosphorylation of Maria Wro´bel and Halina Jurkowska tau is characteristic of Alzheimer’s disease (AD) and the associated tauopathies. So a potent and specific O-GlcNAcase inhibitor can be Chair of Medical Biochemistry, Jagiellonian University Medical used to enhance O-GlcNAc level and downregulate phosphorylation College, Krako´w, Poland of tau and further provide a new drug for AD therapy. Here, using Cu(I)-catalyze ‘‘click’’ cycloaddition reactions between Dialyl trisulfide (DATS), a component of garlic extract, contains three glycosyl azides and alkynes, 16 candidate compounds were synthesized sulfur atoms with two allyl groups. It acts as the direct precursor of

123 S24 12th International Congress on Amino Acids, Peptides and Proteins sulfane sulfur atoms (oxidation state 0 or -1) and exerts a potent acid composition in chelators permits substantial decrease of renal antiproliferative effect on several human cancer cell lines. The cel- retention of radioactivity. lular sulfane sulfur levels depend on cysteine availability and the In conclusion, the use of peptide based chelators for technetium activity of two sulfane sulfur-generating enzymes: c-cystathionase and rhenium radioisotopes permits not only stable coupling of nuc- and 3-mercaptopyruvate sulfurtransferase. The response of the two lides to scaffold proteins, but also improvement of their targeting cell lines: SH-SY5Y, a human derived neuroblastoma, and U-87MG, properties. a commonly studied grade IV glioma, to the presence of DATS, 1–5 mM in culture medium, were analyzed. The elevated level of sulfane sulfur was determined in both lines after 48 h treatment with 2 mM DATS. The effect was accompanied by the increased expres- Effects of arsenic exposure from drinking water sion of 3-mercaptopyruvate sulfurtransferase in the U-87 MG cells on the expression of MMP-1 and TIMP-1 in hepatic and c-cystathionase in the SH-SY5Y cells, and the inhibition of tissue in rats proliferation in case of both cell lines. In the SH-SY5Y cells an increased expression of gene for Bcl-2 protein suggested induction of XU Zhao1,2, WANG Zhou3, LI Jian-jun2, ZHANG Ping-chuan1, apoptosis, while in U-87 MG cells, the formation of apoptotic bodies DONG Lu1, ZHANG Xiao-tian1, WANG Shu-mei1, was observed by ﬂuorescence microscopy. In both cell lines, DATS and WANG Zhi-lun1 potency to enhance endogenous levels of GSH was shown; this allowed for maintaining intracellular redox status measured as the 1Xi An Jiao Tong Univ, Sch Med, Minist Educ, Key Lab Environm & concentration ratio of reduced to oxidized glutathione. Increased Genes Related Dis, Xian 710061, People’s Republic of China levels of cystathionine and cysteine after 24 h of culturing the U-87 2Xian Jiaotong Univ, Sch Sci, Dept Chem, Xian 710049, People’s MG cells in the presence of 2 mM DATS suggested that the pathway Republic of China towards the glutathione formation through the cystathionine b-syn- 3Shenzhen Center for Disease Control and Prevention, Shenzhen thase and c-cystathionase reactions was stimulated in these cells. 518055, People’s Republic of China

Objective: Chronic toxicity of arsenic from drinking water has Designing radiolabelling strategies for proteins become a global health problem that affect millions of people. Epi- and peptides. How peptide based chelators can improve demiological studies indicate that chronic exposure to arsenic can cause varying degrees of liver damage, liver fibrosis, cirrhosis and radionuclide imaging and therapy liver cancer, but the exact mechanism of these actions is not very clear. In the present study, our aim was to study the effects of arsenic Vladimir Tolmachev exposure from drinking water on the liver function and the expression of MMP-1 and TIMP-1 in hepatic tissue in rats. Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala Methods: Sprague–Dawley rats were divided into three groups at University, Sweden;Email: [email protected] random, each of which was treated with sodium arsenite (NaAsO2) at 0 mg/L (Group A), 2.4 mg/L (Group B), 12 mg/L (Group C) in An increasing number of scaffold proteins are being evaluated for in drinking water. The rats were sacrificed at 3, 5 and 7 months for vivo tumour targeting. One important application area of scaffold pathologic, total arsenic levels, liver function, RNA and proteins proteins is radionuclide tumour targeting for imaging and therapy. examination. The levels of arsenic in blood and hepatic tissue were Selection of an optimal labelling chemistry for scaffold proteins is determined by atomic fluorescence spectrometry. The serum were essential, since it can influence their affinity, cellular processing of the collected and the glutamic–pyruvic transaminase (ALT), aspartate tracer by cancer cells and cellular retention of a radionuclide, bio- transaminase (AST), lactic dehydrogenase (LDH), and gamma- distribution of radiolabeled tracers and predominant excretion glutamyl transpeptidase (GGT) were measured by automatic bio- pathway. chemistry analyzer. The RNA and proteins were extracted by Trizol A promising approach for labelling chemistry is the use of pep- reagent. Then the RNA were reversed transcription into cDNA, the tide-based chelators for radionuclides 99mTc for radionuclide imaging mRNA expression of MMP-1, TIMP-1 and b-actin were detected and rhenium isotopes 188Re and 186Re for radionuclide therapy. The by real-time fluorescence quantitative PCR. The protein expression use of peptide-based chelators permits site-specific labelling of of MMP-1, TIMP-1 and b-actin were detected by Western Blot recombinant proteins without additional chemical processing. An Assay. experience with Affibody molecules suggests that optimising com- Results: After 5 months of arsenic feeding, the serum ALT, AST, LDH position and order of amino acids in peptide-based chelators might of group B and group C were higher than that of group A, and pathologic appreciably improve radionuclide tumour targeting. For example, the examination showed that there were different degree of inflammatory 99m use of hexahistidine tags for labelling of proteins using Tc(CO)3 is penetration embellish and water-like lesions in hepatic tissue of group associated with elevated liver uptake of radiotracer. However, a B and group C; After 3 months of arsenic feeding, the expression of substitution of every second histidine with glutamate provides MMP-1 mRNA and protein of group B and group C were lower than HEHEHE-tag, which permits IMAC purification and stable labelling that of group A, and the expression of TIMP-1 mRNA and protein of 99m with Tc(CO)3, but does not cause high hepatic uptake. Increasing group B and group C were higher than that of group A. hydrophilicity of amino acids in meracptoacetyl-containing chelators Conclusion: Exposed to arsenic solution could result in hepatic injury at N-terminus of Affibody molecules suppress undesirable hepato- in rats. The TIMP-1 and MMP-1 gene play significant effect in pro- biliary excretion of tracers. Moreover, a careful optimisation of amino cess of hepatic injury.

123 12th International Congress on Amino Acids, Peptides and Proteins S25

Effect of Panax notogino side on the p38MAPK spinal cord damage are not well understood. Since spinal cord injury pathway in lung tissue of hypoxic rats* (SCI) induced widespread alterations in amino acid neurotransmitters e.g., glutamate, GABA, aspartate and glycine, in present investigation

1 2# 2 2 inﬂuence of engineered nanoparticles form metals on amino acid con- LIN Li-na , ZHAO Shan , LIANY Ying-qi , LIU Ya-kun , tent of the spinal cord in normal rats and after SCI was examined. TANG Lan-lan2, WANG Shu-Jun2, LI Guan-Long2, 2 3 2* Rats were administered engineered nanoparticles from Cu and Ag WANG Yuan-yuan , WU Cheng-yun , and WANG Wan-tie (50–60 nm) once daily (50 mg/kg, i.p.) for one week. On the 8th day,

1 one group of rats were subjected to SCI by making a longitudinal Department of anesthesiology, The Frist Affiliated Hospital, incision into the right dorsal horn of the T10-11 segments under 2Department of Pathophysiology, 3 Equithesin anesthesia. Five h after SCI, the spinal cord from the T9 Department of Respiratory Disease, The Second Affiliated Hospital, and T12 segments were taken out and glutamate, aspartate, GAB and Wenzhou Medical College, Wenzhou 325035, China glycine were analyzed using HPLC technique. Another group of Correspondence to: Wang Wan-tie, Department of Pathophysiology, nanoparticles treated rats no injury was made. Normal rats served as Wenzhou Medical College, wenzhou325035, China control. Nanoparticles treatment resulted in a marked increase in (E-mail:[email protected]; Tel: 0577-86689817) Glutamate and aspartate (+50 to 70%) content in the T9 and T12 segment of the cord where as GABA and glycine showed a significant Objective: To observe the effect of PNS (Panax notogino side) on the decline (-20 to 40%). Control from the control value. This effect was pulmonary artery pressure and the p38MAPK (p38 Mitogen activated most pronounced with Ag nanoparticles. Subjection of rats to SCI in protein kinase) in lung tissue of rats treated with hypoxia and there- nanoparticles treated animals resulted in further enhancement of fore to explore the mechanisms of PNS on HPH (hypoxic pulmonary Glutamate and aspartate content in the cord (+150 to 180%) whereas, hypertension). GABA and glycine showed marked decline (-60 to 80%). This effect Methods: Thirty adult male SD rats were randomly divided into 3 on amino acid contents was most marked in Ag treated rats after SCI. groups. One group was exposed to normal conditions (N group), the Neurological dysfunction and cord pathology were also exacerbated second group was exposed to isobaric hypoxia (H group), and the in nanoparticles treated arts after SCI. These observations are the first third group was treated with PNS under the hypoxia (HP group), after to show that nanoparticles induced exacerbation of cord pathology 4 weeks, cardiac catheterization was used to measure the mPAP following SCI is mediated through amino acids neurotransmission. (mean pulmonary arterial pressure). The heart was isolated, and the RV (right ventricle), LV + S (left ventricle plus ventricular septum) were weighed to calculate the ratio RV/(LV + S). The quantity of p-p38MAPK (phosphorylation p38 Mitogen activated protein kinase) From single gene to pathway analysis: high-throughput in rat pulmonary arterioles was determined by immunohistochemistry screening for drug dependence and the content of p38MAPK mRNA was tested by RT-PCR. Results: The mPAP, RV/(LV + S) in HP group were higher than N Ju Wang group, The expression of p-p38MAPK in rat pulmonary arterioles and p38MAPK mRNA in the lung were higher than that in N group Department of Psychiatry and Neurobehavioral Sciences, University (P \ 0.05). The mPAP, RV/(LV + S). The expression of of Virginia p-p38MAPK in rat pulmonary arterioles and p38MAPK mRNA in the lung were significantly lower than those in the H group (P \ 0.05). Drug dependence is a prevalent neurological disorder characterized Conclusion: PNS was shown to prevent hypoxic pulmonary hyper- by the structural, biochemical or electrical abnormality in the nervous tension, the underlying mechanisms may relate to the decrease on system, which affects millions of people worldwide and is one of the p-p38MAPK and down regulation of p38MAPK mRNA. most serious threats to public health. Although our understanding on Keywords: Panax notogino side, Hypoxia, Pulmonary arterial the nature of drug dependence has greatly advanced in the past dec- hypertension, p38MAPK ades, the underlying molecular mechanisms are still not clearly defined. The major reason for such gap is that as a complex neurological disease, hundreds of genetic and environmental risk factors Engineered nanoparticles from metals influence may be involved in the development of drug dependence, with each glutamate, aspartate, GABA and glycine factor only has a relatively small effect. In recent years, the rapidly growing field of functional genomics and proteomics has played an neurotransmission in the normal and injured spinal cord increasingly important role in drug addiction research because of their ability of analyzing a large number of molecular targets simulta- 1Jose´ Vicente Lafuente*, 2Ranjana Patnaik, 3Aruna Sharma, neously. In this study, we summarize the application of a multitude of and 3Hari S Sharma high-throughput genomic and proteomic technologies, such as gene expression microarray, microRNA array, 2-D gel electrophoresis, and 1Lab Neurociencias Clıńicas y Experimentales (LaNCE), Dpto. de genome-wide association studies (GWAS), toward indentifying and Neurociencias, Universidad del Paı´s Vasco—EuskalHerriko characterizing the molecular factors involved in drug dependence. We Unibertsitatea, Apdo. 699, 48080-Bilbao, Espanã, first provide an overview of genomics and proteomics technologies 2Department of Biomaterials, School of Biomedical engineering, and bioinformatics tools available to analyze the data harvested via Institute of technology, Banaras Hindu University, Varanasi-221005, these approaches. Then we summarize the recent applications of these India technologies to profile the gene/protein expression pattern in animal 3Department of Surgical Sciences, Anesthesiology & intensive Care or human brain tissues in response to substances of abuse, as well as Medicine, University Hospital, Uppsala University, SE-75185 identify the genes genetically associated with drug dependence in Uppsala, Sweden. Email: [email protected] human. Further, we detect the biological pathways enriched in these genes/proteins. Our study demonstrates the promise and potential of Previous reports from our laboratory show that engineered nanoparticles high-throughput screening technology in drug dependence study that from metals exacerbate spinal cord pathology following injury. How- aims at deciphering the underlying molecular mechanisms and finding ever, the possible mechanisms of nanoparticles induced exacerbation of better targets for developing therapeutic intervention.

123 S26 12th International Congress on Amino Acids, Peptides and Proteins

Genetics analysis of complex traits in human: what we 1 month into the plateau increased significantly (P \ 0.01), AVP have learned from studying genetics on smoking mRNA expression levels Quick access to the plateau in the early (2d) increased slightly (not statistically significant) after no significant dependence? change. Conclusion altitude chronic hypoxia, the plasma concentration of AVP with the time change hypoxia, coexist in the PVN large Ming D. Li and small cells of the AVP gene transcription by different mechanisms, this may be related to degree of hypoxia and high altitude Department of Psychiatry and Neurobehavioral Sciences, University hypoxia Acclimatization processes. of Virginia Project: Tibet, the Office of Science and Technology Research Fund (2010), the State Ethnic Affairs Commission research project (2011). Addiction to smoking is a common chronic brain disorder that is extremely costly to the individuals and to society. Although many years of twin and family studies reveal that genetics contribute sig- High dose simvastatin administration ınduces changes nificantly to vulnerability to this complex trait, the susceptibility in the structure and concentration of liver microsomal genes underlying it are largely unknown. To identify susceptibility genes for smoking dependence, almost all approaches commonly used membrane proteins in the genetic studies on complex traits such as genome-wide linkage analysis, candidate gene-based association analysis and genome-wide 1Kumsal OZGUN, 1Nihal SIMSEK OZEK, 2Mete SEVERCAN, association analysis have been employed. These analyses have and 1Feride SEVERCAN implicated several common genomic regions and genes in the etiology of smoking addiction. Although a significant number of reported 1 Department of Biological Sciences, Middle East Technical genomic regions did not reach the level of ‘‘suggestive’’ or ‘‘signifi- University, Ankara, Turkey cant’’ linkage and failed to be replicated in other independent studies, 2Department of Electrical and Electronics Engineering, Middle East thirteen regions, located on chromosomes 3–7, 9–11, 17, 20, and 22, Technical University, Ankara, Turkey have been found to be suggestive or significant in at least two independent samples. Among them, the regions on chromosomes 9, 10, Simvastatin is one of the most frequently prescribed cholesterol 11, and 17 have received the strongest support. In addition, several reducing drugs, due to its higher efficacy in reducing LDL cholesterol genome-wide association analyses including three meta analyses of levels and its tolerability. The possible hepatoxic effect of this drug smoking related behaviors have been conducted. A gene cluster on may be related to drug induced alterations in structure and function of chromosome 15q24/q25.1 that encompasses the genes for nicotine liver microsomal membrane proteins. This is because it contains many acetylcholine receptor subunits a5, a3, and b4 was implicated in enzymes that are involved in drug, cholesterol and fatty acid metab- addiction to tobacco and other substances as well. Current efforts aim olism and any changes in the structure and amount of these enzymes not only to replicate these findings in independent samples but also to may lead to protein malfunction. In this study, the simvastatin-induced determine the functional mechanisms of these associations. alterations in the structure and concentration of liver microsomal membrane proteins were investigated by Fourier transform infrared (FTIR) spectroscopy utilizing amide I and II bands (1,700–1,480 cm-1 High altitude hypoxia environment changes region). FTIR results revealed structural alterations in protein secondary structure and an increase in total protein amount in simvastatin of the content of AVP and expression treated group, deduced from changes in the band area, bandwidth, band frequency values of these bands and also their ratio values. Li Wenhua, Yuan Dongya, Zhang Min, Sun Fangyun, Sun Zhenqi, Detailed protein secondary structural changes were obtained from Zhao Fengcang intensity calculations from second derivative spectra and neural network (NN) analysis, using the amide I band of FTIR spectra. Chronic Medical School, Tibet Institute of Nationalities, Xianyang, Shanxi simvastatin treatment induced a reduction in b-sheet and an increase in 712082, China; Email: [email protected] the random coil and aggregated b content of liver microsomal membrane. Moreover, based on these alterations in proteins, successful Objective: To study the environment of high altitude hypoxia in rats discrimination of control and simvastatin treated groups were obtained plasma arginine vasopressin (AVP) levels and the expression of gene by hierarchical cluster analysis. The results of this study approved the transcription and its relationship with the relationship between alti- higher efficiency of FTIR technique in determination of alterations in tude hypoxic acclimatization. Methods 50 male SD rats were protein structure and content induced by simvastatin administration. randomly divided into 5 groups, namely groups 1d, 2d group, 3d group, 1 w group and 1y group, while the control group (in Xi’an, elevation 5 m). From Xi’an, were brought 1d Golmud, Qinghai Hypoxic preconditioning induced neuroprotection (altitude 2,700 m), 2d to the Tibetan Tanggula (elevation 5,000 m), 3d to the Naqu (elevation 4,500 m), 1 w Nagqu (elevation 4,500 m) against cerebral iachemic injury of mice and 1 y Nagqu (elevation 4,500 m), were sacrificed at different time and its cPKCc-mediated molecular mechanism points, application of radioimmunoassay of plasma AVP concentration, and compared with the control group. Using [35S] CTPRNA Nan Zhang, and Junfa Li* probe by in situ hybridization histochemistry was used to detect within the PVN of rats AVPhnRNA, AVPmRNA expression. Results Department of Neurobiology and Beijing Institute for Neuroscience, Plasma AVP concentration increased after entering the plateau, then Capital Medical University, #10 You An Men Wai Xi Tou Tiao, decreased to the lowest in the 2d, after rising gradually to 1w close to Beijing 100069, People’s Republic of China the level of control when, a month reached the highest level. Hypo- *E-mail: [email protected] thalamic paraventricular nucleus (PVN) of small cells and AVPmRNA AVP hnRNA expression was positively correlated with As of yet, pharmacological treatments of stroke are only met with the above results, large cells AVP hnRNA expression levels in mediocre results, which are either ineffective or confounded by adverse

123 12th International Congress on Amino Acids, Peptides and Proteins S27 effects, thus calling for a better understanding of endogenous neuro- Keywords: Breast cancer, PLAC1, Cytotoxic T lymphocyte, protective mechanism. Previously, we have demonstrated that the Epitope, Immunotherapy translocated activation of conventional protein kinase Cc (cPKCc)is involved in the development of cerebral hypoxic preconditioning Influence of cholesterol redistribution on APP (HPC), one of the most profound neuroprotective strategies. This study was designed to substantiate the role of cPKCc and its signaling mol- processing and its relevance to Alzheimer’s disease ecules in HPC-induced neuroprotection against subsequent middle pathology cerebral artery occlusion (MCAO)-induced permanent cerebral ischemic injuries. The effects of HPC and cPKCc on cerebral ischemic Satyabrata Kar injuries were studied by observing the changes in neurological deficits, infarct volume and neural cell apoptosis. cPKCc membrane transloca- Departments of Medicine and Psychiatry, University of Alberta, tion and its interacting protein synapsin in the ischemic brain were Edmonton, Canada examined by Western blot analysis. Proteomic approaches were employed to identify the cPKCc-interacting proteins. We found that Evidence suggests that cholesterol by regulating amyloid precursor HPC could markedly attenuate MCAO-induced brain injuries and the protein (APP) processing can influence the generation of b-amyloid (A decrease of cPKCc membrane translocation, but cPKCc inhibitor b) peptide—the key player in Alzheimer’s disease (AD) pathogenesis. Go6983 could block HPC-induced neuroprotection. Among the 41 However, most of these observations arise either from cell culture identified cPKCc-interacting proteins, 17 up- and 6 down- regulated studies using drugs having multiple effects or animal models with proteins were observed in cytosol or particulate fraction during HPC. In dietary modulation of cholesterol, which does not cross the blood–brain addition, the up-regulated synapsin could reciprocally co-precipitated barrier. At present, it remains unclear how cells/neurons genetically with cPKCc both in cytosol and particulate fractions, and Go6983 modified to accumulate cholesterol can influence APP processing and abolished HPC-induced inhibition on synapsin dephosphorylation in A b production. To address this issue, we developed novel in vitro and ischemic core and penumbra. This study is the first to report multiple in vivo genetic models overexpressing APP in the absence of Niemann- cPKCc-interacting proteins in HPC mouse brain and suggested that Pick type C1 (Npc1) protein, required for intracellular cholesterol cPKCc-synapsin pathway might be responsible for HPC-induced neu- transport, and analyzed the effects of cholesterol accumulation on APP/ roprotection against cerebral ischemic injuries of mice. A b metabolism. The newly generated bigenic ‘‘ANPC (APP+/-/ Npc1-/-)’’ mice revealed that intracellular cholesterol accretion can significantly increase mortality rate, trigger early motor and object recognition memory impairments, accelerate degeneration of neurons, Identification of a novel HLA-A2-restricted cytotoxic T exacerbate glial pathology and to some extent influence extracellular A lymphocyte epitope from cancer-testis antigen PLAC1 b deposition. Additionally, cholesterol accumulation in brain neurons in breast cancer of ANPC mice and in cultured N2a cells can differentially regulate levels and distribution of APP C-terminal fragments (CTFs) and Wei Liu, Mingxia Zhai, Zongyin Wu, Yuanming Qi, Yahong Wu, Ab1–40/1–42 with no evident alterations in APP mRNA levels. This is Chao Dai, Meng Sun, Lu Li, and Yanfeng Gao* accompanied by changed levels of APP processing enzymes in ANPC mouse brains as well as in cultured N2a cells. Collectively, these results Department of Bioengineering, Zhengzhou University, Zhengzhou suggest that cholesterol sequestration in neuronal cells can reduce 450001, China longevity, exacerbate degeneration of neurons possibly via the pro- b *Corresponding author: Dr. Yanfeng GaoDepartment duction/accumulation of A and APP CTFs which can influence AD- of Bioengineering, Zhengzhou University, 100 Science Road, related amyloid pathology. Zhengzhou 450001, ChinaTel.: +86-371-67739057; (Supported by funding from CIHR). fax: +86-371-67783235. E-mail: [email protected] Interactions of acidic pharmaceuticals with human Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor serum albumin: insights into the molecular toxicity antigens is essential to the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis of the emerging pollutants (CT) antigens are the most widely-studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel J. B. Chen1,2, Y. L. Zhang1, X. F. Zhou2, Y. J. Qian2, member of caner-testis antigen, which expressed in a wide range of and H. W. Gao2,* human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were 1State Key Laboratory of Pollution Control and Resources Reuse, predicted by T cell epitope prediction programs including SYFPEI- College of Environmental Science and Engineering, Tongji THI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in University, Shanghai, 200092, China T2 cells showed that two native peptides, p28 and p31, and their 2Key Laboratory of Yangtze River Water Environment for Ministry analogues (p28-1Y9 V, p31-1Y2L) had more potent binding activity of Education College of Environmental Science and Engineering, towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs Tongji University, Shanghai, 200092, China induced by these four peptides could release IFN-c. The CTLs *Corresponding author: [email protected] induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02+ healthy donor could lyse MCF-7 Acidic pharmaceuticals such as diclofenac, clofibric acid, ketoprofen breast cancer cells (HLA-A*0201+, PLAC1+) in vitro. When immu- have been frequently detected in the environment. In order to investi- nized in HLA-A2.1/Kb transgenic mice, the peptide p28 could induce gate the toxicity of such emerging pollutants, their interactions with the most potent peptide-specific CTLs among these peptides. There- human serum albumin (HSA) were investigated by capillary electro- fore, our results indicated that the peptide p28 (VLCSIDWFM) could phoresis and molecular spectrometry. The binding constants and sites serve as a novel candidate epitope to the development of peptide of these acidic pharmaceuticals with HSA were obtained. The ther- vaccines against PLAC1-positive breast cancer. modynamic parameters, e.g. enthalpy change (DH) and entropy change

123 S28 12th International Congress on Amino Acids, Peptides and Proteins

(DS) of these interactions were calculated in order to characterize the (C. albicans) causes oral thrush and vaginal candidiasis. On the cell acting force between acidic pharmaceuticals and HSA. From the fluo- walls of C. albicans, Xog1p serves as the major b-1,3-exoglucan- rescence and UV–Vis spectra, the fluorescence quenching of HSA is ase, which participates in the metabolism of b-glucan. The previous due to the formation of ground-state complex with the pharmaceuticals. studies showed antimicrobial peptides (AMPs) such as LL-37 and The three-dimensional fluorescence confirmed that the conformation of hBD-3 could inhibit the cell adhesion and growth. We proposed HSA changed after the interactions with the pharmaceuticals. Thus, the that Xog1p is a target for AMPs binding, and involves in the C. interactions between acidic pharmaceuticals and HSA may influence on albicans adherence. Sixty percent of the adherence of C. albicans the normal activity of HSA. on the plastic surface would be inhibited by LL-37 and hBD-3 in a dose dependent manner. Pull down assay and ELISA demonstrated Investigation of the effects of different epileptic activities that recombinant Xog1p-6H and deletion fragments could interact with both AMPs in vitro. Moreover, the b-1,3-exoglucanase activity on cell membrane proteins reveals diagnostic information of Xog1p-6H would be enhanced around twofolds by LL-37 and hBD-3. Thus, the elevated b-1,3-exoglucanase activity of Xog1p Sevgi Turker1, Mete Severcan2*, Gul Ilbay3, and Feride Severcan4 might destroy the cell wall integrity and decrease the C. albicans adhesion. To further investigate the hypothesis, the C. albicans was Department of Biology1, treated with 2 lM Xog1p-6H for 2 h at room temperature, and we Physiology3 Medical School of Kocaeli University, Kocaeli, Turkey found a decrease of the C. albicans adherence for 3.5-folds. Department of Electrical and Electronics Engineering2, Altogether, Xog1p is a cell wall protein serving as a target inter- Biological Sciences4, Middle East Technical University, Ankara, Turkey acting with LL-37 and hBD-3, and the enhanced b-1,3- exoglucanase activity leads to reduce the C. albicans adherence In the current study, the effects of convulsive (pentylenetetrazol trea- onto plastic surface. ted) and mixed form (audiogenetically susceptible) epileptic activities on the structure and concentration of brain cell membrane proteins were investigated by Fourier Transform Infrared (FTIR) spectroscopy. This technique enables analysis of proteins in a variety of environments even Mixed linear approaches of mapping QTL, without isolation procedure, requiring less time and sample. The QTS and QTT with gene–gene interaction information is deduced by monitoring the amide I (1700–1600 cm-1) and amide II (1600–1500 cm-1) bands. The peak areas of both amide I and gene-environment interaction for complex traits and amide II modes give information about total protein concentration which were dramatically decreased in the mixed group. The alteration Jun Zhu, Zhihong Zhu, and Chenhao Zhang in the amide I to amide II ratio implies structural variations in the proteins of the system. Since amide I band arises from overlapping Institute of Bioinformatics, Zhejiang University, Hangzhou, 310058 individual peaks assigned to different protein conformations like a- helix and b-sheet, this band was used to determine secondary structural It has been a challenge to develop efficient statistical methods for changes in protein using neural networks (NNs). This chemometric mapping genes underlying complex traits. Here, we report the method has been previously shown to be a powerful tool for secondary development of mixed linear approaches that integrate the detection structure prediction. The findings revealed that for both epileptic groups of gene-by-gene and gene-by-environment interaction for quantita- there was a significant increase (p \ 0.05*) in random coil and sig- tive trait loci (QTLs) based on microsatellite markers, for nificant decrease (p \ 0.05*) in beta sheet structures. Moreover, cluster quantitative trait nucleotides (QTNs) based on SNPs, and for analysis based on amide I band provided successful differentiation of quantitative trait transcripts (QTTs) based on variation in expres- the epileptic groups in between and the control samples. Consequently, sion of transcripts. The genetic models include cofactors (i.e., sex, FTIR spectroscopy together with NNs method introduces promising age), genetic main additive (A) and dominant (D) effects, epistasis approach for diagnosis of epilepsy by detecting the impacts of different effects including additive by additive (AA), additive by dominance epileptic models on brain cell membrane proteins. (AD), dominance by dominance (DD), and gene-by-environment interaction (AE, DE, AAE, ADE, and DDE). Mixed linear model approaches are used for unbiased prediction of all these genetic LL-37 and hBD-3 elevate Xog1p activity resulting main effects, epistasis effects, and gene-environment interaction in adherence decrease of Candida albicans onto plastic effects. The variation contributing to these effects can be estimated. Mapping software (QTLNetwork V3.0) has also been developed, surface which can be used under different operating systems. To illustrate our proposed approach, both phenotypic and transcriptome data on Hsin-Hui Huang1,2, Pei-Wen Tsai3, Ting-Jia Chang1, anxiety assay with alcohol and stress treatments from 71 strains of Tzu-Shan Chien1, Chuan-Yi Lan3, and Hao-Teng Chang1,4,5* the BXD family were analyzed. Using 506 microsatellite markers, we detected eight QTLs including three main-effect QTLs 1Graduate Institute of Molecular Systems Biomedicine, (h2 = 0.27) and three pairs of epistasis QTLs (h2 = 0.23). By using 2Department of Medical Laboratory Science and Biotechnology, 2,320 SNPs, 17 QTNs were detected for seven main-effect QTNs 3Institute of Molecular and Cell Biology & Department of Medical (h2 = 0.25) and six pairs of epistasis QTNs (h2 = 0.22). Further, Science, National Tsing Hua University, Hsinchu, Taiwan, Republic we also used a small mapping population (188 individuals in 5 Of China.Correspondence: [email protected] treatments), and detected one main QTT (h2 = 0.07) and two pairs 4Graduate Institute of Clinical Medical Science, of epistasis QTTs with relatively small effects. The mapping results 5Graduate Institute of Basic Medical Science & Ph.D. Program by three methods were compared for chromosomes 1 and 11. Three for Aging, China Medical University, Taichung, Taiwan, QTSs were mapped onto the flanking marker intervals of three Republic Of China QTLs detected. The QTT was mapped into the QTLs on chromosome 1. In conclusion, the approaches we developed here are Candidiasis is the common cause of opportunistic fungal infection capable of identifying causal genes/loci associated with complex in immunocompromised patients. For instance, Candida albicans traits.

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Nanowired cerebrolysin restores imbalance cop_20e with diseases and it could be named as nanomedicine applied of excitatory and inhibitory amino acid to difficult-to-treat diseases. As known, in this field of research, the most important goal to be neurotransmitters in the CNS following hyperthermia reached is an increase in selectivity and specificity of drug action. induced brain pathology Several results with stimulating findings in preclinical or clinical phases have been reached by using nanocarriers, delivering agents to 1Dafin F Muresanu*, 2Ranjana Patnaik, 3Aruna Sharma, targeted pathologies, and among them, it is known that neuro- and 3Hari S Sharma pathologies represent a stimulating issue. In fact, the pharmaceutical treatment of Central Nervous System (CNS) disorders is the second 1Department of Neurology, University of Medicine & Pharmacy, largest area of therapy, following cardiovascular diseases. Nowa- Cluj-Napoca, Romania days, non-invasive drug delivery systems for CNS are actively 2Department of Biomaterials, School of Biomedical engineering, studied. In fact, the development of new delivery systems (nano- Institute of technology, Banaras Hindu University, Varanasi-221005, particles and liposomes) started with the discovery that properly India surface-engineered colloidal vectors, with a diameter around 3Department of Surgical Sciences, Anesthesiology & intensive Care 200 nm, were shown to be able to cross the blood–brain Barrier Medicine, University Hospital, Uppsala University, SE-75185 without apparent damage, and to deliver drugs or genetic materials Uppsala, Sweden. Email: [email protected] into the brain. During this talk, an overview will be presented considering the most recent literature results of nanomedicine Previous reports from our laboratory showed that hyperthermia induces applied to brain diseases, carried out with all the most popular kinds marked increase in excitatory amino acid neurotransmitters, e.g., glu- of nanoparticulate systems, focusing in particular on immune- tamate and aspartate in the cerebral cortex, hippocampus, thalamus, and nanoparticles and peptide-decorated nanosystems able to target the hypothalamus and in spinal cord at the time of neuronal damages and CNS, with in vivo and in vitro evidences investigating the pathway blood–brain barrier (BBB) dysfunction. At this time, inhibitory amino for BBB crossing and CNS localization of engineered nanoparticles. acids such as GABA or glycine showed marked decrease in the above The brain localization and the multi-modal pathways for BBB CNS regions. This suggests that an imbalance between excitatory and crossing highlighted the endocytosis as preferential pathway; inhibitory amino acids in the CNS causes brain damage. Since cerebr- moveover, in vitro test on hippocampal neurons showed the pres- olysin is a mixture of various neurotrophic factors and induces marked ence of cell-to-cell transport of nanoparticles. neuroprotection in hyperthermia, in present investigation the influence of cerebrolysin with or without TiO2 nanowiring was examined on the glutamate, aspartate, GABA and glycine levels in the CNS following Nanowired delivery of HOE-140 enhances hyperthermia in relation to brain damage. neuroprotection in spinal cord injury Rats were subjected to 4 h heat stress (HS) in a biological oxygen and downregulates heat shock protein expression demand (BOD) incubator and amino acids were analyzed using HPLC in the above brain regions. Separate groups of rats were treated with 1 2 2 cerebrolysin (2.5 ml/kg, i.v.) with or without nanowiring after 30, 60 Linyuan Feng*, Aruna Sharma, and Hari S Sharma and 90 min of HS. Rats received cerebrolysin after 30 min markedly 1 thwarted the increase in glutamate and aspartate and reduction in Department of Neurology, Norman Bethune International Peace GABA and glycine in the CNS resulting in neuroprotection. However, Hospital Army, 398 Zhong Shan Road West, Shi Jia Zhuang, Hebei 60 or 90 min after cerebrolysin administration did not affect the Province, China 50082, Email: [email protected] 2 amino acid levels and/or brain damage. On the other hand nanowired Department of Surgical Sciences, Anesthesiology & intensive Care cerebrolysin if given at 60 or 90 min after HS, thwarted this amino Medicine, University Hospital, Uppsala University, SE-75185 acid imbalance and induced profound neuroprotection. These results Uppsala, Sweden. Email: [email protected] show that nanowiring of cerebrolysin enhances its neurotherapeutic efficacy in hyperthermia induced neuroregeneration probably through Previously, we reported that blockade of bradykinin B2 receptor with modulating amino acid neurotransmission, not reported earlier. H-140 prior to spinal cord injury (SCI) in low dosage induced neuroprotection and downregulated neuronal nitric oxide synthase (nNOS) expression in the cord. However, HOE-140 administration Nanomedicine in neuro-diseases: engineered after 30 min SCI did not affect nNOS expression or cell injury. Since SCI induces profound oxidative and cellular stress, in this investi- nanoparticulate systems for the drug brain delivery gation we examined effects of HOE-140 on heat shock protein expression in SCI. Furthermore, the role of nanodrug delivery of Giovanni Tosi*1, Lucia Bondioli1, Barbara Ruozi1, HOE-140 in attenuating cell damage and stress reaction was also Andreas Grabrucker2, Antonietta Vilella3, Michele Zoli3, examined in our rat model. Francesco Rivasi4, Maria Angela Vandelli1, and Flavio Forni1 SCI was produced by a longitudinal incision over the right dorsal horn of the T10-11 segments of the cord under Equithesin anes- 1Department of Pharmaceutical Science, Univ. of Modena and Reggio thesia. The animals were allowed to survive 5 h after injury. In Emilia, Modena, Italy separate group of rats HOE-140 was delivered tagged with TiO2 2Department of Psychiatry and Behavioral Sciences, Stanford Univ., nanowires (0.1 mg/kg, i.v.) 30 min or 60 min after trauma and heat Stanford, CA, US shook protein (HSP 72 kD), nNOS expression, cell injury and 3Department of Biomedical Sciences, Univ. of Modena and Reggio edema formation was examined using standard procedures. HOE- Emilia, Modena, Italy 140 without nano-labeling was used as control. Treatment with 4Department of Morphological Sciences, Univ. of Modena HOE-140 without tagging with nanowires did not influence massive and Reggio Emilia, Modena, Italy upregulation of HSP 72 kD at 5 h after trauma or cellular damage. On the other hand, nano-drug delivery of HOE-140 markedly In the last years, the application of ‘‘nanotechnology ‘‘to the field of attenuated HSP expression, nNOS upregulation, edema formation ‘‘medicine’’ surely represented the most innovative strategy to and cell injury when given 30 or 60 min after injury. However,

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TiO2 nanowires alone did not influence pathophysiology of SCI or Olfactory ensheathing cell neurorestorotherapy HSP expression. These observations are the first to show that for amyotrophic lateral sclerosis patients: Benefits nanodrug delivery of HOE-140 even at a later stage of SCI is able to thwart stress protein response, nNOS expression and cell injuries. from multiple transplantations This indicates that nanowiring of drugs could enhance their therapeutic potentials in SCI. Hongyun Huang

Beijing Hongtianji Neuroscience Academy, Beijing, 100144, People’s Republic of China Center for Neurorestoratology, Beijing Novel ionic polymer-platinum nano-composite Rehabilitation Center, Beijing, People’s Republic of ChinaDivision of Neurorestoratology, Yuquan Hospital, Tsinghua University, for real-time control of drug metabolism Beijing, People’s Republic of China E-mail address: [email protected] Jun Lu1, Sang-Gyun Kim3, Sunwoo Lee4 and Il-Kwon Oh2 Our previous series studies have proved that OEC transplantation 1Key Laboratory of Advanced Technologies of Materials, Ministry appears to be able to slow the rate of clinical progression after of Education, School of Materials Science and Engineering, olfactory ensheathing cell (OEC) transplantation in the first four Southwest Jiaotong University, Chengdu 610031, Sichuan, China, months and cell intracranial (key points for neural network restoration, corresponding author e-mail: [email protected] (J. Lu) KPNNR) and/or intraspinal (impaired segments) implant are benefit 2Division of Ocean System Engineering, School of Mechanical for the patients (both subtype of bulbar onset and limb onset) with Aerospace and Systems Engineering, Korea Advanced Institute amyotrophic lateral sclerosis (ALS). Here we report the results of cell of Science and Technology, 335 Gwahangno (373-1 Guseong-dong), therapy in patients with ALS on basis of the long-term observation Yuseong-gu, Daejeon, 305-701, Republic of Korea, following multiple transplants. From March of 2003 to January of Corresponding author e-mail: [email protected] (I.K. Oh) 2010, 507 ALS patients received our cellular treatment. Among them, 3Membranes & Separation Research Center, Korea Research Institute 42 patients underwent further OEC therapy by the route of KPNNR for of Chemical Technology, P.O. Box 107, Yuseong, Daejon 305-600, 2 or more times (2 times in 35 patients, 3 times in 5 patients, 4 times in Republic of Korea 1 patient, and 5 times in 1 patient). The time intervals are 13.1 (6–60) 4Department of Chemistry, Chonnam National University, 300 months between the first therapy and the second one, 15.2 (8–24) Yongbong-dong, Buk-gu, Gwang-Ju, 500-757, Republic of Korea months between the second therapy and the third one, 16 (6-26) months between the third therapy and the fourth one, and 9 months NADPH plays an important role in drug metabolism, which is an between the fourth therapy and the fifth time. All of the patients got obligatory requirement for cytochrome P450-dependent drug oxi- partial neurological functional recovery after each cell-based admin- dation. NADPH levels vary periodically in time in neutrophils, istration. Firstly, the scores of ALS Functional Rating Scale macrophages, and certain tumor cells, and it oscillates in an (ALS-FRS) and ALS Norris Scale increased by 2.6 ± 2.4 (0–8) and approximate sinusoidal pattern with a period between 3 and 4 min 4.9 ± 5.2 (0–20) after the first treatment, 1.1 ± 1.3 (0–5) and in all of these cells. In this study, a novel nano-composite with 2.3 ± 2.9 (0–13) after the second treatment, 1.1 ± 1.5 (0–4) biocompatibility, composed of styrene-maleimide alternating poly- and 3.4 ± 6.9 (0–19) after the third treatment, 0.0 ± 0.0 (0–0) and mer, vinylidene fluoride polymer and platinum, was fabricated, and 2.5 ± 3.5 (0–5) after the fourth treatment, and 1 point after the fifth its electromechanical properties were investigated at ultra-low fre- cellular therapy, which were evaluated by independent neurologists. quency. Under the stimuli of sinusoidal wave signals with Secondly, majority of patients have achieved improvement in EMG frequency from 0.1 to 0.005 Hz, the nano-composite with glycerol assessments after the first, second, third and fourth cell transplantation. as a solvent displayed excellent harmonic response, and its defor- After the first treatment, among the 42 patients, 36 (85.7%) patients’ mation was observed to be improved continuously by a self- electromyogram (EMG) test results improved, the rest 6 (14.3%) activating process along with the time increased. The experiments patients’ electromyogram results showed no remarkable change. After also showed that the treatment conditions by glycerol had signifi- the second treatment, of the 42 patients, 30 (71.4%) patients’ EMG cant effect in determining its final response properties. The best results improved, 11 (26.2%) patients no remarkable change, 1 (2.4%) harmonic response to the signals with ultra-low frequency was patient worse. After the third treatment, out of the 7 patients, 4 (57.1%) achieved when the nano-composite was immersed in anhydrous patients improved, the remaining 3 (42.9%) patients no change. glycerol at 100°C for 4 days and then exposed to air for 8–10 days Thirdly, the patients have recovered breathing ability partially which at ambient temperature. The current study on ionic polymer-plati- proven by pulmonary functional tests. After the first treatment, 20 num composite suggested it could be used in monitoring the (47.6%) patients’ pulmonary function ameliorated. After the second metabolic oscillations so as to realize the real-time control of drug treatment, 18 (42.9%) patients’ pulmonary function improved. After metabolism. the third treatment, 2 (28.6%) patients’ some pulmonary function Acknowledgments: Supported by the Korea Science and Engineering recovered. After the fourth treatment and the fifth treatment, patients’ Foundation NRL Program grant funded by the Korea government pulmonary function did not reveal significant change. The results show (MEST) (No. R0A-2008-000-20012-0), National Natural Science that the multiple cellular therapy definitely serves as a positive role in Foundation of China (No. 50973089), and the Project Sponsored by the treatments of ALS, and the repeated and periodic cell-based the Scientific Research Foundation for the Returned Overseas Chinese therapy which patients benefit from is strongly recommended for Scholars, State Education Ministry. better controlling this progressive deterioration disorder.

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Pain Neurorestoratology: a new idea for intractable relationship between PPRs, especially RAGE, and b cell failure in ache? type 2 diabetes. Methods and results: Pancreatic islets were isolated and dispersed

1-3 1-3 3 into single cells, from diet-induced obesity (DIO), ob/ob (leptin- Lin Chen *, Hongyun Huang , Huancong Zuo , -/- 4 deficient), db/db (leptin receptor-deficient), RAGE-null (RAGE ), and Hari Shanker Sharma and the control (WT) mice, and then analyzed by flow cytometry (BD

1 FACS Aria II) with insulin, RAGE and TLR2 and 4 antibodies. Center for Neurorestoratology, Beijing Rehabilitation Center, RAGE expression was detected in insulin-positive b cells from ob/ob Beijing, China; and db/db mice, but not from DIO and WT as well as RAGE-/- mice, 2Beijing Hongtianji Neuroscience Academy, Beijing, China; 3 suggesting a possible linkage between inadequate leptin receptor Division of Neurorestoratology, Yuquan Hospital, Tsinghua signalling and RAGE expression. TLR2 was only detected in non b University, Beijing, China; 4 cell clusters. The expressions of RAGE and TLR2 were induced by Laboratory of Cerebrovascular Research, Department of Surgical palmitate in MIN6 cells, a mousebcell line, in a dose-dependent Sciences Anesthesiology & Intensive Care Medicine, University manner. Hospital, Uppsala University Summary: RAGE expression on pancreaticb cells was demonstrated in diabetic ob/ob and db/db mice. FFA elevation with concomitant Pain Neurorestoratology (PN) is an important branch of Neuroresto- AGE formation may causeb cell damage through induction of RAGE ratology, which study how to intervene pain, especially refractory expression in type 2 diabetes. neuropathic pain. The fundamental principles of PN are to promote nerve repair, immune regulation, improve microcirculation, and les- sen clinical symptoms. New treatment options for PN includes: cellular therapies, close Pressure-crystallized polymer micro-spheres injection of neurorestorative drugs, ozone, local muscle deep as delivery systems for therapeutic proteins hyperthermia, and neurotransmitter modulating agents. Cell medicine and transplantation is the core of this comprehensive PN Jun Lu1, Jiaojiao Tian1, Daopeng Zhang1, Daikun Xi1, therapy. Cells for nerve repair include glial cells (olfactory en- and Rui Huang2 sheathing cells, Schwann cells, etc.), mesenchymal cells (umbilical cord/blood mesenchymal stromal cells, bone marrow stromal cells, 1Key Laboratory of Advanced Technologies of Materials, Ministry etc.), and chromaffin cells. The ways of transplanting cells include of Education, School of Materials Science and Engineering, local parenchymal implanting, intrathecal injection and or intra- Southwest Jiaotong University, Chengdu 610031, Sichuan, China, vascular routes. corresponding author e-mail: [email protected] (J. Lu) Four main directions of PN: (1) to treat chronic intractable pain 2College of Polymer Materials Science and Engineering, Sichuan with no way available currently, (2) to improve the outcome of tra- University, Chengdu 610065, Sichuan, China ditional pain therapy, (3) to relief pain together with nerve system repair and vascular and blood circulation reconstruction in the Micrometer-sized polymer spheres are promising in controlled- patients with central pain after spinal cord trauma, stroke, neurode- release drug delivery systems, which protect protein and peptide generative impairments, etc., (4) to provide new ideas for current structures from rapid degradation in the bloodstreams and capture by effective treatments. For instance, can we treat the trigeminal neu- non-specific receptors. In this study, two polymers, bisphenol-A ralgia by cell therapy instead of the surgical vascular decompression? polycarbonate (BAPC) and poly (ether ether ketone) (PEEK), were Could we control spinal cord pain using cell therapy with electric crystallized at high pressure with a piston-cylinder apparatus, and stimulation? Can we treat the acute pain by cell therapy? Here, we were investigated using wide-angle X-ray diffraction (WAXD), review the most recent clinical results of the PN therapy in patients differential scanning calorimetry (DSC) and scanning electron with pain due to spinal cord injury, stoke, diabetes, aging, muscle microscopy (SEM). The results showed that crystalline micro- strain, and sports injuries. Taken together, the proposing and imple- spheres of BAPC and PEEK with folded-chain substructures were menting of the concept should be helpful for greatly improving the easily exposed and isolated with selective etching techniques. The level of pain therapeutics. mesoporous spheres of BAPC with three-dimensional organization, which were obtained by a stepwise double-heat treatment at high pressure, retained their entity without being broken even though the Pancreatic b cell dysfunction in diabetes through amorphous region was totally etched away. As for PEEK micro- spheres, they were endowed with an elliptical outline, and were induction of RAGE expression consisted of flake-like lamellae with rugged surfaces. Further morphological observations suggested that such PEEK macro-spheres Dong Han*, Yasuhiko Yamamoto, Seiichi Munesue, Soh Motoyoshi, might be evolved from a novel dendritic crystal, which was iso- Hidehito Saito, Takuo Watanabe, and Hiroshi Yamamoto thermally crystallized and then accumulated into the final agglomerations. The as-prepared polymer micro-spheres, with high Department of Biochemistry and Molecular Vascular Biology, surface area, large pore volume, hydrophilic character, chemical and Kanazawa University Graduate School of Medical Science thermal stability, unique morphologies and toxicological safety, may diversify niche applications in achieving a controlled and sustained Objective: Elevated free fatty acids (FFA) and uncontrolled release system of proteins, as well as their adsorption and hyperglycemia could contribute to pancreaticb cell failure in dia- separation. betes. Pattern recognition receptor (PPRs) including receptor for Acknowledgments: This work was supported by the National Natural advanced glycation end-products (RAGE) and toll-like receptor Science Foundation of China (Grant Number 50973089),, as well as (TLR) 2 and 4 could be the main sensors of the triggers and danger the Project Sponsored by the Scientific Research Foundation for the signals inb cell dysfunction. In this study, we examined the Returned Overseas Chinese Scholars, State Education Ministry.

123 S32 12th International Congress on Amino Acids, Peptides and Proteins

Proteins from Erwinia asparaginase ErwinaseÒ puriﬁed and the structure of the protein will be determined as a and E.coli asparaginase 2 MEDACÒ for treatment potential biocontrol candidate. Acknowledgments: This work was supported by grants from of human leukemia, show a multitude of modiﬁcations the NKBRPC (2003CB114204 and 2006CB101902), NKPC for which the consequences are completely unclear (2004BA901A36), RFDP (20104601110004), CARS-34-GW8 and KYQD1006 (China). Narkhyun Bae, and Gert Lubec

L-Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; Uni- Ò ProtKB accession number P06608 [Erwinase ]) and L-asparaginase 2 Real-time studies of ultrafast electron transfer reactions from E.coli (ASPG2_ECOLI; UniProtKB accession number P00805 of carbon tetrachloride with amino acids (tryptophan Ò [Medac ]), both L-asparagine amidohydrolases, are widely used for and tyrosine) and dGMP: free radical formation the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the and implications for medicinal chemistry protein chemistry of the medical products sold. Mass spectrometry data on ASPG_ERWCH and ASPG2_ECOLI have not been published Ting Luo and Qing-Bin Lu* so far and herein a gel-based proteomics study was performed to provide information on sequence and modifications of the Departments of Physics and Astronomy, and Departments of Biology commercially available medical products. ASPG_ERWCH and and Chemistry, University of Waterloo, Ontario, Canada ASPG2_ECOLI were applied onto two-dimensional gel electrophoresis, spots were in-gel digested with several proteases and resulting Femtomedicine is an exciting emerging frontier, which involves a -15 peptides and protein modifications were analysed by nano-ESI-LC– fusion of ultrafast (femtosecond, 1 fs = 10 s) laser techniques with MS/MS. Four spots were observed for ASPG_ERWCH, six spots biomedical methods to address important biological processes with were observed for ASPG2_ECOLI and the identified proteins showed close relevance to diseases, diagnosis and treatment. We have recently high sequence coverage without sequence conflicts. Several protein applied this innovative approach to obtain real-time observations of modifications including technical and posttranslational modifications molecular reactions forming free radicals, which are highly relevant to were demonstrated. Protein modifications are known to change the molecular pathways for DNA damage and cell death, the molecular physicochemical, immunochemical, biological and pharmacological mechanisms of cancer therapies and the development of new anti- properties and results from this work may challenge re-designing of cancer agents. Some halogenated compounds with a high affinity to the product including possible removal of the modifications by the DNA have been explored as radiosensitizers for cancer therapy, while manufacturer because it is not known whether they are contributing to carbon tetrachloride (CCl4) has served as a model substance for the serious adverse effects of the protein drug. studying the mechanisms of hepatoxicity. It is generally believed that the mechanism of CCl4 hepatoxicity involves the cleavage of a carbon- chlorine bond by the enzyme cytochrome P450 (CYP), generating free Purification and preliminary function of disease- radicals such as CCl3 and CCl3OO . However, the mechanism for the resistance protein from Brevibacillus brevs A57 generation of free radicals in CCl4 metabolism is still not clear, and no real-time studies on the reaction dynamics of CCl4 with amino acids and nucleotides have been reported. Here, we will present real-time fs- Tingya Yang, Wenbo Liu, Xiaoxi Lan, Lin Li, Xiang Li, Liang Sun, TRLS studies of the reaction dynamics of CCl4 with amino acids Xiaoyan Wu, Weiguo Miao*, and Fucong Zheng* (tryptophan and tyrosine) and nucleotides. Our results show direct evidence of electron-transfer reactions of CCl4 with amino acids, College of Environment and Plant Protection, Hainan University, leading to the formation of free radicals. This observation unravels a Haikou 570228 China molecular pathway for CCl4 activation, i.e., through direct targeting *Corresponding author Weiguo Miao the proteins at their Trp/Tyr sites, and provides a mechanistic under- E-mail: [email protected]; Fucong Zheng standing of the negative genotoxic and mutagenic effects of CCl4. E-mail: [email protected]

Brevibacillus brevs A57 was isolated from cotton rhizosphere soil. It has been confirmed for A57 to have stronger antagonism against Redistribution of calcitonin-gene related peptide several important plant pathogens, such as Verticillium dahliae, (CGRP) in the brain and heart tissues after cardiac Fusarium oxysporum f.sp. infectum, and Colletotrichum gloeospo- rioides Penz. The examination of antagonistic mechanism under light arrest. Neuroprotection by methylene blue treatment microscope showed that the antagonistic substance of A57 appeared to inhibit pathogens by leading to mycelium rupture, distortion, 1Lars Wiklund*, 2Ranjana Patnaik, 1Aruna Sharma, abnormality, and inhibition bourgeon of conidiophore, and leading to and 1Hari S Sharma abnormality of conidiophore. Antifungal factor from A57 was further identified as a kind disease-resistance protein by ammonium sulfate 1Department of Surgical Sciences, Anesthesiology & intensive Care precipitation method. This protein enable to accumulation in PDA Medicine, University Hospital, Uppsala University, SE-75185 (potato dextrose agar) medium, or in PDA medium against a patho- Uppsala, Sweden. Email: [email protected] gen. The accumulation content of A57 protein indicated higher peak 2Department of Biomaterials, School of Biomedical Engineering, in about 3 days, producing clear blank band between paper disk Institute of technology, Banaras Hindu University, Varanasi-221005, containing the protein and a pathogen. The protein could be isolated India and purified from the medium with the blank band by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). It was Calcitonin-gene related peptide (CGRP) is altered in the brain, aorta, able to inhibit the growth of pathogen, and promote plant develop- heart muscles and liver in spontaneously hypertensive rats. Further- ment. In the future, the novel antifungal protein will be further more, low dose of CGRP treatment in cardiac arrest results in

123 12th International Congress on Amino Acids, Peptides and Proteins S33 improvement of heart function. However, the role of CGRP in cardiac normal and disease states in vivo, is demonstrated. These recent arrest induced brain pathology is not understood. experiments also support the potential of RAGE and its S100 ligands In present investigation, we measured CGRP in the cerebral cortex as attractive theragnostic markers and targets, respectively. as well as in the left and right cardiac ventricles after different periods of cardiac arrest in a porcine model. Separate groups of pigs were treated with methylene blue and CGRP was determined in identical brain and heart tissues using radioimmunoassay procedures. Serum amino acid profiles and clinical purpose The normal CGRP content in the cortex was 55 ± 2 pg/mg tissue, in patients undergoing CAPD whereas the right and left ventricles were 15 ± 1 and 18 ± 2 pg/mg tissue respectively. Cardiac arrest of 30 min resulted in a significant Zheng li and Huang fengxian decline in cortical CGRP content (14 ± 3 pg/mg) whereas, cardiac tissues showed a slight but significant increase (left 27 ± 4 pg/mg; The First Affiliated Hospital of Sun-Yatsen University right 23 ± 2 pg/mg). These changes in CGRP were further altered following 60 min after restoration of spontaneous circulation (ROSC) Fasting serum amino acid levels were measured in seven diabetic (brain CGRP 8 ± 2 pg/mg, ventricles left 34 ± 4; right 30 ± 2 pg/ male, fourteen nondiabetic male, six diabetic female, and nondiabetic mg). At 180 min after ROSC the heart tissues showed significant female patients on CAPD (RRF C 2 ml/min). Comparison of their decline (left ventricle 8 ± 2; right 12 ± 4 pg/mg) whereas, cortical serum amino acid values with fourteen controls showed that CAPD CGRP showed a mild elevation (64 ± 6 pg/mg) from the control did not restore the serum amino acid levels of these patients to nor- values. Pretreatment with methylene blue significantly attenuated these mal. High levels of phenylalanine and low levels of tryptophane changes in CGRP at 180 min as compared to the control cardiac arrest showed up in essential amino acids of four group patients; among group (brain 40 ± 6 pg/mg; left ventricle 23 ± 4; right 33 ± 6 pg/ non-essential amino acids, taurine, glycine, aspartic acid, citrulline, mg). Since neuronal changes are significant following 30–180 min glutamic acid, proline, ornithine were higher significantly, but tyro- after cardiac arrest and methylene blue markedly reduced these neu- sine was lower significantly. Other 21 species of amino acids: ronal damages at 180 min, our results are the first to demonstrate that asparamide, hydroxyproline, ethanolamine, 1-methyl-histidine, ASA alterations in CGRP in brain and heart are crucial for neurological arginine succinic acid, 3-methyl-histidine, Carnosine, c-aminobutyric impairments. Further studies are needed to understand the molecular acid, hydroxylation citrulline, a-amid adipic acid, b-aminoisobutyric mechanisms of CGRP induced brain pathology in cardiac arrest. acid were higher, a-amid- o-butyric acid, methionine were lower than healthy subjects. The concentrations of total 42 amino acids diabetic female patients on CAPD were to approach normal level. We presume S100 proteins and RAGE in cancer clinical causes: deficient nutrition intake, transducer process blockage and urotoxin accumulation. To supply vitamins, low-protein diet compounding a-keto acid will improve amino acids equilibration. Jens Pietzsch

Helmholtz-Zentrum Dresden-Rossendorf, Institute Stochastic resonance therapy (SRT) and tryptophan of Radiopharmacy, Dresden, Germany E-mail: [email protected] metabolism RAGE, the receptor for advanced glycation endproducts, is a pattern recognition receptor that belongs to the immunoglobulin superfamily. Berthold Kepplinger1,2, Halina Baran2, Brenda Sedlnitzky-Semler2, In homeostasis, RAGE is expressed ubiquitously high only in the Nagy-Roland Badawi1 and Helene Erhart1 lung, and moderate to low in a wide range of cells such as endothelial cells, mononuclear phagocytes, smooth muscle cells, mesangial cells, 1Department of Neurology, Neuropsychiatric Hospital Mauer, Mauer- and certain neurons. It is found either as a membrane-bound or sol- Amstetten uble protein that is markedly and quickly upregulated by stress in both 2Neurochemical Laboratory, Karl Landsteiner Research Institute epithelial and inflammatory cells. RAGE binds multiple ligands, for Pain Treatment and Neurorehabilitation, Landesklinikum including advanced glycation end products (AGEs), amyloid fibrils, Mauer-Amstetten, Austria amphoterin, and various members of the S100 family of EF-hand Corresponding author: [email protected] calcium-binding proteins such as S100A4, S100A8/A9, S100A11, S100A12, and S100B. Activation and upregulation through a positive Stochastic resonance therapy (SRT) is applied for treatment and feedback loop of RAGE and, subsequently, perpetual RAGE S100 rehabilitation of patients with various neurological and mental dis- engagement effects the activation of diverse signaling cascades that orders e.g. Parkinson disease, Alzheimer’s, Multiple Sclerosis, stroke, initiate and stimulate chronic stress and survival pathways, depending depression and schizophrenia, also for the treatment of low back pain on the distinct interacting S100 protein, environment, and develop- and also for prophylaxis of osteoporosis. The effectiveness of SRT in mental stage. This can result in chronic inflammation and is supposed treatment of these diseases has been reported however the mecha- a setting in which predominantly epithelial malignancies can arise. nism(s) of action is complex and needs further explanations. An Therefore, exploring the function of RAGE and its panoply of S100 involvement of tryptophan metabolism along kynurenine pathway has ligands in the setting of inflammation is critically important in been demonstrated in various experimental and/or human neuroin- understanding the role of this receptor in carcinogenesis and metas- flammatory and neurodegenerative diseases and during the aging tasis, but also in other pathological conditions such as radiation process. Kynurenine metabolites show an ability to modulate the therapy-related vascular dysfunction. In this review, we summarize neuro-glia biochemical processes and they are involved significantly novel findings on RAGE S100 protein interaction and subsequently in neurophysiological and neuropathological events. The aim of this triggered signaling cascades from published reports and own ongoing study was to search the influence of SRT as an exercise activity, on studies. In particular, a comprehensive evaluation of S100 protein tryptophan metabolites in the serum of healthy subjects. The con- metabolism in rodent models using fluorine-18 radiolabeled recom- centration of L-tryptophan, L-kynurenine, kynurenic acid and binant S100 proteins and small animal positron emission tomography, anthranilic acid was searched in the serum of volunteers 1 min before further underlining the role of RAGE S100 protein interaction in SRT and 1, 5, 15, 30 and 60 min after SRT application. We found that

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SRT altered significantly tryptophan metabolism in the serum. mitomycin C, known antitumor agents are identified as novel inhib- L-tryptophan, L-kynurenine and kynurenic acid were time dependent itors and subversive substrates. Possible mechanism of subversive significantly lowered up to 60 min of SRT. Changes of anthranilic substrate is investigated. Both of these compounds show significant acid were characterized by transient decrease accompanied by up a effect on redox homeostasis of the parasite and high leishmanicidal mild enhancement at 60 min. Revealed data demonstrate that exercise activity. Moreover, reactive oxygen species (ROS) determination using SRT method affects tryptophan metabolism in the periphery using CM-H2DCFDA probe employing fluorescence spectroscopy significantly and it is likely that these influences take places in the and flow cytometric analysis show increase ROS generation in pres- central nervous system, as well. The lowering of tryptophan would ence of these compounds. Additionally, our flow cytometric analysis suggest generally an activation of serotonin, protein and bone bio- also indicates change in cell morphology due to ROS. Our toxicity synthesis and might affect positively the immune system. The studies as well as available toxicity data in literature indicate these lowering of kynurenic acid due to exercise might increase the neu- compounds to have acceptable toxicity in limited dose. The current rotransmission of dopamine- and cholinergic systems and therefore report points out toward potential application of doxorubicin and activates the anti-depressive, anti-dementive and anti-aging processes mitomycin C as therapeutics against leishmaniasis. as well support the physiological condition. These mechanisms of actions observed after SRT might be relevant for the improvement of symptoms in Parkinson’s and Alzheimer’s diseases also in patients with mental depression and in patients with chronic low back pain. Targeted delivery of IDUA across the blood–brain barrier for MPS I disease

Study on effects of Baicalin Bai on the repaired Daren Wang1, Alex Kuan2, David Hui3 and Dao Pan1,4 of injured podocytes in diabetic nephropathy rats 1Division of Experimental Hematology and Cancer Biology 2Division of Developmental Biology, Cincinnati Children’s Hospital SU Ning, ZHAO Ping, ZHOU Le-quan, DU Biao-yan, LUO Hui, Medical Center, Cincinnati, OH 45229, USA and LANG Jian-ying 3Department of Pathology 4Department of Pediatrics, University of Cincinnati, Cincinnati, School of Basical Science, Guangzhou University of TCM, OH 45219 Guangzhou 510006, China Mucopolysaccharidosis (MPS) type I, resulting from the deficiency of Objective: To Study Effects of Baicalin Bai on the repaired of injured the enzyme a-L-iduronidase (IDUA), is one of the most common podocytes and FN and IV-C expression in diabetic nephropathy (DN) lysosomal storage disorders affecting the central nervous system Rats, which to explore its preventive and therapeutic effect on treating (CNS) that cannot be cured. We investigate in this study if the fusion DN. of receptor binding domain (Rb) of Apolipoproteins (apo) to IDUA Methods: DN rat model was established substances in kidney of rat can enable the modified protein to bind members of low-density of streptozotocin. DN model rats were randomly divided into model lipoprotein receptor family (LDLRf) on BBB and transcytose to the group and Baicalin treatment group, Normal control group was set up CNS. To determine the accessibility of IDUA protein for genetic additionally. Rats in Baicalin treatment group were injected Baicalin modification, we first evaluated myc-tag IDUA using Western blot Bai solution 40 mg/kg/day abdominally, rats in the model group were analysis, immunoprecipitation and IDUA enzyme assay, and identi- injected water 1 ml/rat/day abdominally, 6 weeks later, the weight of fied IDUA3’Myc not only retained normal biological function, their bodys and kidneys was monitored. The right kidney tissues was lysosomal enzyme trafficking, and endogenous receptor-mediated fixed in 3% glutaraldehyde solution, and electronic scopy was per- uptake pathway, but also acquired additional myc-antibody binding formed. The left kidney tissues were fixed in 10% formalin solution, ability. We then constructed seven fusion IDUA-Rb candidates con- and pathological scopy was performed. Observe the morphology taining various amino acid residues within the receptor-binding changes in podocytes and FN and IV-C expression in kidneys. domains of apolipoproteins, and evaluate receptor-specific up-take Results: The kidney index decreased in the Baicalin treatment group, pathway in cells that overexpressed LDLR-associated protein 1 and there was significant difference compared to the model group, (LRP1) (highly expressed on BBB). Two out of seven fusion protein (p \ 0.05); under electric scopy, incrassation injured podocytes; candidates introduced LRP1-specific protein uptake. The specific expression of FN and IV-C ware decreased, and there was significant binding of fusion IDUA-Rb to LRP1 was further confirmed by a difference compared to the model group, (p \ 0.05). pause-chase assay. Using an in vitro BBB model, these two candi- Conclusion: Baicalin reduce the expression of FN and IV-C in glomer- dates could introduce IDUA levels significantly higher than ular or tubule, to repaired podocytes, witch prefect the kidneys in DN Rats. IDUA3Myc control (up to 3.2 fold) after pause-chase assays. Finally, we evaluated the potential brain delivery of IDUA-Rb in vivo by hydrodynamic injection into MPS I mice with plasmids introducing Subversive substrates of trypanothione reductase liver-specific expression of fusion proteins. Elevated IDUA activities of Leishmania parasite: an alternative approach (22–120-fold of normal plasma IDUA levels) were found in plasma of all MPS I mice 2 days after injection. Moreover, capillary-depleted for chemotherapy brain tissues from well-perfused animals injected with IDUA-Rb exhibited significantly higher IDUA activities than those from Anil Kumar Shukla and Vikash Kumar Dubey* IDUA3Myc controls. Immunofluorescent study has identified IDUA- Rb positive cells mostly in non-endothelium perivascular cells, neu- Department of Biotechnology, Indian Institute of Technology rons, and less so in astrocytes of cerebra of injected mice. In Guwahati, Assam, India- 781039, E-mail: [email protected] summary, we have identified two Rb candidates by in vitro and in vivo evaluation that could mediate efficient IDUA delivery across the Trypanothione reductase is a key enzyme of unique redox metabolism BBB via LRP1 receptor mediated transcytosis, thus open a door for of Leishmania parasite and validated drug target. Doxorubicin and novel and non-invasive approaches in treating brain diseases.

123 12th International Congress on Amino Acids, Peptides and Proteins S35

Targeting COX-2 for anticancer drug development, especially HLA-DPB1*0201 (DP2) and other alleles that contain a tumor radiosensitization, and molecular imaging glutamic acid residue at position 69 of the b-chain (b69Glu). Importantly, the HLA-DP alleles that can present Be to T cells of cancer match those implicated in the genetic susceptibility, confirming that the HLA contribution to disease is based on the ability of those Frank Wuest molecules to bind and present Be containing compounds to T cells. Here we show a 3.25 A˚ crystal structure of DP2 whose antigen- Department of Oncology, University of Alberta, Edmonton, Canada. binding groove is occupied by a self peptide derived from the HLA- E-mail: [email protected] DR a-chain. The most striking feature of the structure is a unique large solvent exposed pocket formed between the peptide backbone Two isoforms of cyclooxygenase (COX-1 and COX-2) control the and the DP2 b chain a helix. This pocket is acidic due to the complex conversion of arachidonic acid to prostaglandins and presence of three glutamic acids from the b chain including b69Glu. thromboxanes, which trigger as autocrine and paracrine chemical In the crystal packing, this pocket has been filled with the guanid- messengers many physiological and pathophysiological responses. inium group of an arginine from a neighboring molecule. This As a housekeeping enzyme, COX-1 is constitutively expressed in positively charged moiety has formed an extensive H-bond network resting cells of most tissues, whereas COX-2 is an inducible iso- that offers a plausible model for how Be containing complexes form which is significantly upregulated as part of acute and chronic might occupy this site. This idea is strengthened by the demon- inflammatory conditions. Moreover, various immunohistochemical stration that mutation of any of the three glutamic acids in this studies have confirmed COX-2 overexpression in several malignant pocket results in loss of the ability of DP2 to present Be to T cells. or pre-malignant human tumors including colon, breast, lung, gas- tric and esophageal, prostate and pancreatic cancers. COX-2 overexpression is not only linked to tumor formation alone, but also with invasiveness and the metastatic potential of the tumor. The effects of high altitude hypoxia environment The evident role of COX-2 in carcinogenesis has stimulated research activities towards molecular targeting of COX-2 and the on ultrastructure and the expression of HIF-1a COX-2 pathway as a promising strategy for the prevention and in the rat lung treatment of solid tumors. This included various preclinical and clinical studies using combined treatments of either chemotherapy Li Wenhua, Yuan Dongya, Zhang Min, Sun Fangyun, Zhao Fengcang or radiotherapy with COX-2 inhibitors. Another recent development is aimed at the development of non-invasive imaging probes for Medical School, Tibet Institute of Nationalities, Xianyang, Shanxi assaying COX-2 expression in vivo as a potential valuable diag- 712082,China; Email: [email protected] nostic tool to identify and select cancer patients for combined tumor therapy with selective COX-2 inhibitors. In addition, Objective: To study the effects of high altitude hypoxia environment molecular imaging of COX-2 could also be applied to monitor on rat lung ultrastructure and the expression of HIF-1a in the rat lung. disease progression and to evaluate the efficacy of therapeutic Methods: 50 SD rats were randomly divided into 5 groups, namely interventions. The present review will survey recent research group 1 d Golmud (altitude 2,700 m), 2 d Tanggula group (altitude activities towards molecular targeting of COX-2 for molecular 5,000 m), 3 d Nagqu group (altitude 4,500 m) and 30 d Nagqu group imaging and therapy of cancer. This will include the discussion of (altitude 4,500 m), the control group (in Xi’an, elevation 5 m). 4 combined chemo- and radiotherapy with COX-2 inhibitors, and the time-consuming experimental animals 1 d from Xi’an to the Golmud, development of molecular probes for imaging COX-2 expression in Qinghai (altitude 2,700 m), 2 d to the Tibetan Tanggula (altitude vivo. 5,000 m), 3 d to the Naqu (altitude 4,500 m), 30 d in Tibet Nagqu. Light and electron microscopy of the lung tissue samples, Western Blot method to detect the lung tissue of hypoxia inducible factor-1a The critical role of glutamic acid in chronical beryllium expression, RT-PCR method for detection of high altitude hypoxic group, lung tissue expression of hypoxia inducible factor-1amRNA disease change. Results: 2d Tanggula acute hypoxia in lung tissue microstructure and Shaodong Dai, Guinevere A. Murphy, Frances Crawford, ultrastructure of apparent high altitude pulmonary edema, and after Douglas G. Mack, Michael T. Falta, Philippa Marrack, hypoxic group after 30d Nagqu significantly reduce high altitude John W. Kappler, and Andrew P. Fontenot pulmonary edema, lung tissue was no Canon of Western blotting, HIF-1a Protein expression, RT-PCR detection of HIF-1amRNA 30d Integrated Department of Immunology. National Jewish Health Nagqu group in the expression of a certain amount, the remaining and School of Medicine of University of Colorado, Denver, CO three experimental groups the expression of HIF-1amRNA not sig- 80206 nificant (P [ 0.05), 30d expression of HIF-lamRNA Nagqu group were significantly higher (P \ 0.01). Chronic beryllium disease (CBD) is a fibrotic lung disorder caused Conclusion: The hypoxia acclimatization for improved HIF-la by beryllium (Be) exposure, characterized by granulomatous mRNA expression is beneficial to the hypoxic altitude pulmonary inflammation and the accumulation of Be-responsive CD4+ T cells edema. in the human lung. Genetic susceptibility to CBD has been associ- Keywords: SD Rat, High altitude hypoxia, Transmission electron ated with certain alleles of the MHCII molecule, HLA-DP, microscopic, HIF-1a

123 S36 12th International Congress on Amino Acids, Peptides and Proteins

The key residues of active sites located on beta-tubulin angiography. All patients were prospectively followed up. The pri- forming an active pocket for paclitaxel binding mary end point was the combined occurrence of major adverse cardiovascular events (MACE) including cardiovascular and noncardiac death, nonfatal myocardial infarction (MI), nonfatal Sichuan Xu1,2,*, Shaoming Chi1, Yi Jin1, Qiang Shi2, Maofa Ge2, 2 2 ischemic stroke, rehospitalization for unstable angina, and revascu- Shu Wang and Xingkang Zhang larization with percutaneous coronary intervention or coronary

1 artery bypass grafting. There were 34 MACEs including 1 nonfatal Key Laboratory of Education Ministry for Medicinal Chemistry MI, 7 ischemic stroke, 11 noncardiac death, and 15 rehospitalization of Natural Resource, College of Chemical Science and Technology, for unstable angina in a duration of 88 ± 30 months. Patients with Yunnan University, Kunming 650091, People’s Republic of China 2 MACE had lower baseline serum bilirubin levels (P = 0.002). All State Key Laboratory for Structural Chemistry of Unstable patients were stratified into the high-, normal-, and low-bilirubin and Stable Species, Beijing National Laboratory for Molecular group. The patients in high-bilirubin group had the lowest incidence Science, Institute of Chemistry, Chinese Academy of Sciences, of MACE (P = 0.004). In a multivariate Cox regression analysis, Beijing 100190, People’s Republic of China serum bilirubin was the only independent predictor of MACE (HR, 0.066; 95% CI 0.008–0.562; P = 0.013). Accordingly, in patients Paclitaxel (PTX) is used to treat various cancers but causes heavy with CSX, baseline serum bilirubin level was associated with long- side effect and resistance as well. To better design similar com- term outcomes, suggesting that serum bilirubin may be a predictive pounds with less toxicity and more activity against drug-resistant and protective biomarker for disease progression and the develop- tumors, a significant issue is to clear understand the PTX-binding ment of cardiovascular events in patients with atherosclerosis as pocket organized by key residues as active sites on b-tubulin. well as cardiometabolic diseases. Using docking method, molecular dynamics (MD) simulation and density functional theory (DFT), we have identified some residues such as Arg278, Asp26, Asp226, Glu22, Glu27, His229, Arg369, Lys218, Ser277 and Thr276 locate on b-tubulin as the potential Thymosin beta4 treatment improves neurological sites responsible for interaction with PTX. Other two residues functional recovery in experimental autoimmune Leu371 and Gly279 also likely serve as the active sites. Most of them contact with the ‘‘southern hemisphere’’ of PTX. Only one encephalomyelitis mice key residue interacts with the ‘‘northern hemisphere’’ of PTX. These key residues are composed of four groups and serve as Jing Zhang1, Zheng Gang Zhang1,YiLi1, Mei Lu2, Dan Morris3, active compositions to form an active pocket for PTX binding to Stanton B. Elias1, and Michael Chopp1, 4 the surface of b-tubulin. This active binding pocket provides the very potent interaction, 1Department of Neurology whose strength is predicted to be in the range of -327.8 to 2Biostatistics and Research Epidemiology -365.7 kJ/mol between b-tubulin and PTX using different orientated 3Department of Emergency Medicine, Henry Ford Health System, conformations. The potent interaction makes PTX have high activity Detroit, MI, 48202; against cancer cells, which is in good agreement with the clinical 4Department of Physics, Oakland University, Rochester, MI, 48309 mechanism of PTX. The disclosed PTX pocket associated with the key residues can be applied to probe the mechanism of tumor cell Multiple sclerosis (MS) is the demyelinating disease of the central resistant to PTX and design novel analogues with superior properties. nervous system (CNS), which results from damage to oligodendro- Acknowledgments: The work was supported by One-Hundred-Tal- cytes that make myelin to encase axons. There is no cure for MS. ents Project of Chinese Academy of Sciences, and Academic Talent Thymosin beta4 (Tbeta4) is a highly conserved 43-amino acid acidic Foundation of Yunnan Province, China (2006PY01-29). peptide; its prominent activity is cell motility and organogenesis. Recently, studies found that Tbeta4 facilitates wound healing and promotes angiogenesis. Therefore, in the present study, we hypothesized that Tbeta4 stimulates oligodendrocyte progenitor cells The role of bilirubin as a nature antioxidant in clinical (OPCs) and thereby to enhance remyelination in MS. SJL/J mice outcomes in patients with cardiovascular were subjected to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated with saline and cardiometabolic diseases or Tbeta4 (6, 18, 30 or 60 mg/kg) daily starting on the day of disease onset for a total of 30 days. Neurological function, the Shao-Sung Huang, Shing-Jong-Lin, and Jaw-Wen Chen number of OPCs and mature oligodendrocytes, proliferated and differentiated OPCs were measured using antibodies against National Yang-Ming University School of Medicine and Taipei bromodeoxyuridine (BrdU), NG2 and 20,30 cyclic nucleotide 30 Veterans General Hospital, Taipei, Taiwan phosphodiesterase (CNPase), respectively. Triple immunofluorescent staining with antibodies against BrdU, CNPase and neurofilament Accumulating in vitro or in vivo evidence suggest that oxidative heavy chain was employed to examine whether newly generated stress and inflammation could contribute to both the development mature oligodendrocytes myelinate axons. Tbeta4 treatment (30 mg/ and progression of atherosclerosis. Increased oxidative stress and kgbw) significantly improved functional recovery after EAE. NG2+ vascular inflammation have been demonstrated in patients with OPCs, CNPase+ mature oligodendrocytes, BrdU+ with NG2+ OPCs, cardiac syndrome X (CSX). Bilirubin, once considered simply the BrdU+ with CNPase+ mature oligodendrocytes were significantly metabolic end product of heme degradation, has emerged as a increased in the EAE CNS with Tbeta4 treatment compared to those potential endogenous inhibitor of atherosclerosis. We conducted this of saline controls. The alignment of newly differentiated OPCs study to evaluate the prognostic role of serum bilirubin in disease along axons indicated the induction of remyelination. These data progression and clinical outcome in patients with CSX. A total of indicate that Tbeta4 treatment improved functional recovery and 108 consecutive CSX patients were enrolled. Serum bilirubin levels stimulated oligodendrogenesis after EAE. Tbeta4 is a potential were examined from blood samples collected before coronary therapy for MS.

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TNF-a in carcinogenesis and cancer therapy progesterone concentration, and IgG and PRV-specific antibody levels on day 9 of pregnancy were all increased dose-dependently by Trp Xia Wang inclusion in the diet of PRV-challenged mice. Increased Trp levels in PRV challenged mice promoted the upregulation of uterine and Laboratory of Molecular and Translational Medicine, West China embryonic indoleamine 2, 3-dioxygenase expression, but attenuated the Second University Hospital, Sichuan University, Chengdu, upregulation of uterine and embryonic Toll-like receptor 3 (TLR3) and People’s Republic of China; E-mail: [email protected] TLR9 expression and increased serum interferon-gamma concentration. Collectively, Trp supplements might improve reproductive performance Tumor necrosis factor-a (TNF-a) was identified in the late 1970 s as a of PRV-challenged pregnant mice by downregulating TLR expression cytokine produced by immune cells capable of suppressing tumor cell and proinflammatory cytokine synthesis, by upregulating PRV-specific proliferation and inducing tumor regression. TNF-a is a multifunc- antibody and immunoglobulin synthesis and by elevating the concen- tional cytokine that plays important roles in diverse cellular events trations of anti-inflammatory cytokines and progesterone. such as cell survival, proliferation, differentiation, and death. Upon binding to its specific receptor, TNF-a activates distinct signaling pathways including nuclear factor-jB (NF-jB), c-Jun N-terminal Metabolism kinase (JNK), and apoptosis. NF-jB is a major cell survival signal that exerts its anti-apoptotic effect by activating a number of anti- apoptotic target genes, whereas transient JNK activation contributes A characteristic urinary metabolic profile in citrin to cell survival and sustained JNK activation contributes to cell death. deficiency Thus the fate of TNF-a exposed cells is determined by the balance of TNF-a-induced survival and death signaling. In view of cancer, TNF- a is a double-edged sword. As it is an important pro-inflammatory Chunhua Zhang cytokine, TNF-a may be involved in inflammation-associated carcinogenesis by serving as an endogenous tumor promoter. It could Dept. of Research and Development, MILS International, stimulate the growth, proliferation, invasion and metastasis, and 3-1-1, Heiwa Machi, Kanazawa, 921-8105 Japan tumor angiogenesis of cancer cells, almost all aspects of carcinogenesis, which is closely related to activation of NF-jB and JNK Citrin deficiency, which is due to the mutation in SLC25A13 gene, is pathways. On the other hand, TNF-a is a potential cancer therapeutic characterized clinically by intrahepatic cholestasis in early infancy agent as it has the property of inducing cancer cell death. However, (neonatal intrahepatic cholestasis by Citrin deficiency; NICCD) and using TNF as a chemotherapeutic drug has been hampered by its CTLN2 in adulthood. In NICCD, the clinical feature present jaundice, deleterious side effects, including systemic shock and widespread hypoglycemia, galactosemia, and multiple aminoacidemias. Gas inflammatory responses. In addition, many cancer cells are resistant to chromatograph/mass spectrometry (GC/MS) analyzing, enables the TNF-induced cytotoxicity. Therefore, much work is needed to reduce simultaneous measurement of multiple categories of compounds and its side effects and maximize the tumor-selective cytotoxicity. In this offer reliable and quantitative evidence for the screening or chemical lecture, a comprehensive introduction about TNF-a and it’s roles in diagnosis of more than 130 inborn errors metabolism (IEMs). Using cancer development will be given, with focus on the role of TNF-a in GC/MS analyze patient urine, we can gait difference urinary metabolic cancer therapy including data from our lab. profiles base on each difference IEMs. In our study, NICCD patient shown the characterized urinary metabolic profiles, that contains (1) multiple amino aciduria (high level of urinary excretion of serine, Tryptophane supplements promote pregnancy success threonine, methionine, phenyalanine, tyrosine) like Funconi syndrome profile, (2) high level of galactose, galactitol and galactonate like in mice challenged with pseudorabies virus (PRV) galactosemie profile, combine (3) high level of 4-hydroxyphenyllac- by regulating the expression of systemic cytokines, tate and 4-hydroxyphenylpyruvate like tyrosinemia profile, and (4) immunoglobulins, PRV-specific protein profiles decreased ratio of serine/threonine in the metabolic profile pattern. and toll-like-receptors Also we found above metabolic profile dynamical changes with patient clinical features and diet menu until age 5 months to 1-year-old. When the clinical feature disappear, the urinary metabolic profile present De Wu *, Zhengfeng Fang, Shixiu Qiu, Yan Lin, and Lianqiang Che within normal pattern. Therefore, the metabolic profile of Citrin deficiency depend sample collective condition. The final diagnosis of Key Laboratory for Animal Disease Resistance Nutrition Citrin deficiency need refer genetic mutation analysis. However, uri- of the Ministry of Education of China, Animal Nutrition Institute, nary metabolic profile analysis is the effective procedure for screen of Sichuan Agricultural University, Ya’an 625014, People’s Republic Citrin deficiency. The detail should be presentation. of China *To whom correspondence should be addressed Email: [email protected] Biochemical management in hepatorenal tyrosinemia: Tryptophane (Trp) plays an important role in regulating the maternal a report of 18 cases in Algerian children immune response, a key determinant of the success or failure of pregnancy, but whether Trp supplements can prevent a pseudorabies virus L. Yargui, S. Brahimi, L. Douaibia, S. Meherhera, and M. Berhoune (PRV)-induced failure of pregnancy remains unknown. This study examined the effect of three dietary Trp levels (0.25, 0.35 and 0.5%) on Laboratoire Central de Biochimie, CHU Mustapha Alger. the immunity and reproduction of PRV-challenged pregnant mice. PRV Place 1er mai. 16000 Alger, Algerie challenge resulted in decreased live embryo numbers, live litter sizes, serum progesterone and interleukin-10 (IL-10) concentrations, but Hepatorenal tyrosinemia (TH1) is an autosomal recessive inborn increased the levels of serum immunoglobulins (PRV-specific antibody, disease caused by deficiency of fumarylacetoacetate hydrolase, which IgG, IgA, IgM) and IL-1b. Live embryo numbers, live litter sizes, serum is the last enzyme in the tyrosine degradation.

123 S38 12th International Congress on Amino Acids, Peptides and Proteins

This disorder is clinically severe and affects liver, kidney, and Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mito- peripheral nerve. Hepatocarcinoma is usually a long term complica- chondrial multifunctional enzyme encoded by the HSD17B10 gene. tion. TH1 was diagnosed in 18 cases (ages 4–36 months) on screening Missense mutations in this gene result in HSD10 deficiency (for- 1,268 children for aminoacidopathies, between December 2009 and merly MHBD deficiency) attributed to an inborn error of isoleucine January 2011. The commonest presenting features were hepatomeg- metabolism. In fact, the detection of elevated levels of organic aly (37%) and cholestasis (35%). Other features included, hepatic acids in patients’ blood and urine is a clinical marker for the failure (20%), rickets (3%), acute porphyria (3%) and renal mani- screening of HSD10 deficiency, but not a critical pathogenic factor. festations (2%). Missense mutations of the HSD17B10 gene especially the most Diagnosis was confirmed by an elevated serum tyrosine and common one, p.R130C, abolished the HSD10 activity, interfering urine succinylace´tone levels. Other investigations revealed mildly with not only isoleucine but also neuroactive steroid metabolism. deranged liver functions and a markedly raised alpha feto protein. 8 Since affinities of HSD10 mutants to other peptides are probably tyrosinemic infants were treated with nitisinone plus diet restricted altered, the pathogenesis of HSD10 deficiency may be also related in tyrosine. to the formation of protein complexes by HSD10, which serves as We report the biochemical findings and the results of 1 year fol- a core of the mitochondrial RNase P for instance. These recent low-up in tyrosinemic patients, in our laboratory. findings provided an explanation why the benefit of the protein- restriction regimen, a prevailed treatment, to a HSD10 deficiency patient is questionable despite the curtailing of an accumulation of isoleucine metabolites. Moreover, a blockade of isoleucine degra- Comparative mutagenicities of PAHs metabolites dation was not found in another X-linked mental retardation with induced by peroxynitrite/Fe(III)porphyrin system choreoathetosis and abnormal behavior (MRXS10) resulting from a through Ames test silent mutation of the same gene. Symptoms of these patients appear to be, in some extent, similar to those of a mild form of HSD10 deficiency. Both are X-linked disorders. HSD10 deficiency Yunjing Luo1*, Jing Dai1, Yuanbin She2, and Rugang Zhong1 affects male and some female patients, but female has usually milder phenotype. The establishment of our theory would lead to a 1College of Life Science and Bioengineering, Beijing University new therapeutic strategy of this disease for ameliorating intellectual of Technology, Beijing 100124, China, disability. 2College of Environmental and Energy Engineering, Beijing University of Technology, Beijing, China, [email protected]

Polycyclic aromatic hydrocarbons (PAHs) are widespread contami- Lysine/ornithine decarboxylase: an enzyme catalyzing nants in the environment because of their high potential toxicity, the first step of quinolizidine alkaloid biosynthesis mutagenicity and carcinogenicity. Most of PAHs have no or less mutagenicity, which require activation to exert their mutagenic or in legume plants carcinogenic effects. In our previous work, we took peroxynitrite/ Fe(III)porphyrin system instead of traditional enzyme system to Somnuk Bunsupa1, Kae Katayama1, Akira Oikawa2, Kazuki Saito1,2, induce their metabolic activation, then we successfully identified and Mami Yamazaki1,3 three major metabolites through high performance liquid chromatography coupled with mass spectrometry which were the quinone 1Graduate School of Pharmaceutical Sciences, Chiba University, group, OH group and nitro group. The present study was undertaken 2RIKEN Plant Science Center, 3CREST, JST to isolate three main PAHs metabolites and compare their mutagenicities through Ames test (Salmonella typhimurium TA98) without Lysine decarboxylase (LDC) is the key enzyme involved in the first using the activation of S9 mix. At dose levels ranging from 5 to 50 lg step of quinolizidine alkaloids (QAs) biosynthesis. We have cloned per plate, quinone group, nitro group, PAHs metabolic mixtures all the lysine/ornithine decarboxylase (L/ODC) from alkaloid-containing showed dose correspondence with their mutagenicities, while PAHs cultivar of L. angustifolius by using PCR-select-subtraction and 50/30- parent and OH-group were nonmutagenic. Moreover, it is clear that RACE techniques. The purified recombinant protein expressed in nitro group has the highest mutagenic level, PAHs metabolic mixtures E. coli exhibited decarboxylase activities towards both L-ornithine rank the second, quinone group is the lowest one. It is suggested, and L-lysine with similar Km value. The decarboxylase activities therefore, nitro-PAHs plays the key role in mutagenicity of PAHs toward both substrates were competitively inhibited by DL-a-difluo- metabolic activation. romethylornithine, which is a specific inhibitor of ornithine This work was supported by National Natural Science Foundation decarboxylase. We also characterized L/ODC genes from legume of China (No. 20875006), and Beijing Natural Science Foundation plants Sophora flavescens and Echinosophora koreensis which pro- (No. 2102005). duce QAs. Kinetic study of these two purified recombinant L/ODCs showed the decarboxylase activity toward both substrates with similar Km as same as L/ODC from L. angustifolius. The comparison of the catalytic efficiency (kcat/Km) of these three L/ODCs revealed that the Is hydroxysteroid (17beta) dehydrogenase X (HSD10) preference for L-ornithine over L-lysine is only 0.5–1.5 times. The deficiency an inborn error of isoleucine metabolism? heterologous expression of L/ODC in Arabidopsis and tobacco resulted in the enhanced accumulation of cadaverine and tobacco Song-Yu Yang alkaloids derived from lysine, indicating the actual function of this enzyme for the formation of cadaverine and subsequent production of NYS Institute for Basic Research in Developmental Disabilities, alkaloids. This is the first report on an L/ODC involved in QAs Staten Island, NY 10314, USA biosynthesis from plants.

123 12th International Congress on Amino Acids, Peptides and Proteins S39

Neonatal intrahepatic cholestasis caused by citrin Supported by Grants: SAF 2010-17573 (Spanish Ministry of deficiency (NICCD): clinical analysis of 50 cases Science and Innovation), CVI-6656 (Regional Government of Andalusia) and RD06/0001/1012 (Spanish Health Institute Carlos III). Yuan-Zong Song1, Mei Deng1, Xin-Jing Zhao1, Zhi-Gang Yang1, and Feng-Ping Chen2 On the role of the mitochondrial 2-oxoglutarate 1 Department of Pediatrics, The 1st Affiliated Hospital, Jinan dehydrogenase complex in the amino acid metabolism University, Guangzhou 510630, China, 2 Department of Laboratory Science, The 1st Affiliated Hospital, Jinan 1 2 3 1 University, Guangzhou 510630, China W. L. Araujo , L. Trofimova , Karavaeva Yu. , D. Steinhauser , L. Krall1, A. R. Fernie1, and V. I. Bunik3,4

Aims: Citrin deficiency is resulted from dysfunction of citrin, a 1 mitochondrial aspartate/glutamate carrier encoded by SLC25A13 Max-Planck-Institut fu¨r Molekulare Pflanzenphysiologie, 14476 Potsdam-Golm, Germany, gene. This study aims to investigate the clinical and laboratory fea- 2 tures of Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency Department of Biophysics, 3Department of Bioengineering and Bioinformatics, and (NICCD). 4 Methods: Fifty NICCD cases confirmed by SLC25A13 analysis A.N. Belozersky Institute of Physico-Chemical Biology, were enrolled as research subjects. Major clinical manifestations and Lomonosov Moscow State University, 119991 Moscow, Russia the features of blood biochemistry, hepatopathology, medical imaging and metabolome were analyzed by means of a cross-sec- Providing not only energy, but also intermediates for the de novo tional study. synthesis of cellular compounds including amino acids, mitochondria Results: Clinical presentations included jaundice, abnormal coagu- are tightly linked to cellular nutrient sensing. Mitochondrial meta- lation tests, chubby face, failure to thrive, steatorrhea, motor bolic enzymes as generators and/or targets of signals could therefore retardation, hepato/hepatosplenomegaly, anemia, echinocytosis, and be important players in the distribution of intermediates between the light stool. Besides the elevated biochemical indices of cholestasis catabolic and anabolic pathways. The aim of this work was to char- and dyslipidemia, reduced fibronectin, retinol binding protein and acterize the specific impact of regulating the 2-oxoglutarate ceruloplasmin along with zinc deficiency were also revealed. Diffused dehydrogenase complex (OGDHC), an essential system in both plant fat deposition, cholestasis in hepatocytes and canaliculi, and varying and animal mitochondria. Participating in the glucose oxidation via degrees of inflammation and fibrosis were observed, suggestive of the tricarboxylic acid (TCA) cycle, this highly regulated complex hepatopathological features of non-alcoholic fatty liver disease occupies an amphibolic branch point of the cycle, where the energy- (NAFLD). Ultrasound, CT and MRI revealed fatty livers in some producing reaction of the irreversible decarboxylation of 2-oxoglu- cases, respectively. Metabolome features included coexistence of tarate competes with glutamate synthesis via nitrogen incorporation indices of tyrosinemia type I and markers for galactosemia at urine into 2-oxoglutarate. A synthetic analog of 2-oxoglutarate, succinyl samples, and abnormal amino acid spectrum and acylcarnitine profile phosphonate (SP), forming a tight inhibitory complex with the in blood specimens, with dramatically elevated free, myristyl and coenzyme of the starting OGDHC component, was applied to living palmityl carnitine. systems from different Kingdoms in situ and in vivo. Using a high- Conclusions: This study revealed motor retardation, reduced serum throughput mass-spectrometry-based approach, we show that the fibronectin, retinol binding protein and ceruloplasmin and zinc defi- organisms possessing complete TCA cycle including OGDHC, ciency as novel clinical and biochemical features for NICCD, and respond to SP by significant changes in their amino acid pools. proposed, for the first time, that NICCD might be a specific etiology Increased glutamate and 4-aminobutyrate represent the most universal for non-alcoholic fatty liver disease (NAFLD). Moreover, the changes change accompanying the 2-oxoglutarate accumulation upon the of acylcarnitine profile in this study further expanded the metabolome OGDHC inhibition. Other amino acids were affected in a species- feature of NICCD. specific manner, suggesting specific metabolic networks mediating secondary changes. In contrast, cyanobacteria which display an incomplete TCA cycle lacking OGDHC, do not show perturbations in amino acids following SP treatment. The data provide specific evi- New insights into the roles of brain glutaminases dence of a considerable role of OGDHC in amino acid metabolism.

Javier Ma´rquez

Glutamine/glutamate homeostasis must be exquisitely regulated in The central nitrogen metabolism in Escherichia coli: mammalian brain and glutaminase is one of the main enzymes coordinated regulations everywhere involved. Glutaminase is considered as the main glutamate-producer enzyme in brain and, consequently, it is essential for both glutama- Dalai Yan tergic and gabaergic transmissions. The classical pattern of glutaminase expression in mammals has been recently challenged by Department of Microbiology and Immunology, Indiana University the discovery of novel isoforms and subcellular locations with par- School of Medicine, Indianapolis, Indiana 46202, USA ticular relevance in brain. Furthermore, the interactome of brain glutaminases is also starting to be uncovered adding a new level of The central nitrogen metabolic circuit in Escherichia coli is a com- regulatory complexity. All these experimental evidences suggest new pact system that consists of only three enzymes. While either functions for brain glutaminases. In this talk, we summarize recent glutamate synthase and glutamate dehydrogenase may synthesize ﬁndings that point consistently towards glutaminase as a multifaceted glutamate, glutamine synthetase (GS) catalyzes the sole pathway for protein able to perform different tasks. Some controversial issues glutamine biosynthesis, plays a central role in the system, and is under about brain glutaminases will be also discussed. elaborate regulations. Ammonium is preferred sole nitrogen source,

123 S40 12th International Congress on Amino Acids, Peptides and Proteins supporting the fastest growth. Ammonium assimilation by the circuit identification of clinically important bacteria on the genus and species yields the two central nitrogen intermediates that in turn supply level. nitrogen to all cellular nitrogen components such as amino acids and nucleotides. Through quantitative studies, my group has revealed extensive regulatory coordination throughout the system. The first is on the reversible covalent modification of GS, catalyzed by a The fingerprint of microbial lipopeptides and their bifunctional adenylyltransferase/adenylyl-removing enzyme. We identification demonstrated that the opposing activities are dynamically balanced during cell growth. The study suggests that counterbalance by Shi-Zhong Yang, Jin-Feng Liu and Bo-Zhong Mu* reversible covalent modification may be a general strategy for controlling the activity of enzymes like GS, whose physiological output State Key Laboratory of Bioreactor Engineering and Institute allows adaptation to environmental fluctuations. The second is a of Applied Chemistry, East China University of Science differential but partially overlapping regulatory scheme governing GS and Technology, 130 Meilong Road, Shanghai 200237, People’s expression and modification, achieved by two key metabolic effectors Republic of China. School of Chemistry and Molecular Engineering, through regulatory cascades. We discovered that both GS expression East China University of Science and Technology, Shanghai 200237, and modification respond to a unique scaling combination of the China. internal concentrations of the metabolites, (glutamine)/(2-oxoglutar- *Corresponding to: Tel: +86-21-64252063; Fax: +86-21-6425 2485; 1/3 ate) . The third is a ‘‘kinetic’’ coordination for cell growth during E-mail: [email protected] ammonium depletion. We designed a new culturing apparatus which can continuously monitors cellular behavior during the nutrient The molecular weights of about 90 reported lipopeptides were excess to depletion transition with high temporal precision. With its calculated and the fingerprint of the lipopeptides, which can be application, we show that cells simultaneously elevate enzyme used to distinguish microbial lipopeptides, was established. Based expression (GS) and concentrate the substrate (ammonium) internally, on the fingerprint, a lipopeptide could be identified through simple by the function of a unique active ammonium channel AmtB, to and easy methods, the ESI MS and the amino acid analysis. As an compensate the depleting external ammonium thus sustain the fastest example, a kind lipopeptide from Bacillus subtilis was determined possible growth before ammonium exhaustion. All the cases here through ESI MS and amino acid analysis, the molecular weight and demonstrate the power of quantitative studies for understanding the the amino acid composition of the lipopeptide revealed that the fine print of metabolic regulation. lipopeptide was surfactin. The ESI–MS and amino acid analysis are very useful for identifying the lipopeptides in virtue of the figerprint. Microbiology

Vitreoscilla hemoglobin gene vgb improves the growth Rapid genus- and species-specific identification rate of Corynebacterium glutamicum ATCC13032 of clinically important bacteria by MALDI-TOF with atp gene inactivation in a routine laboratory LI Tie-Min, Huang Ye-peng, and DU Bo Zhao Ning-wei*, and Huang Cheng-cai School of Life Science, Liaoning University, Shenyang 110036, China Life Science and Clinical Medicine Department, Shimadzu China. [email protected]; [email protected] Previously we reported that Corynebacterium glutamicum ATCC13032 with atp gene inactivation, designated as C.glutamicum - The human bowel contains a large and diverse bacterial community, Datp, showed an approximately 1.4 times higher L-glutamate pro- termed as gut flora. These microorganisms perform a number of duction than the wild type strain, but manifested a low growth rate. useful functions, such as making energy, training the host immune In this study, vgb gene, which increases oxygen supply in Vitre- system, preventing growth of pathogenic bacteria, regulating the oscilla sp., was introduced into C. glutamicum-Datp via constructed development of the gut, producing vitamins and hormones for the shuttle vector pJC1-vgb to enhance oxygen uptake. To determine the host. However, under certain conditions, some species are capable of characteristics of recombinant C. glutamicum-Datp harboring vgb causing diseases or increasing cancer risk for the host. Here we gene,namely C. glutamicum-Datp–pJC1-vgb, the fermentation with it evaluated the application of MALDI-TOF MS for rapid genus and as starter was performed in 25 L fermentor, and vgb activity were species identification of some clinically important members in gut detected using CO-difference spectra. flora. The strains provided mass spectra profiles covering a wide Under low dissolved oxygen (DO), vgb gene was expressed on molecular mass range (2,000–30,000 Da) with high reproducibility vector pJC1 and regulated by the DO. vgb gene expression level and specificity. Genus- and species-specific biomarker protein mass increased rapidly as DO dropped. In 100 g/L glucose culture medium, patterns were determined, and validated by Spectral Archive and the cell density of recombinant C. glutamicum-Datp-pJC1-vgb was Microbial Identification System (SARAMIS) SuperSpectrum. For more 22% than the original strain C. glutamicum-Datp. example, the mass spectrometry-based identification scheme yielded The results demonstrated that increasing oxygen supply via vgb identical results of E. coli as with a PCR-based identification system, gene expressed in recombinant C. glutamicum-Datp remarkably which was correctly identified. More importantly, the SuperSpectrum enhanced cell growth rate.It is suggested that using vgb in C. glu- could be constructed manually, and will be imported into SARAMIS tamicum-Datp could be an effective approach to improve the database for the optimal validations. This study demonstrated that production of L-glutamate fermentation in high density culture MALDI-TOF MS was a reliable and powerful tool for the rapid involved in oxygen-limited bioprocess.

123 12th International Congress on Amino Acids, Peptides and Proteins S41

Neurobiology there is significant shortage of knowledge on disease biology. For example, the etiology of AD and PD is far from understood. CNS drugs are known to have a high attrition rate. Compared with other medicines, CNS drugs need to pass blood–brain barriers, a daunting Carbon nanofiber (CNF) electrode and wireless task for drug development. Moreover, there is no good animal model instantaneous neurotransmitter concentration sensor that truly mimics human disease situations. In addition, lack of gen- uine biomarker and good clinical readout for AD or PD makes it (WINCS) system for fast scan cyclic voltammetry extremely challenging for proof of concept studies in humans. detection of neurochemicals To meet the challenges in ND drug discovery, many different approaches have been attempted in the biopharmaceutical industry. At Jessica E. Koehne1, Michael Marsh2, Adwoa Boakye1, GSK-R&D China, we have adopted three basic principles to guide our Brandon Douglas1, Christopher J. Kimble3, Kevin E. Bennet3, drug development efforts. First, our pipeline driver is strategic. Our M. Meyyappan1, and Kendall H. Lee2,4 ND programs are strategically positioned to address multiple disease mechanisms including, inflammation, neuronal cell death pathways 1Center for Nanotechnology, NASA Ames Research Center, Moffett and neuroprotection/repair. Second, our science is game-changing. Field, CA We focus on cutting-edge science and emphasize on differentiation. 2Department of Physiology and Biomedical Engineering, Mayo Third, our approach is innovative. We do not constrain ourselves by Clinic, Rochester, MN the traditional ways of drug discovery, but try novel and creative 3Division of Engineering, Mayo Clinic, Rochester, MN approaches. These three principles are the bases of our overall strat- 4Department of Neurologic Surgery, Mayo Clinic, Rochester, MN egy for the ND drug discovery program.

Deep brain stimulation (DBS) is a state-of-the-art neurosurgical therapy for treating Parkinson’s disease, epilepsy, tremor, dystonia, depression and obsessive compulsive disorder. Carbon nanoﬁber Efﬁcacy of GluK1 receptor antagonists against soman- (CNF) nanoelectrodes have shown great promise as stimulating induced seizures and neuropathology: a potential new electrodes due to their high surface area, low impedance, biocom- emergency treatment for nerve agent exposure patibility and capacity for highly localized stimulation. In addition,

CNF nanoelectrodes have been used as sensing electrodes for the 1 detection of many small biomolecules including DNA, proteins and T. H. Figueiredo, F. Qashu, J. P. Apland , V. Aroniadou-Anderjaska, neurotransmitters. This work describes recent developments in a A. P. Souza, and M. F. Braga novel approach based on CNF nanoelectrodes for localized neurochemical recording. Our approach combines a multiplexed CNF Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD electrode chip, developed at NASA Ames Research Center with the 1 wireless instantaneous neurotransmitter concentration sensor US Army Medical Research Institute of Chemical Defense, (WINCS) system, developed at the Mayo Clinic. The multiplexed Aberdeen Proving Ground, MD electrode chip contained a 3 9 3 array of 200 lm by 200 lm sensing pads. Each sensing pad contains 3–5 lm tall, 100 nm The possibility of mass exposure to nerve agents by a terrorist attack diameter CNFs that are spaced at 1 lm intervals. The CNFs are necessitates the availability of antidotes that can be effective against nerve agent toxicity even when administered at a relatively long embedded in SiO2 so that only the CNF tips are exposed for sensing. The CNF nanoelectrode chip and a traditional carbon fiber latency after exposure, because medical assistance may not be microelectrode were utilized for neurochemical recording in a immediately available. Nerve agents induce status epilepticus (SE), flowcell system and successfully recorded both dopamine and which can cause brain damage or death. Antagonists of kainate adenosine concentrations by fast scan cyclic voltammetry. The CNF receptors that contain the GluK1 (formerly known as GluR5) sub- nanoelectrode chip demonstrated similar performance to the CFM unit (GluK1Rs) are emerging as a new potential treatment for SE and has advantage in flexible multiplexed design architectures for and epilepsy from animal research, whereas clinical trials to treat highly localized neurochemical recording. In the future, combining pain have shown that the GluK1/a-amino-3-hydroxy-5-methyl-4-iso- CNF based stimulating and recording electrodes with WINCS may xazolepropionic acid receptor antagonist LY293558 [(3S,4aR,6R, lay the foundation for an implantable ‘‘smart’’ DBS system that 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-car- utilizes neurochemical feedback control while likely resulting in boxylic acid] is safe and well tolerated. Therefore, we tested improved battery life and increased DBS application in various whether LY293558 is effective against soman-induced seizures and neuropsychiatric disorders. neuropathology, when administered 1 h after soman exposure, in rats. LY293558 stopped seizures induced by soman and reduced the total duration of SE, monitored by electroencephalographic recordings within a 24-h period after exposure. In addition, LY293558 Challenges in neurodegeneration drug discovery prevented neuronal loss in the basolateral amygdala (BLA) and the CA1 hippocampal area on both days 1 and 7 after soman exposure Bai Lu and reduced neuronal degeneration in the CA1, CA3, and hilar hippocampal regions, entorhinal cortex, amygdala, and neocortex on GlaxoSmithKline, R&D China, Building 3, 898 Halei Road, day 1 after exposure and in the CA1, CA3, amygdala, and neocortex Zhangjiang Hi-tech Park, Pudong, Shanghai 201203 China on day 7 after exposure. It also prevented the delayed loss of glutamic acid decarboxylase-67 immuno-stained BLA interneurons on Despite huge progress in neuroscience research, the number of day 7 after exposure. LY293558 is a potential new emergency approved drugs remains unchanged. Neurodegeneration (ND) is one treatment for nerve agent exposure that can be expected to be of the most challenging areas in drug discovery. This is not only effective against seizures and brain damage even with late because the brain is the most complex organ in the body, but also administration.

123 S42 12th International Congress on Amino Acids, Peptides and Proteins

Linolenic acid, a dietary supplement, is an efficacious methylpiperidin-2-yl)(thiophen-3-yl)methyl)-4-(trifluoromethyl)pico- 3 neuroprotective agent against soman-induced brain linamide ([ H]CHIBA-3007), for studying GlyT-1 in the brain. The presence of a single saturable high-affinity binding component for damage: Its possible use as a preventative strategy [3H]CHIBA-3007 binding to the rat brain membranes was detected. Scatchard analysis revealed an apparent equilibrium dissociation 1 1 1§ 2 Hongna Pan , Cynthia Chen , David Jacobowitz , Kerry Van Shura , constant (Kd) of 1.61 ± 0.16 nM and a maximal number of binding 2 2 1 Megan Lyman , John McDonough , and Ann M. Marini sites (Bmax) of 692.8 ± 22.8 fmol/mg protein (mean ± SEM, n = 3). The specific binding of [3H]CHIBA-3007 was inhibited by a number 1Department of Neurology and §Department of Anatomy, Physiology of GlyT-1 inhibitors, such as CHIBA-3007, desmethyl-CHIBA- and Genetics, Uniformed Services University of the Health Sciences, 3007, CHIBA-3008, SSR504734, NFPS/ALX5407, LY2365109 Bethesda, MD and Org24598, consistent with the pharmacological profiles of 2US Army Medical Research Institute of Chemical Defense, GlyT-1 inhibitors. Interestingly, the potency of eight GlyT-1 inhi- Aberdeen Proving Ground, MD bitors (CHIBA-3007, desmethyl-CHIBA-3007, NFPS/ALX5407, LY2365109, Org24598, SSR504734, sarcosine, and glycine) for Nerve agents represent a key threat to the United States military and blocking in vitro specific binding of [3H]CHIBA-3007 was signifi- civilian populations. The major mechanism of acute toxicity is the cantly correlated with the potency of these inhibitors for inhibiting inhibition of acetylcholinesterase resulting in the accumulation of [14C]glycine uptake in the rat brain membranes. In contrast, the acetylcholine (ACh). Excessive ACh levels in synapses lead to the GlyT-2 inhibitor ALX1393 exhibited very weak at [3H]CHIBA-3007 progression of toxic signs including hypersecretions, tremors, seizure binding. Furthermore, the regional distribution of [3H]CHIBA-3007 activity, respiratory distress and ultimately death. Prolonged seizures binding in the rat brain was similar to the previously reported dis- induce severe brain damage and long-term cognitive impairment that tribution of GlyT-1. The present findings suggest that [3H]CHIBA- is commensurate with the extent of damage caused by the nerve 3007 would be a useful new radioligand for studying GlyT-1 in the agent. The current treatment of nerve agent-induced seizures is only brain. efficacious if given within a very short period of time after the induction of seizures. Therefore, strategies to protect neurons against nerve agent-induced neuropathology would reduce brain damage and Activities of kynurenine aminotransferase I, II and III improve outcome. Linolenic acid is an essential omega-3 fatty acid that can be in the rat brain and heart tissues during the aging obtained over the counter as a dietary supplement. The compound is process safe, has no known side effects and exerts neuroprotective, antidepressant and anti-convulsant properties. Therefore, we tested Halina Baran1 and Berthold Kepplinger1,2 whether linolenic acid may protect against soman-induced brain damage. Here, we report that linolenic acid is a highly efficacious 1Neurochemical Laboratory, Karl Landsteiner Research Institute, neuroprotective agent against soman-induced neuronal cell death in Landesklinikum Mauer-Amstetten four brain regions known to be damaged by soman: piriform cortex, 2Department of Neurology, General Hospital, Landesklinikum hippocampus, amygdala and prefrontal cortex. Pretreatment or post- Amstetten, Amstetten, Austria treatment administration of linolenic acid to rats reduces neurode- Corresponding address: [email protected] generation 24 h after exposure to soman. Surprisingly, subcutaneous injection of linolenic acid afforded much higher levels of neuropro- The pattern of kynurenine aminotransferase (KAT) I, II and III, the tection compared with the intravenous route. This is the first study to biosynthetic enzymes of the excitatory glutamate amino acid receptor show significant protection against soman-induced neuropathology by and nicotine cholinergic receptor antagonist kynurenic acid was linolenic acid. Its wide safety margin and neuroprotective efficacy examined in brain and heart tissues of 3, 12 and 26 months old male against soman-induced neuropathology suggests its use as a pre- Wistar rats. KAT I activity at pH 9.6, KAT II at pH 7.2 and KAT III treatment strategy. at pH 8 were measured in the presence of 1 mM pyruvate and 100 lM L-kynurenine in homogenates and mitochondrial suspensions prepared from brain and heart tissues of rats. Synthesized kynurenic Neuroscience acid was purified and determined by high performance liquid chromatography system. We found that KAT II and KAT III activities were increased progressively and significantly in the brain and in the 3 heart homogenates between 3 and 26 months, whereas in the mito- [ H]CHIBA-3007: a new radioligand for glycine chondrial suspensions of brain and heart the KAT II and KAT III transporter 1 in rat brain activities were moderately increased. Interestingly, no alteration of KAT I activities was found in the homogenates and mitochondrial Jichun Zhang, Jin Wu, Jun Toyohara, Yuko Fujita, Hongxian Chen, suspensions prepared from brain and heart of 3, 12 and 26 months and Kenji Hashimoto old animals. Obtained data indicate that under physiological condition the age- Division of Clinical Neuroscience, Chiba University Center dependent increase of kynurenic acid levels in the brain is rather due for Forensic Mental Health, Chiba, Japan to an involvement of an age-dependent increase of KAT II and KAT III activities but not KAT I. Our data would suggest that the increase Glycine transporter-1 (GlyT-1) in glial cells regulates extracellular of KAT I activities seen in Alzheimer’s brain is not related to aging levels of glycine, which acts as an obligatory co-agonist at the process but probably predominantly to the pathological events. ¨ N-methyl-D-aspartate (NMDA) receptors in the brain. In the present Supported by Osterreichische Nationalbank Jubilaümsfonds Pro- study, we developed a novel radioligand, [3H]3-chloro-N-((S)-((R)-1- ject: Nr. 12316.

123 12th International Congress on Amino Acids, Peptides and Proteins S43

APPL1 mediates synaptic NMDA receptor-dependent Ser845 and Ser831 in the presence of a VPAC1 antagonist than in neuroprotection control conditions. Correlation between LTP and GluR1 phosphorylation at Ser 831 was also smaller. LTP was not correlated with GluR1

1 1 phosphorylation at Ser 845. Yubin Wang , Shuang Qiu , JieJie Wang, Shaohua Wang, This suggests that additional molecular mechanisms are recruited and Jianhong Luo for LTP expression when VIP VPAC1 receptors are blocked. Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China Corresponding author E-mail: [email protected] D-Aspartate and D-glutamate in the brain: from formation and localization to function N-methyl-D-aspartate (NMDA) receptors are glutamate gated ion channels and play important roles in both physiology and pathophysiology of central neurons. Excessive activation of NMDA receptor can Jonathan V. Sweedler, Liping Wang, Ting Shi, Nobutoshi Ota, cause neuronal loss in acute trauma such as ischemia, as well as certain Lee Replogle, and Stanislav S. Rubakhin neurodegenerative diseases such as Huntington’s disease. However, physiological levels of synaptic NMDA receptor activity can promote Departments of Chemistry and Neuroscience, University of Illinois neuronal survival through different signaling pathways under different at Urbana-Champaign, Urbana, IL 61801, USA circumstances. Among them, PI3K (phosphoinositide-3-kinase)/Akt chemistry.illinois.edu/faculty/Jonathan_Sweedler.html kinase cascade is a key signaling pathway responsible for neuroprotective effects of NMDA receptor activity. It is still unknown how In the nervous system of animals ranging from mollusks to mammals, NMDA receptors are coupled with PI3K-Akt pathway. APPL1 D-amino acids are present. As an important example, D-aspartate (adaptor protein containing pH domain, PTB domain, and Leucine (D-Asp) and D-glutamate (D-Glu) are present at high levels in specific zipper motif), is an endosomal adaptor protein which are associated brain regions, although their functions are not well understood. with various transmembrane receptors. Furthermore, APPL1 has been Because neurochemistry is well conserved across metazoan life, we reported to interact with, and regulate the activity of, the kinase Akt. In have selected the well-known physiological model Aplysia californica the present study, using biochemical analysis and immunostaining, we to probe the formation and functions of these D-amino acids in cell to found that APPL1 associates with NMDA receptor complex through its cell signaling. Specifically, using capillary electrophoresis with laser C-terminal PDZ binding motif. More importantly, APPL1 is involved induced fluorescence and radionuclide detection, we have determined in synaptic NMDA receptor mediated neuroprotective effect. Inter- which neurons synthesize D-Asp, and have measured the formation, ruption of APPL1-NMDA receptor complex interaction by peptide transport and release of D-Asp in a stimulation dependent manner. We against APPL1 C-terminal PDZ binding motif dissociates PI3K and even make these measurements from individual indentified neurons. Akt from NMDA receptor, blocks activation of Akt by synaptic NMDA Once we determine which specific neurons synthesize D-Asp, we have receptor, and consequently, blocks NMDA receptor-dependent neu- located and characterized the enzyme responsible for D-Asp forma- roprotection from starve-induced apoptosis. It suggests that in central tion. The novel racemase has the ability to convert both L-Ser and nervous system APPL1, as an adaptor protein, connects synaptic L-Asp to their D-counterparts. We have also probed the electrophys- NMDA receptor with intracellular PI3K/Akt cascade and down-stream iological effects of D-Asp and L-Asp on the activity of specific prosurvival signaling pathway. postsynaptic neurons. When combined with physiological measurements, we are able to show that D-Asp acts as a neurohormone and as a neurotransmitter in our system. We have also measured the presence of a unique population of D-Glu containing neurons and are currently Correlation of VIP VPAC1 modulation of GluR1 studying the effect of D-Glu on neurotransmission, as well as facets of phosphorylation and inhibition of protein kinases its formation and release. in theta-burst LTP experiments with data mining tools

Cunha-Reis D1,2 and Carmo AJ2 Decreased number of oxytocinase/vasopressinase- containing neurons in the hypothalamus of patients 1Enzymology Group, CQB, FCUL and 2Autonomic Nervous System Unit, IMM, University of Lisbon, with schizophrenia Lisbon, Portugal. [email protected] Hans-Gert Bernstein1, Susan Mu¨ller1, Henrik Dobrowolny1, VIP inhibits hippocampal CA1 long-term potentiation (LTP) through Uwe Lendeckel2, Johann Steiner1, and Bernhard Bogerts1 VPAC1 receptor activation in PKA and PKC independent process that may involve changes in AMPA GluR1 phosphorylation. We used data 1 Department of Psychiatry, Medical School, University mining techniques to correlate VPAC1 modulation of LTP with of Magdeburg. Leipziger Str. 44, 39120 Magdeburg GluR1 phosphorylation and activity of PKA, PKC and CamKII. 2 Institute of Medical Biochemistry and Molecular Biology, Two independent data sets of experiments were used: G1-VPAC1 University of Greifswald, Germany modulation of LTP, GluR1 levels and phosphorylation at Ser845 and Ser831; G2-impact of PKA, PKC and CamKII inhibitors on VPAC1 The insulin-regulated aminopeptidase (IRAP) is a membrane-span- action on LTP. Grouping and distribution of data sets was studied by ning protein predominantly located in intracellular vesicles. We data clustering (k-means and hierarchical methods). Tree decision suggest that alterations of IRAP-levels in the brain could be involved models targeting GluR1 phosphorylation versus of VPAC1 activation, in the pathophysiology of schizophrenia and depression, because: (1) LTP and kinase inhibition, were generated by supervised classiﬁca- its catalytic function as oxytocinase and vasopressinase. Altered tion methods with Rattle 2.13.0. levels of these neuropeptides are found in schizophrenia and Clustering analysis targeting theta-burst stimulation evidenced depression. (2) The inhibition of IRAP by AngIV and LVV-H7 is weaker data clustering of LTP versus phosphorylation of GluR1 at associated with memory enhancement. Cognitive dysfunction is a

123 S44 12th International Congress on Amino Acids, Peptides and Proteins common symptom in both disorders. (3) IRAP is colocalized with sources can all affect the brains efficiency in this behavioral task. Glut 4 and thus involved in the regulation of the glucose transport. (4) Studies showed clear effect of altered energy intake on cognitive Polymorphisms in and close to the IRAP encoding gene are associ- function. In this study we tested learning and memory by Morris ated with schizophrenia and depression. water-maze in males and females groups of rats after acute and We studied the densities of IRAP-immunoreactive neurons in the chronic food restriction. paraventricular and the supraoptic nuclei in post-mortem brains of 11 Methods: Sprague–Dawley rats were used. The daily need of food was schizophrenic patients, 8 subjects with major depression, 7 patients measured using a metabolic cage. Each animal was then fed with 40% suffering from bipolar disorder and 11 matched controls. of its daily need. Control animals were fed ad libitum. The rats were Our study shows a significant decrease in cell densities of IRAP- tested by Morris-water maze after 2 h (acute) and 2 weeks (chronic containing neurons in the paraventricular nucleus of schizophrenics in groups) of food restriction. During a series of trials the rats were trained comparison with controls. In the supraoptic nucleus of individuals to locate and reach a hidden platform to escape the necessity of with schizophrenia a tendency towards reduced cell densities was swimming. The latency and distance swam by the animals to reach the seen. Depressive patients do not demonstrate significant alterations of platform, and the average speed of swimming, were all measured. IRAP-immunoreactive neurons densities in both nuclei. Results: Our results showed that after acute stress of 2 h FR the male The decrease in IRAP cell density is most probably related to the rats were learning to locate the platform better than the control ani- disturbed oxytocin and/or vasopressin metabolism seen in schizophrenia. mals. These rats were reaching the platform more quickly, and swam shorter distance for this purpose. The female rats on the other hand, were taking more time, and swam longer distances to reach the Evidence of emergent patterns in GluR1 platform. Interestingly, the female animals showed higher speed of phosphorylation in theta-burst LTP experiments swimming than the male rats. After 2 weeks of FR, both males and females were performing significantly inferior to the corresponding with data mining tools control rats. Conclusion: Acute stress by 2 h FR affected differently the cognitive A. J. Carmo2 and D. Cunha-Reis1,2 behavior of male and female rats. The male animals showed enhanced learning and memory in the water maze tests. Both groups were 1 Enzymology Group, CQB, FCUL and performing inferior to control animals after chronic FR. 2 Autonomic Nervous System Unit, IMM, University of. Lisbon, Lisbon, Portugal. [email protected]

Hippocampal CA1 long-term potentiation (LTP) involves phosphor- From potent AMPA/KA agonists to either selective ylation of AMPA GluR1 subunit that varies with stimulus strength NMDA antagonists or to inhibitors of the glutamate and pattern. Kinases mediating this phosphorylation do not always transporters affect LTP. We now used data mining techniques to correlate GluR1 phosphorylation patterns with proposed mechanisms for its Carlo De Micheli, Paola Conti, Andrea Pinto and Lucia Tamborini occurrence. Two independent data sets of experiments were used: G1-LTP Dipartimento di Scienze Farmaceutiche, Universita` di Milano, elicited by mild theta-burst stimulation (MTBS) versus GluR1 levels via Mangiagalli 25, 20133 Milan, Italy and phosphorylation at Ser845 and Ser831; G2-impact of PKA, PKC and CamKII inhibitors on LTP. Grouping and distribution of data was Taking as a model kainic acid we designed a series of aspartic and studied by data clustering (k-means and hierarchical methods). Tree glutamic acid analogues where the amino acid skeleton was embed- decision models targeting GluR1 phosphorylation as a function of ded into the bicyclic isoxazolin-proline motif. Some compounds (i.e. MTBS, LTP and kinase inhibitors, were generated by supervised 3-carboxy-isoxazolin-prolines, CIPs) proved to be very potent classification methods with Rattle 2.13.0. AMPA/KA agonists. Worth noting, the 3-hydroxy-isoxazolin-prolines Clustering analysis targeting MTBS evidenced stronger data (HIPs) turned out to be totally inactive at the glutamate receptors but clustering when comparing LTP and both phosphorylation of GluR1 efficient blockers of the excitatory amino acid transporters (EAATs). at Ser845 and Ser831 (but not GluR1 levels). A stronger correlation The EAATs under physiological conditions remove glutamate from was found between LTP and GluR1 phosphorylation at Ser831 than its synapses but in pathological conditions (e.g. ischemia, neuro- for Ser845. Phosphorylation at those two sites were not correlated trauma), due to the reduction of energy levels and the collapse of the neither LTP versus GluR1 levels. Na+ transmembrane gradient, release additional glutamate through the These observations suggest that MTBS causes LTP through reversed mode of operation, thus contributing to neuronal cell death. phosphorylation of Ser831 and Ser845 of GluR1 subunits through Inhibitors of EAATs have been proposed as promising molecular independent pathways, and that the first is preponderant in this effect. targets for the development of novel neuroprotective agents. The design, synthesis and pharmacological characterization of two potent Food restriction in male and female rats: effects EAATs inhibitors HIP-A and HIP-B will be presented and their potential usefulness in the post-ischemic therapy will be pointed out. on the spatial memory of the hippocampus It is well known that overactivation of NMDA-type glutamate receptors generates an uncontrolled influx of calcium that triggers an Amer Al-Ansari, Tarik Al- Shaibani, Hassan Al Aali, excitotoxic cascade leading to neurodegeneration, a phenome- Alaa AbdulAmeer, Sahar Ashoor, Sara Al-Ghareeb, Reem Al Aradi, non associated to several acute and chronic neurological disorders (e.g. and Fatima Ahmed cerebral ischemia, epilepsy, Parkinson’s and Alzheimer’s diseases). Competitive NMDA antagonists are typically conformationally Arabian Gulf University, College of Medicine, Manama, Bahrain constrained glutamic acid homologues in which the spacer between the a- and the x-acidic groups is a 4-6 carbon atom chain, and the x- Purpose: Food restriction represents specific challenge for animal’s carboxylate may be fruitfully replaced by the bioisosteric phosphonate behavior. Strategies for food seeking, stress and availability of energy group to increase the receptor affinity. According to these rules and

123 12th International Congress on Amino Acids, Peptides and Proteins S45 taking advantage of the results obtained with the above mentioned CIP NMDA receptors (NMDARs) subserve numerous neurophysiologi- derivatives, we have designed a series of new amino acids acting as cal and neuropathological processes in the cerebral cortex. Their potent and selective NMDA receptor antagonists. In particular, two activation requires the binding of glutamate and also of a co-ago- highly potent and selective NMDA receptor antagonists have been nist. Whereas glycine and D-serine (D-ser) are candidates for such a identified, characterized by a very good in vivo anticonvulsant activity role at central synapses, the nature of the co-agonist in the cerebral and by a promising in vitro neuroprotective activity. cortex remains unknown. We first show that the glycine-binding site of NMDARs is not saturated in acute slices preparations of medial prefrontal cortex (mPFC). Using enzymes that selectively Functional coupling between D-serine synthesis degrade either D-ser or glycine, we demonstrate that under the and vesicular transport in astrocytes present conditions D-ser is the principle endogenous co-agonist of synaptic NMDARs at mature excitatory synapses in layers V/VI of mPFC where it is essential for long term potentiation (LTP) Magalie Martineau1, Ting Shi2, Jonathan Sweedler2, Reinhard Jahn3, 4 induction. Furthermore, blocking the activity of glia with the and Jean-Pierre Mothet metabolic inhibitor, fluoroacetate, impairs NMDAR-mediated syn-

1 aptic transmission and prevents LTP induction by reducing the Institute for Medical Physics and Biophysics, University extracellular levels of D-serine. Such deficits can be restored by of Muenster, Muenster, Germany exogenous D-ser, indicating that the D-amino acid mainly originates 2Department of Chemistry, University of Illinois, Urbana, IL, USA 3 from glia in the mPFC, as further confirmed by double-immuno- Department of Neurobiology, Max-Planck-Institute for Biophysical staining studies for D-ser and GFAP. Our findings suggest that D-ser Chemistry, Go¨ttingen, Germany 4 modulates neuronal networks in the cerebral cortex by gating the Magendie Neurocenter, INSERM U862, Bordeaux, France activity of NMDARs, and that altering its levels is relevant to the induction and potentially treatment of psychiatric and neurological Neuron-astrocyte reciprocal communication at synapses has emerged disorders. as a novel signaling pathway in brain function. Astrocytes sense the level of synaptic activity and, in turn, influence its efficacy through the regulated release of gliotransmitters such as glutamate, ATP or D- serine. We focused on the molecular determinants of gliotransmission with a specific interest in D-serine, the endogenous co-agonist of N- Glutamate attenuated survival promoting effect methyl-D-aspartate receptors. First, we showed that D-serine is confined to the regulated secretory pathway in cultured astrocytes and its and signaling of IGF-1 in neuronal cells in vitro release is dependent on calcium and on SNARE proteins, hence and in vivo arguing in favour of an exocytotic release. Then, to explore the existence of a vesicular transport mechanism for D-serine, we devel- Chengming Sun, Dejun Wang, Yannan Ren, Lang Zhang, oped a procedure to immunoisolate synaptobrevin 2-containing and Wenhua Zheng* vesicles from astrocytes. The purified organelles are clear vesicles which possess the molecular machinery to undergo membrane fusion, Neuropharmacology, School of Pharmaceutical Sciences, and contain large amount of D-serine and glutamate. Furthermore, Sun Yat-Sen University, Guangzhou 510006, China they can actively transport these two amino acids. Finally, we pro- *Corresponding author E-mail: [email protected] vided direct evidence for the presence of serine racemase, the D-serine biosynthetic enzyme, at the membrane of astroglial vesicles, and for Impairing intracellular signallings induced by excess glutamate play the coupling between D-serine synthesis and its subsequent vesicular important roles in the process of neurodegenerative diseases. transport. Our results highlight for the first time the existence of a However, the underlying mechanism(s) and its interrelationships glia-specific vesicular transporter for D-serine yet to be discovered. with neurotrophinc factors signaling are most unknown at present. We have shown here that glutamate attenuated the tyrosine phosphorylation of the IGF-1 receptor and the survival effect of IGF-1 in hippocampal cultured neurons. Pre-treatment of cultured hippo- Glial D-serine gates NMDA receptors at excitatory campal neurons with glutamate concentration-dependently inhibited synapses in prefrontal cortex the tyrosine phosphorylation of IGF-1 receptors and the phosphorylation of Akt, GSK3b and FOXO3a. These inhibitory effects Fabrice R. Turpin1,2, Pascal Fossat1,2, Silvia Sacchi3, of glutamate were blocked by NMDA receptor antagonist MK801 Je´roˆme Dulong1,2, Ting Shi4 Jean-Michel Rivet5, but not by blockers of other glutamate receptor sub-types verifying Jonathan V. Sweedler4, Loredano Pollegioni3, Mark J. Millan5, the involvement of the NMDA receptor. These findings demon- Ste´phane H. R. Oliet1,2 and Jean-Pierre Mothet1,2 strate that glutamate can block the effect of IGF-1 by decreasing IGF-1 receptor survival signalling in vitro. To verify this hypoth- 1INSERM U862, Neurocentre Magendie 33077 Bordeaux, France esis further in vivo, similar experiments were performed in mouse 2Universite´ de Bordeaux, 33077 Bordeaux, France brain and in ischemia mouse brain. Our results demonstrated that 3Dipartimento di Biotecnologie e Scienze Molecolari, Universita` glutamate also inhibited the survival signaling of IGF-1 in ischemia degli Studi dell’Insubria, and The Protein Factory, Centro brain via NMDA receptor. Put together these results suggest a Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano novel mechanism by which glutamate can reduce cell viability and and Universita` degli Studi dell’Insubria, Varese, Italy induce neurotoxicity via affecting the survival signaling of growth 4Department of Chemistry and Beckman Institute, University factors. of Illinois, Urbana, USA Supported by National Natural Science Fund of China (No. 5Institut de Recherches Servier, 78290 Croissy-sur-Seine, France 30711120565; No. 30970935).

123 S46 12th International Congress on Amino Acids, Peptides and Proteins

Modulation of nociceptin cleavage, b-endorphine PKC inhibitor staurosporine that does not effectively inhibit PKMf.We liberation, glutamate release and ATP secretion also found that inhibiting PKMf activity in the infralimbic cortex, but not prelimbic cortex, disrupted the expression of the extinction memory by non-opioid analgesic drug metamizol of CPP and CPA. These results indicate that PKMf activity in accumbens core is a critical cellular substrate for the maintenance of drug M. Vlaskovska1, S. Surcheva1, A. Tsakova1, and L. Kasakov2 reward memory, whereas PKMf in the basolateral amygdala is required for the maintenance of both drug reward and aversion memories, and 1Department of Pharmacology, Medical University Sofia, 1143 Sofia, PKMf in the infralimbic cortex is required for the maintenance of Bulgaria extinction memory of reward and aversion cues. 2Institute of Neurobiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Role of low-conserved areas of extracellular vestibule Metamizol (Analgin) is a widely used non-opioid analgesic drug with a strong antinociceptive effect that involves possibly central and in function of purinergic P2X4 receptor peripheral mechanisms. Aim: Study of central/peripheral neurotransmitter mechanisms of V. Tvrdonova1, M. Rokic1, P. Kuzyk1, S. S. Stojilkovic2, Metamizol. and H. Zemkova´1 Methodology: Nociceptin cleavage was studied in primary brain cortical cell cultures from neonatal (\12 h) Sprague–Dawley rats by 1Department of Cellular and Molecular Neuroendocrinology, Institute electrospray ionization mass spectrometry. b-Endorphine liberation was of Physiology of the Academy of Sciences of the Czech Republic, quantified by RIA in hypophyseal slices and plasma before and after i.m. Prague 4, Czech Republic injection of metamizol in mature male Wistar rats. K+-evoked release of 2Section on Cellular Signaling, Program in Developmental glutamate and aspartate was quantified in primary brain cortical cell Neuroscience, The Eunice Kennedy Shriver National Institute cultures from neonatal (\12 h) Sprague–Dawley rats by HPLC analysis. of Child Health and Human Development, National Institutes Urothelial release of ATP was quantified in whole urinary tract prepa- of Health, Bethesda MD 20892, USA ration with in situ urinary bladder in mature male Sprague–Dawley P2X3 + + - - / and matching P2X3 / rats by chemiluminiscence. Purinergic P2X receptor channels (P2XR) are formed by three subunits Results: (i) metamizol stimulated nociceptin biotransformation each containing two transmembrane domains (TM1 and TM2). Crys- increasing the levels of fragments 1–6, 1–9, 1–12, 1–13, 1–17, (ii) tallographic data showed that the channel pore is formed by TM2 and metamizol increased b-endorphine liberation with fivefold maximum surrounded by TM1 helices which are connected with a large ectodo- at 45 min, which faded to control levels at 240 min, (iii) metamizol main by the W50-V61 and F324-I337 linkers forming an extracellular diminished K+-evoked release of glutamate and aspartate, (iv) vestibule. The aim of this study was to identify residues in this region metamizol did not change distension-induced ATP release. However that are of importance for the P2XR function. All residues of external metamizol inhibited pelvic sensory nerve distension-induced firing in vestibule segments of rat P2X4R were substituted one by one with + + - - P2X3 / but not P2X3 / rats. alanine or cysteine, the wild-type and mutant receptors were expressed Conclusion: metamizol analgesic action involves central and in HEK293 cells, and examined using whole-cell recording. This peripheral mechanisms. scanning analysis revealed that V49, Y54, Q55, F324 and G325 mutants were low-responsive, whereas all other mutants were functional. The expression of these mutants was examined by biothinylation that PKMf maintains drug reward, aversion and extinction showed absence of V49 mutants in the membrane. The receptor function was preserved in V49L, Y54F and G325P mutants, whereas Q55E, memories Q55T and Q55N mutants were non-functional. Ivermectin, a positive allosteric modulator of P2X4R, was unable to restore of the function of Yan-qing Li, Yan-xue Xue, Yin-yin He, Fang-qiong Li, Li-fen Xue, Q55 and G325 mutants. These experiments suggest that conserved Chun-mei Xu, and Lin Lu* residues Q55, adjanced to TM1, and G325, distal to TM2, are essential for P2X4R function. Glutamine in position 55 could interact with some National Institute on Drug Dependence, Peking University, Beijing partner in its vicinity to form the proper three-dimensional structure of 100083, China the vestibule in the functional receptor. It is also possible that glycine in *Corresponding author E-mail: [email protected] position 325 operates as a hinge during conformational changes of vestibule, transducing the signal from ectodomain to TM2. During abstinence, memories of drug-associated cues persist for many This study was supported by the grants No. IAA500110910, months and exposure to these cues often provokes relapse to drug use. 305/07/0681, 305/08/H037, the Grant Agency of Charles University The mechanisms underlying the maintenance of these memories are (Grant No. 3446/2011) and the Centrum for Neuroscience (LC554). unknown. Here, we used conditioned place preference (CPP) and conditioned place aversion (CPA) procedures to study the role of PKMf in the maintenance of drug reward and aversion memories in rats. We also investigate the role of PKMf intheextinctionmemoryofrewardand Roles of oxytocin in the control of social recognition aversion cues. We found injections of the PKMf inhibitor ZIP into accumbens core but not shell after CPP training blocked the expression Tatsushi Onaka, and Yuki Takayanagi of morphine, cocaine and high-fat food CPP for up to 14 days after injections but had no effect on CPA induced by naloxone-precipitated Division of Brain and Neurophysiology, Department of Physiology, morphine withdrawal. On the contrary, intra-basolateral amygdala but Jichi Medical University, Shimotsuke-shi, Tochigi-ken 329-0498, not central nucleus of the amygdala injection of ZIP 1 day after CPP and Japan CPA training impaired the expression of CPP and CPA for up to 14 days. The ZIP’s effect was mimicked by the PKC inhibitor chel- Oxytocin and its receptors have been shown to be implicated in social erythrine that inhibits PKMf, but not by the conventional and novel recognition. Mice lacking the oxytocin or oxytocin receptor gene

123 12th International Congress on Amino Acids, Peptides and Proteins S47 show socio-behavioral deficits, including impaired social recognition. The accessibility in the external part Administration of oxytocin improves social recognition, while oxy- of the transmembrane domain 5 of the glutamate tocin receptor antagonists impair social recognition. On the other hand, secretin receptors have also been reported to be involved in transporter EAAT1 is conformationally sensitive social recognition. Secretin receptor-deficient mice show impaired during the transport cycle social recognition. Secretin administration has been shown to increase oxytocin mRNA in the hypothalamus. However, relationship between Xiuping Zhang1 and Shaogang Qu2* oxytocin and secretin concerning social recognition remains to be determined. 1China-America Cancer Research Institute, Guangdong Medical Here, we examined effects of secretin administration upon College, Dongguan, Guangdong, China 2Department of Immunology, expression of Fos protein in oxytocin-secreting neurons. An intra- Southern Medical University, Guangzhou, Guangdong, China cerebroventricular injection of secretin induced expression of c-Fos *Corresponding author E-mail: [email protected] protein in oxytocin-secreting neurons of the hypothalamus and facilitated oxytocin release into the blood. Secretin also facilitated Excitatory amino acid transporter 1 (EAAT1) is a glutamate trans- oxytocin release from the isolated supraoptic nucleus. porter which is a key element in the termination of the synaptic We then investigated whether secretin increases social recogni- actions of glutamate. Moreover, it serves to keep the extracellular tion, and whether the effects of secretin are blocked by an oxytocin glutamate concentration below neurotoxic level. However the change receptor antagonist. Secretion facilitated social recognition and the of accessibility of transmembrane domain (TM) 5 during the transport facilitative effects of secretin were blocked by an oxytocin receptor cycle is not clear yet. We used cysteine mutagenesis with treatments antagonist. These data suggest that secretin facilitates social recog- with MTSET to investigation the change of accessibility of TM5. nition via activation of oxytocin-secreting neurons. Cysteine mutants were introduced from position 291–300 of the cysteine-less version of EAAT1. We checked the activity of the mutants before and after treatments with MTSET, furthermore we also analyzed the effect of the substrate and blocker on the inhibition Spike timing-dependent long-term depression requires of cysteine mutants by MTSET. Inhibition of transport by MTSET presynaptic NMDA receptors was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally Antonio Rodrı´guez Moreno sensitive reactivity pattern. The sensitivity of L296C, G299C and K300C to MTSET was potentiated by glutamate and TBOA. Our University Pablo de Olavide, 41013 Sevilla, Spain results indicate that the accessibility of some positions of the external part of the TM5 of EAAT1 is conformationally sensitive during the Spike timing-dependent plasticity (STDP) is a strong candidate transport cycle. synaptic mechanism involved in cortical development and map plasticity. In STDP, the temporal order and precise timing of pre- and postsynaptic action potentials (spikes) determine the direction The effect of glutathione and its analogue UPF17 and magnitude of synaptic change. Both timing-dependent long- on activity of frontal cortical Na,K-ATPase term potentiation (t-LTP) and timing-dependent long-term depression (t-LTD) depend on NMDA receptors. How the same type of of Alzheimer’s disease in vitro receptor can be involved in opposite changes in synaptic efficacy is not well understood. Whereas it is established that postsynaptic Ceslava Kairane, R. Mahlapuu, U. Soomets, and M. Zilmer NMDA receptors are necessary for t-LTP, we investigated here whether presynaptic NMDA receptors are necessary for timing- Department of Biochemistry, Medical Faculty, University of Tartu, dependent LTD and LTP in layer (L) 4-to-L2/3 excitatory synapses The Centre of Excellence for Translational Medicine, 19 Ravila St, in mouse barrel cortex. We used paired whole-cell recordings of 50411 Tartu, Estonia synaptically connected L4 and L2/3 cells. In five pairs, a prebefore- post pairing protocol was applied with 1 mM MK-801 in the pre- Glutathione (GSH) carries an important role in the human body synaptic pipette. Robust t-LTP was induced (154 ± 5%, n = 5; antioxidant defense system, as the most prominent low-molecular p \ 0.01, t test), of similar magnitude to that seen with extracel- weight thiol that occurs in millimolar ranges in the cells. Various lular stimulation (153 ± 9%, n = 5), suggesting that presynaptic structural modifications in tripeptidic GSH molecule have been car- NMDA receptors are not neccessary for induction of t-LTP. In ried out to improve its stability and cellular uptake. In our lab small contrast, t-LTD was completely blocked when MK-801 was library of glutathione analogues (UPF peptides) was designed and included in the presynaptic pipette (104 ± 6%, n = 6), whilst in synthesized. UPF17 is a tetrapeptide in which O-methyl-L-tyrosine is pairs of cells without MK-801 this protocol induced robust t-LTD added to the N-terminus of GSH and characteristic c-glutamate bond (76 ± 6%, n = 6; p \ 0.01, t test), indicating that presynaptic is changed to the a-glutamate bond. NMDA receptors are necessary for t-LTD. The different sites of In present study we have examined the effects of the GSH and its NMDA receptors necessary for induction of t-LTP and t-LTD may analogue UPF17 to frontal cortical Na,K-ATPase activity in human have important consequences for the computational operation of cortical microcircuits and map.

123 S48 12th International Congress on Amino Acids, Peptides and Proteins brain without and with Alzheimer‘s diagnosis in vitro. Results showed glutamatergic transmission and supplies neurons with precursors of that GSH at physiological concentrations inhibits the activity of healthy the main brain antioxidant, glutathione. This last function may be controls Na,K-ATPase in a time and concentration dependent manner, crucial for dopamine (DA) neurons that degenerate in Parkinson’s but UPF17 inhibits in a concentration dependent and time independent disease. Indeed, we had previously found that EAAT dysfunction manner. In Alzheimer’s disease brain preparations GSH showed only a induced by L-trans-2,4-pyrrolidine dicarboxylate (PDC), a substrate tendency of concentration and time dependent inhibition of Na,K- inhibitor of EAATs, is preferentially toxic for these neurons, as the ATPase activity meanwhile 20% inhibition of Na,K-ATPase by UPF17 subsequent decline in antioxidant defenses increased their vulnera- did not depend on peptide concentration nor incubation time. Coin- bility to NMDA receptor-mediated excitotoxicity. Here, we further cubation of UPF17 with cardiac glycoside ouabain increased inhibition investigate the interactions between PDC and glutamate induced of Na,K-ATPase indicating to the different ouabain and UPF17 binding toxicity in immature (8-day-old) and mature (12-day-old) mesence- sites on protein. phalic cultures. Glutamate (500 lM) was not toxic for immature DA neurons, indicating that they do not rely on the cystine/glutamate exchanger, system xc-, for their antioxidant defenses, even in immature stages. On the contrary, glutamate was toxic for mature DA The inhibitory effect of glutamate on the survival neurons and for both immature and mature non DA neurons. In promoting effect and signaling of IGF-1 in vitro immature non DA neurons, glutamate-induced cell loss was inde- and in vivo pendent of excitotoxicity and of oxidative glutamate toxicity, as not being protected by glutamate antagonists or antioxidants. It was prevented by inhibiting glutamate transport through either EAATs (by Zheng Wenhua PDC or DL-threo-b-benzyloxyaspartic acid) or system xc- (by 4-carboxyphenylglycine), but not by the combination of both treat- Neuropharmacology, School of Pharmaceutical Sciences, ments. In mature mesencephalic cultures, glutamate excess was Sun Yat-Sen University, East Waihuan Road, Higher Education Mega primarily excitotoxic. The protective effect of glutamate transport Center, Guangzhou 510006, Guangdong, People’s Republic of China inhibitors on glutamate toxicity was no longer observed in mature non DA neurons, but surprisingly appeared in mature DA neurons. These Impairing intracellular signalling induced by excess glutamate play data suggest that a moderate glutamate transport is required for the important roles in the process of neurodegenerative diseases. However, viability of immature non DA and mature DA neurons in mesence- the underlying mechanism(s) and its interrelationships with neuro- phalic cultures, and that both excessive glutamate transport and trophinc factors signaling are most unknown at present. We have shown complete blockade are toxic for these cells. This may be linked to here that glutamate attenuated the tyrosine phosphorylation of the IGF-1 oxidative deamination of glutamate into alphaketoglutarate, a TCA receptor and the survival effect of IGF-1 in hippocampal cultured cycle intermediate, by glutamate dehydrogenase as both inhibition neurons. Pre-treatment of cultured hippocampal neurons with glutamate and excessive activation of this enzyme have been found to be toxic concentration-dependently inhibited the tyrosine phosphorylation of for DA neurons. IGF-1 receptors as well as that of IRS-1 and Shc. The effect of glutamate was also evident on the phosphorylation of Akt, as well as its upstream kinase PI3K/PDK1 and downstream targets, GSK3b and FOXO3a. Moreover, these inhibitory effects of glutamate on IGF-1 were blocked Time-dependent alterations of NMDAR function by antagonists of the NMDA receptor but not by blockers of other ionotropic or metabotropic glutamate receptor sub-types verifying the and synaptic plasticity by corticosterone in the adult involvement of the NMDA receptor. These ﬁndings demonstrate that hippocampus glutamate can block the effect of IGF-1 by decreasing IGF-1 receptor suvival signalling in vitro. To verify this hypothesis further in vivo, Tak Pan Wong similar experiments were performed in mouse brain and in ischemia mouse brain. Our results demonstracted that glutamate also inhibited the Department of Psychiatry, McGill University, Douglas Mental Health survival signaling of IGF-1 in ischemia brain via NMDA receptor. University Institute, Canada Moreover, inhibition of NMDA receptor enhanced the phosphorylation of IGF-1 receptor in ischemia brain and the protective effect of IGF-1. Stress has an important impact on learning and memory, in part, Put together these results suggest a novel mechanism by which gluta- through the actions of adrenal corticosterone (CORT). However, the mate can reduce cell viability and induce neurotoxicity via affecting the mechanisms underlying the effects of CORT on synaptic plasticity, a survival signaling of growth factors. cellular model of learning and memory, remain elusive. Here we Supported by National Natural Science Fund of China (No. provide evidence that CORT can mediate time-dependent changes in 30670652; No. 30711120565; No. 30970935). synaptic N-methyl-D-aspartate receptor (NMDAR) function, a key player in synaptic plasticity. We found that stress level CORT applied to adult hippocampal slices (3-month-old) potentiated evoked syn- The puzzling problem of glutamate transport for DA aptic responses mediated by NMDARs within 30 min. Surprisingly, following this fast-onset change, we observed a slow-onset ([1h neurons: when too much glutamate uptake becomes after the end of CORT exposure) change in the GluN2 subunit also toxic composition of NMDARs, so that the GluN2A/GluN2B ratio was increased. To investigate the effects of these distinct fast- and slow- Laurence Had-Aissouni onset changes in NMDARs on synaptic plasticity, we examined the formation of long-term potentiation (LTP) and long-term depression IBDML, UMR 6216 CNRS-Aix Marseille University, Campus (LTD) within these time windows. In parallel to the increased of Luminy, Marseille, France NMDAR function found during CORT treatment, both LTP and LTD were facilitated. However, after the slow-onset change in NMDAR By transporting both glutamate and cysteine, the neuronal excit- subunit composition, synaptic plasticity was no longer facilitated. atory amino acid transporter EAAT3/EAAC1/SLC1A1 regulates These ﬁndings suggest that the delayed NMDAR subunit change after

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CORT treatment is related to the loss of LTP and LTD facilitation. receptors in the rat main olfactory bulb. The blockade of bulbar Our findings indicate that NMDARs in the adult hippocampus show vasopressin 1 receptors causes deficits in conspecific social recogni- remarkable plasticity in response to CORT. These CORT-mediated tion, and vasopressin administration leads to an inhibition of mitral alterations of NMDAR function play a critical role in stress regulation cell firing rate. We now have evidence for a large population of of learning and memory. vasopressinergic neurons and a high density of neurons expressing vasopressin 1a and 1b receptors across multiple subdivisions of the anterior olfactory nucleus (AON), an olfactory cortex that processes odor information and acts as a relay between the main olfactory bulbs Urocortin: past, present and a long road to go and higher processing areas. Unlike the glutamatergic vasopressinergic neurons in the olfactory bulb, neurons expressing vasopressin in A. Fatima1, G. Wolf2, M. Engelmann2, and M. G. Spina 2 the AON are GABAergic and co-express the calcium-associated protein calbindin-D-28K. Adult rats exposed to a conspecific juvenile 1Jamia Hamdard, Department of Pharmacology and Pharmacy show increased immediate early gene expression (early growth Practice, Delhi -110062, India response protein 1; Egr-1) in vasopressinergic neurons in the pars 2Otto-von-Guericke Universita¨t Magdeburg, Institut fu¨r Medizinische lateralis and pars distalis of the AON when compared with animals Neurobiologie, Leipziger Str. 44, 39120 Magdeburg, Germany exposed to no odor or a non-social odor control. Despite causing a general up-regulation of Egr-1 expression in the AON, predator odors Urocortin 1 (Ucn1) belongs to the mammalian corticotropin-releasing do not induce increases in Egr-1 expression in vasopressinergic factor family. In brain, Ucn 1 mRNA and peptide distribution is found neurons of the AON. We hypothesize that vasopressin signalling in highest in the non-preganglionic Edinger-Westphal nucleus (npEW). the MOB, intrinsically or via the AON, acts to filter recognized In addition urocortin is also produced in other areas of the rat brain conspecific social odor information, thereby facilitating the formation including the lateral superior olivary, hypothalamic supraoptic of short-term social odor memories. nucleus (SON), pituitary, and substantia nigra. Ucn 1 immunoreactive neurons project to the dorsal raphe and nucleus of the solitary tract which mainly express CRF2-receptors. In early reports, Ucn 1 was found to potently reduce feeding and increase anxiety after i.c.v Nutrition administration in lab rodents. Further, several studies suggested an involvement of central Ucn1 in neuroprotection, stress adaptation pathway, reproduction, body temperature, alcohol consumption Adipose tissue glutamine synthesis and the development preference as well as learning and memory. Also the roles specific brain regions that produce Ucn1 play were explored to identify the of inflammation and insulin resistance neuronal networks that are involved in the physiological functions of this neuropeptide. Indeed, in these networks Ucn1 was found to co- Malcolm Watford, Roshni Patel and Samantha Dori localise and act in concert with other neuropeptides and neuromod- ulators. We investigated here the role of Ucn1 in the SON. We found Department of Nutritional Sciences, Rutgers University, that in the SON both Ucn1 and neuronal nitric oxide synthase are co- New Brunswick, NJ, USA localised. Administering low doses of Ucn1 suggest a role of neuropeptide in controlling anxiety-related behaviour within the SON Adipose tissue expresses very high glutamine synthetase (GS) area. Further, our results indicate Ucn1 signalling within this hypo- activity that is increased during diet-induced obesity in mice. thalamic nucleus has little impact on feeding behaviour. In contrast, a During the differentiation of 3T3-L1 cells into adipocyte-like reduced immunoreactivity in npEW during pregnancy implies Ucn1 cells, expression of GS is increased [50-fold with higher mounts could contribute to an altered feeding behaviour and energy homeo- of GS protein seen within 24 h of the initiation of differentiation. stasis observed during pregnancy. Plotted on the background of its Exogenous glutamine is not required for the differentiation of selective distribution (versus that of its receptors) in the rodent brain 3T3-L1 cells into adipocytes if they continue to express GS. as well as the functional interaction with other neuropeptides and Knock-down of GS shows that monoclonal expansion, expression signalling pathways our findings provide a complex picture of how of C/EBPb, PPARc and C/EBPa, and maximal rates of lipid Ucn1 might affect in behavioural regulation by controlling physio- storage, all require glutamine. Given the relatively poor blood logical parameters. supply in adipose tissue, we propose that adipose tissue GS provides glutamine that acts in a paracrine manner to maintain adipocyte health. During obesity large adipocytes secrete cytokines, such as MCP1, that result in macrophage infiltration leading Vasopressinergic neurons in the anterior olfactory to inflammation and insulin resistance. Glutamine is the major nucleus of the rat: a role in social odor processing respiratory fuel of macrophages, but they express very low GS activity and cannot survive without large amounts of exogenous Douglas W. Wacker*, Vicky A. Tobin, Julia Noack, glutamine. However, when macrophages are cultured over mature Adrian J. Duszkiewicz, Valerie R. Bishop, Simone L. Meddle, adipocytes they are able to survive in the absence of exogenous Mario Engelmann and Mike Ludwig glutamine providing that the adipocytes express glutamine synthetase. Macrophages do not survive in the absence of exogenous *Centre for Integrative Physiology, University of Edinburgh, glutamine when they are cultured over pre-adipocytes, or over Edinburgh, UK EH8 9XD and Department of Molecular adipocytes where GS has been inhibited. We propose that large and Integrative Physiology, University of Michigan, Ann Arbor, MI adipocytes provide glutamine, not only to maintain adipocyte 48109, USA function but also as a fuel for infiltrating macrophages. Therefore, adipose tissue glutamine synthesis plays a major role in the We recently identified a large population of vasopressinergic neurons, development of obesity induced inflammation and insulin resis- as well as neurons immunopositive for vasopressin 1a and 1b tance.

123 S50 12th International Congress on Amino Acids, Peptides and Proteins

Chinese Meishan pigs: an important resource Methods: The digesta in the GI tract and the feces were subjected to for deﬁning genomic regulation of swine western blotting and the morphological observation using immuno- electron microscopy. Furthermore, by using the in vitro digestion reproduction method with pepsin (2 h) followed by pancreatin (6 h), the treatment of indigestible starch degraded-rice protein (SD-RP) with dithio- J. J. Ford threitol (DTT) and urea was employed to see molecular interactions in protein body-I (PB-I) affecting prolamin digestibility. USDA/ARS/US Meat Animal Research Center, Clay Center, NE Results: In the in vivo digestion method, the band intensity of 13 kDa 68933 prolamin (13P) in the GI digesta, especially the caecum/colon contents, and the feces of RF group was decreased compared with that of Chinese breeds of pigs are phenotypically diverse from European CR group. In the in vitro digestion method, a high dose of DTT breeds. Of the many breeds that exist in China, the Meishan breed (1.5 M) improved prolamin digestibility of SD-RP, while urea (8 M) was imported into other countries for use in research with a pri- could have an enough effect only by combining with a low dose of mary interest being their large litter size. In a 2009 review, Onteru DTT (200 mM). These results suggest that rice prolamin becomes et al. noted 34 quantitative trait loci (QTL) for female reproductive indigestible after cooking and the intermolecular interaction in PB is traits reported in pigs. Of these, 20 were identiﬁed in Meishan important to make prolamin indigestible. crossbred lines. Of the 19 candidate genes that had an association with female reproductive traits, associations were observed for 12 of these in Meishan crossbreds. In boars, QTL for plasma concentrations of follicle stimulating hormone (FSH) and testicular Dietary arginine supplementation altered expression size were observed on the X chromosome, and thyroid-binding globulin (TBG) emerged as the potential gene associated with these of IGFs and IGF receptors in weaning piglets differences. Boars with the Meishan allele for TBG experience pubertal development at a younger age, have smaller mature testes Rongjun Chen1,2*#, Wence Wang2,3#, Yulong Yin2*, Kang Yao2, with fewer Sertoli cells, greater expression of inhibin/activin beta-B Yunling Gao2,3, and Tiejun Li2 subunit and FSH beta subunit in their anterior pituitary glands, and more FSH in their blood than boars with the European allele. A 1Rice Research Institute of Sichuan Agricultural University, model to explain these differences is that fewer Sertoli cells in Chengdu, Sichuan 611134, China boars with the Meishan allele for TBG provide lower secretion of 2Key Laboratory of Animal Nutritional Physiology and Metabolic inhibin thereby providing less negative feedback to the anterior Process and Key Laboratory of subtropical Agro-ecology, Institute pituitary allowing for greater synthesis of activin beta-B that of Subtropical Agriculture, the Chinese Academy of Sciences, stimulates greater synthesis of FSH within the pituitary and its Changsha, Hunan 410125, China greater secretion into the blood. The unusually high concentrations 3The Graduate School of The Chinese Academy of Sciences, Beijing of FSH observed in Meishan boars were the primary stimulus for 100039, China this series of studies. To whom correspondence should be addressed: Yulong Yin. E-mail: [email protected]. Tel:86-73184619703 fax: 86-731-84612685; [email protected] Cooking causes the deterioration of prolamin #Rongjun Chen and Wence Wang contribute equally to this paper [email protected]; [email protected] digestibility in rice [email protected]; [email protected]

Masatoshi Kubota1, Yuhi Saito2, Takehiro Masumura2, Early weaning may lead to the stress syndrome and increased occur- Takehisa Kumagai3, Reiko Watanabe4, Shinobu Fujimura1,5, rence of enteric diseases and diarrhea in nursery pig management. and Motoni Kadowaki1,5 Dietary Arginine supplementation may decrease the severity of the weaned stress syndrome in early-weaned piglets. The insulin-like 1Center for Transdisciplinary Research, Niigata University, Niigata, growth factor (IGF) signaling pathway is an important regulatory Japan factor in regulating fetal and placental growth, proliferation, differ- 2Graduate School of Life and Environmental Science, Kyoto entiation, migration and aggregation, and inhibit apoptosis of Prefectural University, Kyoto, Japan mammalian cells. However, whether insulin-like growth factor system 3Kameda Seika Co., Ltd., Niigata, Japan expression is changed in piglets with Dietary Arginine supplementa- 4Department of Health and Nutrition, University of Niigata tion is unclear. This study was conducted to investigate the effect of Prefecture, Niigata, Japan dietary arginine supplementation in modulation IGF system of wean- 5Graduate School of Science and Technology, Niigata University, Japan ling piglets. Twelve, 21-day-old healthy piglets (Landrace 9 Yorkshire) with a mean body weight (BW) were divided into 2 groups Background and Objective: Prolamin is one of the major storage randomly. The test group was supplemented with 0.6% L-arginine, the proteins in rice and notoriously indigestible. However, there is no control group was fed with 1.23% L-alanine (isonitrogenous control). direct evidence that cooking changes rice prolamin digestibility and At 28 days of age, 12 piglets were killed and longissimus muscle, liver few knowledge about the molecular mechanisms of prolamin indi- and kidney were collected. IGF-I was increased in three tissues of gestibility. Consequently, we attempted to investigate the effect of arginine group (P \ 0.05). IGF-II was increased in muscle of arginine cooking on the in vivo digestibility of rice prolamin by determining group. Both muscle and liver have a higher level of IGFBP5 with the gastrointestinal (GI) transit of rice protein in rats fed either a rice arginine supplementation (P \ 0.05). These results showed that argi- ﬂour (RF)- or cooked rice (CR)-based diet and the mechanism to nine can alleviate weaning stress and improving growth performance in cause prolamin indigestible by an in vitro digestion method. early-weaned piglets through IGFs and IGF receptors.

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Dietary L-Arginine supplementation affects protein The purpose of this study was to test the hypothesis that the dietary L- expression in insulin-sensitive tissues of diet-induced arginine supplementation had beneficial effects on edema disease. 156 KunMing mice were randomly assigned to arginine group 1 (0.6% obese rats arginine + basal diet, n = 44), arginine group 2 (0.6% arginine + basal diet, n = 44), control group 1 (1.22% alanine + basal diet, n = 34) and Jun-Jun Wang1,2, Wenjuan Shi Jobgen2, Cynthia J. Meininger3, control group 2 (1.22% alanine + basal diet, n = 34). After 3 days of and Guoyao Wu1-3 adaptive feeding and a 7 days treatment period with the prepared feed, all mice were challenged by intraperitoneal injection Escherichia coli 1State Key Laboratory of Animal Nutrition, China Agricultural O139 (E. coli) at LD50 (2.53 9 108 CFU/ml). Arginine group 2 and University, Beijing 100193, China control group 2 were used to calculate the mortality after 20 h of 2Department of Animal Science, Texas A&M University, College injection. Serum concentrations of platelet-activating factor (PAF), Station, TX, 77843, USA interleukin (IL)-2, interleukin (IL)-10, secretory immunoglobulin A 3Department of Systems Biology and Translational Medicine, Texas (sIgA), superoxide dismutase (SOD) activity, total antioxidant capac- 0 0 A&M Health Science Center, College Station, TX, 77843, USA ity(T-AOC), cyclic 3 ,5 -adenosine monophosphate (cAMP), cyclic 0 0 3 ,5 -guanosine monophosphate (cGMP) were measured in arginine This study was conducted to test the hypothesis that dietary arginine group 1 and control group 1 in a 10-h interval for three times. The serum supplementation reduces fat mass in diet-induced obese rats. Male concentration of PAF was much lower (P \ 0.01) in arginine group Sprague–Dawley rats were fed either low- or high-fat diets for 15 weeks than alanine group in all times. Additionally, T-AOC and SOD activity (16 rats/diet). Thereafter, lean or obese rats continued to be fed their same in the experiment group were increased significantly (P \ 0.05) in the respective diets and received drinking water containing either 1.51% L- first 10 h after initial injection. Unfortunately, T-AOC and SOD activity arginine-HCl or 2.55% alanine (isonitrogenous control) (n = 8/treat- in arginine group become quiet (P [ 0.05) compared to the control ment group). Twelve weeks after the initiation of the arginine treatment, group after that except T-AOC, was greater (P \ 0.05) in arginine rats were euthanized to obtain tissues for biochemical analyses. Results group than the control group in 20 h of initial injection. Meanwhile, were statistically analyzed as a 2 9 2 factorial experimental design using arginine supplementation had little effect on the mortality of mice, ANOVA. High-fat diet increased (P \ 0.05) the mass of white adipose serum IL-2, sIgA, cAMP, cGMP level. In conclusion, dietary arginine tissues at different anatomical locations by 49–96% compared to the low- supplementation can partially attenuate the damage caused by edema fat diet. L-Arginine supplementation reduced (P \ 0.05) white adipose disease, but have little effect on the clinical result. tissue mass by 20–40% while increasing brown adipose tissue mass by Keywords: Arginine, Edema disease, Escherichia coli, Platelet- 15–20% and enhancing glucose and oleic acid oxidation in skeletal activating factor muscle (P \ 0.05). Proteomics analysis revealed that expression of creatine kinase in skeletal muscle was decreased (P \ 0.05) by high fat diet but increased (P \ 0.05) by dietary arginine supplementation. Ha- patic glutathione peroxidase was more abundant in arginine- supplemented rats, compared with nonsupplemented rats. Collectively, Effect of dietary leucine to lysine ratios on performance these results indicate that arginine regulates energy metabolism and of growing pigs antioxidative responses in a tissue-specific manner. The findings have important implications for treating obesity in humans and companion 1 2 2 animals as well as decreasing fat deposition in livestock species. John K. Htoo ,Hećtor Garcia , Miguel Cervantes , 2 2 Supported by AHA and the China Thousand-People Talent program. Adriana Morales , and Alfonso B. Araiza

1Evonik Degussa GmbH, 63457 Hanau-Wolfgang, Germany 2 ´ ´ Effect of dietary L-arginine supplementation on edema Instituto de Ciencias Agrıcolas, UABC, Mexicali, Mexico disease Leucine (Leu) appears to regulate protein synthesis, but an excess of Leu can reduce feed intake and performance of pigs. To Wenkai Ren1,4,*, Guan Yang2,*, Xiangwei Tang 2, *, Yinghui Li 1,4, determine the optimal dietary standardized ileal digestible (SID) Gang Liu1, ,Sisi Cai 3,YulongYin1, ,XinglongYu5, and Guoyao Wu6 Leu:Lys ratio in growing pigs, a 3-week study was conducted with 90 pigs (28.1 ± 0.4 kg) with 8 pen replicates per treatment. A 1Research Center for Healthy Breeding of Livestock and Poultry, wheat and wheat bran based diet was formulated, using analyzed Hunan Engineering and Research Center of Animal and Poultry ingredient amino acid (AA) contents to meet requirements of SID Science and Key Laboratory for Agro-ecological Processes AA other than Leu (0.68%) and Lys (0.77%). L-Leu was added to in Subtropical Region, Institute of Subtropical Agriculture, the basal diet to create 5 SID Leu:Lys (88, 100, 120, 140 and the Chinese Academy of Sciences, 410125 Hunan, China 160%). Diet 6 was produced to be equivalent to diet 4 but con- 2College of Animal science & Technology, Hunan Agricultural tained 12% more Ile. Samples of intestinal mucosa, liver, and University, 410128 Changsha, Hunan, China muscles were collected from 8 pigs per each of treatments 1, 3 and 3Department of veterinary medicine, Orient Science & Technology 5, respectively. mRNA extraction was performed followed by College of Hunan Agricultural University, 410128 Changsha, Hunan, quantitative expression of cationic AA transporters b0,+ and CAT-1. China There was a linear and quadratic response in ADG and a quadratic 4Graduate School of Chinese Academy of Sciences, Beijing 100039, response in FCR of pigs as the Leu:Lys increased from 88 to China 160% (p \ 0.01). Increasing SID Leu:Lys from 88 to 120% 5College of Veterinarian, Hunan Agricultural University, Changsha, increased (p \ 0.05) the expression of b0,+ and decreased Hunan 410128, China (p \ 0.10) CAT-1; no further response was observed when 6Department of Animal Science, Texas A&M University, increased to 160%. Two-slope linear regression estimated the College Station, TX, USA 77843 SID Leu:Lys of 104 and 108% to optimize ADG and FCR, *[email protected] or [email protected] respectively.

123 S52 12th International Congress on Amino Acids, Peptides and Proteins

Effect of immune system stimulation on whole body group. There wasn’t significant difference about amino acids in the protein deposition of growing pigs fed tryptophan serum but valine, which was significant lower than sedentary group’s. Movement training increased significantly all amino acids limiting diets content except cysteine in the intestinal tissue but failed in the liver and muscle, even though all amino acids were higher in the John K. Htoo1, Crystal L. Levesque2, Karl de Ridder2, muscle than sedentary group but cysteine. Amino acids’ digest- and Cornelis F.M. de Lange2 ibility was lower in exercised group and it became significant lower, when it comes to lysine and histidine. Additionally, 1Evonik Degussa GmbH, 63457 Hanau-Wolfgang, Germany Movement training decreased the numbers of lymphocyte but had 2Department of Animal and Poultry Science, University of Guelph, no effect on the goblet cell, villi height and crypt depth in middle N1G 2W1, Guelph, Canada jejunum.

Immune system stimulation (ISS) induced by disease is associated with reduced availability of tryptophan (Trp) for growth in pig, suggesting increased dietary requirement of Trp. The effect of (ISS) on nitrogen (N) retention in pigs fed Trp limiting diets was Effects of dietary arginine or N-carbamylglutamate evaluated using 36 pigs (20.0 ± 1 kg; 12 pigs/block). Pigs were supplementation on reproductive performance assigned to one of 5 diets and fed restrictively (800 g/day). Diets 1 and immunity of sows infected with porcine to 4 contained decreasing levels of SID Trp (1.30, 1.05, 0.80 and 0.55 g/kg), and Trp was first limiting. Diet 5 contained added Trp reproduction and respiratory syndrome virus (0.89 g/kg), and all other essential AA equivalent to diet 4. Fol- lowing a 5-day adaptation, pigs were injected every 2 days with E. De Wu*, Lianqiang Che, Ping Yang, Zhengfeng Fang, and Yan Lin coli lipopolysaccharide (20 lg/kg BW, increasing 15% each injection). Plasma acute phase protein increased (p \ 0.001) fol- Key Laboratory for Animal Disease Resistance Nutrition lowing ISS. There were no interactions of dietary Trp level and of the Ministry of Education of China, Animal Nutrition Institute, ISS. Nitrogen retention decreased linearly (p \ 0.001) with Sichuan Agricultural University, Ya’an 625014, Peoples Republic decreasing Trp intake. Whole body N retention was lower China (p \ 0.001) compared with Pre-ISS during ISS-1 due to an increase *To whom correspondence should be addressed. (p \ 0.001) in urinary N excretion. Adding Trp to diet 4 increased Tel: +86-835-2885107 Fax: +86-835-2885065 N retention (p \ 0.05; diet 4 vs. 5). Regression of response of E-mail: [email protected] protein deposition (PD) against Trp intake revealed that Trp requirement increased at 9.1% during ISS-1 compared with pre- This study investigated the effects of dietary supplementation with L- challenge for achieving the same PD. Arginine (Arg) or N-carbamylglutamate (NCG) on reproductive performance and immunity of sows infected with porcine reproduction and respiratory syndrome virus (PRRSV). One hundred of sows (Land- race 9 Large white) with body condition score at 3 received corn and Effect of movement training on the amino acids soybean-based control diet during the first 30 days of gestation and distribution and intestines morphosis in rats were allocated to 5 groups receiving control, 1% L-ArginineHCL (Arg114), 0.1% NCG (NCG114) diet until farrowing, whereas the rest 2 Min Gong1,2, Wenkai Ren4, Yulong Yin1,4,* Dehua Wang3, groups received 1% L-ArginineHCL (Arg90), 0.1% NCG (NCG90) Gang Liu 4,* and Guoyao Wu4,5 diet only until day 90 of gestation followed by control diet until farrowing. Litter performance was recorded at parturition. Blood samples 1State Key Laboratory of Food Science and Technology and College collected at days 30, 90 and 110 of gestation were measured for meta- of Life Science and Food Engineering, Nanchang University, bolic and immunological parameters. The RT-PCR detection of serum Nanchang 330031, China PRRSV showed sows were infected with PRRSV prior to experiment. 2Jiangxi Science & Technology Normal University, Nanchang At parturition, total litter size was not markedly affected by dietary Arg 330013, China or NCG supplementation. As a result of less pigs born dead, however, 3Nanchang Ctr Dis Control & Prevent, Nanchang 330038, Peoples sows in Arg114 group had more pigs born alive than sows in control Republic of China group (+1.6 pigs, P \ 0.05), total and live litter weights were increased 4Research Center for Healthy Breeding of Livestock and Poultry, (+1.7*2.3 kg, P \ 0.05) in Arg114 group relative to control and Arg90 Hunan Engineering and Research Center of Animal and Poultry groups. However, no significant litter size response to NCG supple- Science and Key Laboratory for Agro-ecological Processes mentation was observed. Compared with control group, likewise, in Subtropical Region, Institute of Subtropical Agriculture, dietary Arg but not NCG supplementation increased (+12*46%, the Chinese Academy of Sciences, 410125 Hunan, China P \ 0.05) the levels of ornithine, proline and arginine at both days 90 5Department of Animal Science, Texas A&M University, College and 110 of gestation. However, both dietary Arg and NCG supple- Station, TX, USA 77843 *[email protected] mentation decreased (-10*18%, P \ 0.05) the level of plasma urea at or [email protected] days 90 and 110 of gestation. In addition, dietary Arg but not NCG supplementation boosted humoral immunity as indicated by higher This work was conduct to study the effect of high intensity serum immunoglobulin and PRRSV antibody level in Arg-supple- movement training on the amino acids metabolism and distribution mented sows. These findings indicate dietary arginine supplementation in internal organ and intestinal morphosis. 40 Sprague–Dawley can improve litter performance and humoral immunity, and intriguingly male rats were randomly divided into exercised group and seden- the effect of arginine supplementation on fetal growth is evident in late tary group. On day 10 after the initiation treatment, average daily gestation. However, the positive effect of arginine supplementation on feed intake became significant lower, and the average weight pregnancy and immunity can not be substituted by the current dose of became significant lower on day 12, compared with the sedentary NCG.

123 12th International Congress on Amino Acids, Peptides and Proteins S53

Effects of peptides of soy isoﬂavones on the male g/kg CP. Intermediately degradable protein PB2 fraction also reproductive regulators in male Xiang Pigs decreased (P \ 0.05) with a range from 496 to 182 (oats), 579–213 (barley), 490–175 (corn) g/kg CP. The slowly degradable protein PB3 fraction increased (P \ 0.05) with a range from 83-294 (oats), Xiaoxue Yuan 1, Lili Li1*, Yulong Yin1*, Juexin Fan2, 3 3 153–300 (barley), 181–308 (corn) g/kg CP. Undegradable protein Chaowu Xiao , and Franc¸ois Blachier PC fraction increased (P \ 0.05) with a range from 24 to 132 (oats),

1 24–92 (barley), 24–132 (corn) g/kg CP. True protein decreased with Research Center for Healthy Breeding of Livestock and Poultry, a range from 349 to 560 (oats), 821–568 (barley), 815–560 (corn) Hunan Engineering and Research Center of Animal and Poultry g/kg CP. In conclusion, the protein fraction profiles of grains can be Science and Key Laboratory for Agro-ecological Processes significantly modified when combination with wheat DDGS. These in Subtropical Region, Institute of Subtropical Agriculture, changes will result in significant impact on degradable kinetics and the Chinese Academy of Sciences, 410125 Hunan, China 2 nutrient availability. College of Animal Science and Technology, Hunan Agricultural Keywords: Wheat-based dried distillers’ grains with soluble, University, Changsha, Hunan 410128, China Protein and carbohydrate fractions, Nutrient variation and availability 3Hlth Canada, Hlth Prod & Food Branch, Nutr Res Div, Food Directorate, Ottawa, Canada; 3USC 914 INRA/AgroParisTech, Nutrition Physiology and Alimentary Behavior, 16 rue Claude Bernard, 75005 Paris, France *[email protected] or [email protected] Expression and characterization of bovine lactoferrampin-lactoferricin in Pichia pastoris Three different doses of soy isoflavones were fed to male Black Small-eared pigs for 60 days (125, 250, 500 mg/kg diet) to evaluate Xiangshan Tang1,2, Zhiru Tang3 Shengping Wang1,2, the effects of soy isoflavones on male reproductive regulators in Zemeng Feng1,2, Dong Zhou1,2, Tiejun Li1 and Yulong Yin1* Black Small-eared pigs. The results shown that dietary supplementation with 250 mg/kg of soy isoflavones increased (P \ 0.05) 1Research Center for Healthy Breeding of Livestock performance, serum testosterone levels and mRNA expression of and Poultry, Hunan Engineering, Research Center of Animal testosterone biosynthesis regulatory protein StAR. However, a higher and Poultry Science and Key Laboratory for Agro-ecological doses of 500 mg/kg suppressed (P \ 0.05) the reproductive perfor- Processes in Subtropical Region, Institute of Subtropical mance of male pigs. Our finding suggested that soy isoflavones can Agriculture, the Chinese Academy of Sciences, 410125 Hunan, affect the male reproductive hormone secretion, the growth and China development of testis and epididymis, enzyme activity of testosterone 2Graduate University of the Chinese Academy of Sciences, Beijing synthesis, and expression of reproductive hormones’ gene in the 100039, China brain, and in dosage-dependant ways. 3College of Animal Science and Technology, Southwest University, Keywords: Soy isoflavones, Testicle, Reproductive hormone, Chongqing, 400715, China *[email protected] Testosterone synthesis, Male Xiang pig Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N1-domain of bovine Effect of wheat DDGS to grain ratio on changes lactoferrin. The bactericidal activity of fused peptide LFA-LFC was stronger than that of LFA or LFC. The high cost of peptide proof protein subfraction profiles in oats, barley and corn duction from either native digestion or chemical synthesis limited the clinical application of antimicrobial peptides. Expression of Daallkhaijav Damiran1, and Peiqiang Yu1* recombinant peptides in yeast may be an effective approach. At present, the expression of recombinant peptide LFA-LFC in yeast College of Agriculture and Bioresources, University was not reported. Here, we present the expression, purification and of Saskatchewan, Saskatoon (*Email: [email protected]) antibacterial activity of LFA-LFC using Pichia pastoris expression system. In our study, the gene of bovine lactoferrampin-lactoferri- The objective of this study was to investigate the effects of wheat cin (LFA268–284-LFC17–30) was obtained by PCR and cloned into dry distiller grains with solubles (DDGS) to cereal grain ratio on the vector pPICZaA. The SacI-linearized plasmid pPICZaA- LFA- changes of true protein and protein (CP) subfractions. The CP LFC was transformed into P. pastoris KM71 by electroporation and fractions were partitioned according to the Cornell Net Carbohydrate screened by PCR. The expression of LFA-LFC was induced about and Protein System and included True protein (TP), PA, PB1, PB2, 72 h with 0.5% methanol at 28°C, One milliliter samples of the PB3 and PC for protein subfractions. Each subfraction has different expression medium were taken at 24 h intervals for an activity degradation behaviour (degradation rate and extend) which are assay. Antibacterial activity of fifty microliters of each expression highly related to component nutrient availability. Three type of supernatant was assayed by measuring zones of growth inhibition grains with wheat DDGS samples from two bioethanol plants were in thin agar plates with E. coli 0149. Fifty microliters of super- mixed manually to combine in ratios of 4:0, 3:1, 2:2 and 1:3. This natant from cultures was freeze-dried and dissolved in 2 mL sterile has resulted in total of 24 (3 9 2 9 4 combinations of each) mix- water; Cation-exchange chromatography and size-exclusion chro- tures. The results show that the effect of wheat DDGS inclusion had matography were used for Purification of recombinant LFA-LFC. significant effects (P \ 0.05) on CP fractions in oat, barley and LFA-LFC was collected and 16.5% Tricine-SDS-PAGE was per- corn. Within wheat DDGS inclusion rate increased (0:4, 1:3, 2:2, formed. Molecular weight of recombinant LFA-LFC was analyzed 3:1), soluble protein PA fraction increased (P \ 0.05) with a range by MALDI-TOF mass spectrometry. The direct sequencing results from 227 to 308 (oats), 156–301 (barley), 162–308 (corn) g/kg CP. indicated that the integrity of LFA-LFC recombinant was correct. But rapidly degradable protein PB1 fraction decreased (P \ 0.05) Zones of growth inhibition of samples taken from the culture were with a range from 181 to 85 (oats), 89–20 (barley), 144–77 (corn) 1.8 ± 0.3, 5.3 ± 0.7, 8.8 ± 0.7 mm at 24, 48 and 72 h,

123 S54 12th International Congress on Amino Acids, Peptides and Proteins respectively. Tricine-SDS-PAGE and mass spectrometry analyses Glutamate: nutrition, metabolism and signaling demonstrated that the molecular weight of the purified LFA-LFC was 4.0 kDa, which is consistent with the theoretical molecular John T. Brosnan and Margaret E. Brosnan weight (3,953.75 Da). Our results showed that Pichia pastoris was a suitable system for secreting LFA-LFC. Bioactivity assay Memorial University of Newfoundland, St. John’s, NL, Canada proved that the recombinant LFA-LFC had antimicrobial effects. Recombinant antimicrobial peptide LFA-LFC may serve as an Glutamate is a central amino acid, both with regard to carbon and attractive candidate for the development of antimicrobial nitrogen metabolism. It also occurs at very high concentrations in the preparations. brain where it is the major inhibitory neurotransmitter. Glutamate is an Keywords: Bovine lactoferricin, Bovine lactoferampin, Antimi- important component of the taste of protein-rich foods. As monoso- Pichia pastoris crobial peptides, dium glutamate it is widely used to impart a distinctive savory flavor to many foods. Glutamate is an abundant amino acid in most proteins, and therefore ingested in substantial quantities. However, nearly all Functional amino acids in nutrition and health dietary glutamate is metabolized by the intestine in the first pass. The liver is efficient in removing any dietary glutamate that escapes intestinal metabolism. Consequently, almost all tissue glutamate must Guoyao Wu be synthesized within the body. Recent work has centered on glutamate as a signaling agent. The identification of umami receptors Department of Animal Science and Faculty of Nutrition, Texas A&M (requiring both glutamate and a purine nucleotide such as IMP) in taste University, College Station, Texas, USA 77843; State Key laboratory buds may provide insights into a dietary protein receptor. Very of Animal Nutrition, China Agricultural University, Beijing 100193, recently, glutamate receptors have been found throughout the gastro- China intestinal tract. Glutamate dehydrogenase (GDH) plays a key role in the increased insulin secretion that occurs upon ingestion of a protein- Based on nitrogen balance or growth, amino acids (AA) were rich meal. Elevated leucine activates GDH in the beta-cells, causing traditionally classified as nutritionally essential or nonessential for increased ATP production from increased glutamate oxidation. This animals and humans. Nutritionally essential AA (EAA) are those increased [ATP] results in increased insulin secretion. A genetic dis- AA whose carbon skeletons are not synthesized by animal or order, HI/HA Syndrome, in which GDH is constitutively active causes human cells and, therefore, must be provided in the diet. Nones- hyperinsulinemia and hypoglycemia in children. sential AA (NEAA) are those AA which are adequately synthesized de novo to meet the needs by the organisms. Synthesis and catabolism of AA depend on species and developmental stages. Therefore, dietary requirements of AA vary greatly among mam- Highly efficient production and product design mals (including humans, pigs, cats, and dogs), birds, and fish. It of VECYGPNRPQF, a potent antioxidative peptide can had long been tactically assumed that animals or humans could synthesize sufficient amounts of all NEAA and did not need them efficiently quench a variety of free radicals in diets for optimal nutrition or health. However, a careful examination of the literature does not substantiate this assumption. Zemeng Feng1,2, Xiaoli Zhou3, Tiejun Li1, and Yulong Yin 1* Growing evidence from animal studies shows that: (1) some of the NEAA (e.g., arginine, glutamine, glutamate, glycine, and proline) 1Research Center for Healthy Breeding of Livestock and Poultry, and leucine play important roles in multiple signaling pathways, Hunan Engineering and Research Center of Animal and Poultry thereby regulating gene expression, intracellular protein turnover, Science and Key Laboratory for Agro-ecological Processes nutrient metabolism, and oxidative defence; (2) young or gestating in Subtropical Region, Institute of Subtropical Agriculture, mammals cannot synthesize sufficient amounts of all NEAA to the Chinese Academy of Sciences, 410125 Hunan, China support maximum neonatal growth or embryonic/fetal survival; and 2Graduate University of the Chinese Academy of Sciences, Beijing (3) some NEAA (e.g., arginine) can reduce adiposity and amelio- 100039, China *[email protected] rate cardiovascular dysfunction in genetically obese and diet- induced obese animals. Beneficial roles for arginine and glutamine Oxygen is essential for the survival of aerobic organisms. Through in improving metabolic profiles, cardiovascular function, immune oxidative phosphorylation, organisms obtain large amounts of energy responses, and intestinal health have also been reported for supply for metabolism, with producing reactive oxygen species (ROS) humans. Clearly, cell- and tissue-specific functions of AA beyond as a price. ROS have roles in cell signaling, bits of ROS are benefit for protein synthesis should be taken into consideration in recom- the health. But overmuch ROS can induce oxidative stress. When in mendation of nutrient requirements for animals and humans. oxidative stress, an uncontrolled fashion causes significant reversible Recognizing that the long-standing classification of AA as EAA or or irreversible damage to a wide range of biological molecules causing NEAA has major conceptual limitations in protein nutrition, we a cascade of chain reactions resulting in cellular damage and disease. have recently developed the concept of functional AA (FAA) to Tissue damage caused by oxidative stress has a role in a number of capitalize on the new developments of this field. FAA are defined pathophysiological conditions including neurodegenerative disorders as those AA that regulate key metabolic pathways to improve (Alzheimer’s and Parkinson’s diseases), diabetes mellitus, athero- health, survival, growth, development, lactation, and reproduction sclerosis, autoimmune disease, and ischaemia/reperfusion injury. A of organisms. Recent advances in FAA are the focus of the session potent anti-oxidative peptide (VECYGPNRPQF, in this paper was on ‘‘Functional Amino Acids in Nutrition and Health’’ at the 12th named AOP) was isolated from microalgae, Chlorella vulgaris,by International Congress on Amino Acids, Peptides and Proteins at Sheih in 2009, which efficiently quench a variety of free radicals, Beijing, China between August 1 and 5, 2011. including hydroxyl radical, superoxide radical, peroxyl radical, etc. In This mini-symposium was supported, in part, by Ajinomoto Inc. our research, AOP and recombinant Tat-AOP were highly efficient (Tokyo, Japan) and National Renderers Association (Alexandria, expression using Pichia pastoris KM71H fermentation. To avoid USA). generating excrescent amino acid, Red/ET recombination was applied

123 12th International Congress on Amino Acids, Peptides and Proteins S55 to construct the expression vector pPICZaA-AOP and pPICZaA-Tat- signalling of food-derived AGE from soy sauce, a Japanese condiment AOP by insertion a PCR amplified DNA fragment coding AOP or Tat- produced by fermenting soybeans. We first fractionated soy sauce into AOP into plasmid pPICZaA, at the position right after the cleavage higher ([5,000) and lower (\5,000) molecular-weight fractions. Lower sequence of Kex2. The vector pPICZaA-AOP and pPICZaA-Tat-AOP molecular-weight fractions (LMF) was found to inhibit the AGE- were transformed into KM71H yeast cells, and positive colonies induced activation of NFjB in a dose-dependent manner. In contrast, harbouring genomic integration of multi-copy AOP or Tat-AOP gene higher molecular-weight fractions (HMF) activated the RAGE signal- were screened out and used for fermentation. Tricine-SDS-PAGE and ling. A plate binding assay revealed that the LMF could inhibit and MALDI-TOF mass spectrometry were used to ensure the peptides compete AGE-RAGE association. In addition, LMF significantly were correctly and successfully expressed. AOP and Tat-AOP were reduced AGE-dependent MCP-1 secretion from peritoneal macro- purified by two-step column chromatography. The bioactivity of AOP phages. These results thus indicate that soy sauce-derived small and Tat-AOP were tested in both in vitro an in vivo. Several coated molecular AGE components are rather able to antagonize RAGE sig- methods containing cyclodextrin, alpha amino boric acid derivatives nalling and could exhibit beneficial effects on our health. (ACD), polyorthoester (POE). The conclusion is AOP and Tat-AOP could effectively remove free radicals and enhance intestinal health of weaning pig. Tat-AOP have a broader distribution and more efficient to retard oxidative stress. Coating could effective reduce degradation Integrative application of sequential digestion of AOP and Tat-AOP. Both the two peptide can be used in feed and elution to the membrane proteome analysis additive industrial and pharmaceutical application. of rat hippocampal cells

Xia Xiong1, Jian-ying Shen, Yulong Yin1*, Xian-chun Wang*, Identification of the protein composition of five and Song-ping Liang* different feed stuffswith Tricine-SDS-PAGE system 1Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Li Ai-ke, ZHOU Nai-ji, and Han Fei Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Academy of State Administration of Grain, 100037, the Chinese Academy of Sciences, 410125 Hunan, China Peoples Republic China 2College of Life Sciences, Hunan Normal University, Changsha 410081, China *[email protected] Objective: The feasibility of the identification of protein and the analysis of the quality of different feedstuffwith Tricine-SDS-PAGE The hippocampus is a distinct brain structure that is crucial in system was discussed. memory storage and retrieval. To identify comprehensively plasma Method: The protein composition of five feedstuffs, rapeseed meal, membrane (PM) especially the low-abundance plasma membrane cottonseed meal, extract from rapeseed meal cottonseed meal fer- (PM) proteins from hippocampal cell, the subsequent study employed mented by Monilia tropicalis and cottonseed meal fermented by black multiple analytical strategies, including the sucrose density gradient mold, was identified with Tricine-SDS-PAGE system. centrifugation and sequential washing. The remaining membranes Results: The better isolation efficiency could be produced with 4.0%C were then extracted by Sodium deoxycholate (SDC) and subjected to and 16.5%T. The protein band of rapeseed meal was showed in the SDC-assisted on membrane digestion with trypsin. Finally, tube-gel central part (14.4–60.0 kDa) and cottonseed meal, end part (about digestion method was treated the remaining membrane. A total of 712 90 kDa), which contain was higher among all of experimental materials non-redundant proteins were identified including receptor protein, and the protein in the method was easy to be effectively separated. The transport protein, signaling protein, structural protein, etc. The result result indicated that the effect of the cottonseed meal treated fermented indicated that the integration and application of fraction digestion and by black mold was better that fermented by Monilia tropicalis. elution method could successfully dissipate the membrane and pro- Conclusion: There was significant different in protein image among moted the digestion efficiency. five feedstuffs and the method of Tricine-SDS-PAGE ought to be the Keywords: Sequential digestion, Proteome, Hippocampus effective method in identification of the protein in feedstuff.

Inhibitory effects of food-derived components Intrauterine growth restriction and age alter expression 0,+ on AGE-RAGE signaling of amino acid transporter b AT in suckling Huanjiang mini-piglets Seiichi Munesue, Yasuhiko Yamamoto, So Motoyoshi, Saito Hidehito, Takuo Watanabe, and Hiroshi Yamamoto W. C. Wang, D. Z. Fu, X. F. Kong*, M. M. Geng, H. S. Yang, X. Y. Song, and Y. L. Yin* Department of Biochemistry and Molecular Vascular Biology, Kanazawa University Graduate School of Medical Science 1Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Advanced glycation end-products (AGE) have been implicated in aging Science and Key Laboratory for Agro-ecological Processes and the pathogenesis of diabetic complications, inﬂammation, Alzhei- in Subtropical Region, Institute of Subtropical Agriculture, mer’s disease, and cancer. AGE engage the cell surface receptor for the Chinese Academy of Sciences, 410125 Hunan, China AGE (RAGE), which in turn elicits intracellular signalling, leading to [email protected] or [email protected] activation of NFjB to cause deterioration of tissue homeostasis. AGE are absorbed into the body partly through the consumption of foods rich Intrauterine growth restriction (IUGR) is a major problem in human in AGE. However, it is still controversial whether food-related AGE are medicine and animal production. Efﬁciency of nutrient utilization is high harmful to health or not. In this study, we tested the effects on RAGE in neonates with normal birth weights (NBW) but is low in those with

123 S56 12th International Congress on Amino Acids, Peptides and Proteins

IUGR. However, the underlying mechanisms are largely unknown. Keywords: Neutral amino acids, Body weight, Transporter, Amino acid transporters take a pivotal role in the small intestine Huanjiang mini-piglets absorption system and growth, especially b0,+AT mediating apical Funded by 30901040, 30928018, 2007EA790004. uptake of basic amino acids (including lysine, arginine, cysteine and etc.). This study was conducted with piglet model and technologies of real-time RT-PCR and Western blot to test the hypothesis that IUGR and Molecular cloning and expression profiling of excitatory age affect expression of b0,+AT in small intestine, the major organ amino acid carrier 1 in Huanjiang mini-piglets involved in the digestion and absorption of dietary nutrients. Suckling with different body weight Huanjiang mini-piglets (five with IUGR and five with NBW) were selected on days 0, 7, 14 and 21 of age, respectively, and the jejunum was Dezhi Fu, Huansheng Yang, Xiangfeng Kong*, Wence Wang, collected for analysis. Data showed that mRNA abundance and protein and Yulong Yin* amount of the b0,+ AT in both NBW piglets and IUGR piglets decreased (P \ 0.01) with the increasing of age (from day 0 to 21); the mRNA Research Center for Healthy Breeding of Livestock and Poultry, abundance and protein amount of the b0,+ AT in piglets with IUGR was Hunan Engineering and Research Center of Animal and Poultry lower (P \ 0.05) compared with the piglets with NBW in each time- Science and Key Laboratory for Agro-ecological Processes point. These findings suggested that the suckling piglets with IUGR did in Subtropical Region, Institute of Subtropical Agriculture, not have a well-developed intestine absorption system due to the trans- the Chinese Academy of Sciences, 410125 Hunan, China portation of intestine amino acid was not sufficient for their growth. [email protected] or [email protected] Keywords: Intrauterine growth restriction, Amino acid transporter, Huanjiang mini-pigs Dietary glutamate, as a major oxidative fuel for gut and an important Funded by 30901040, 30928018, 2007EA790004. precursor for bioactive molecules, is extensively metabolized during its transcellular journey in the intestine. High energy requirement of the epithelium suggests the key role of glutamate in the metabolism Metabolism and transporter expression of neutral and development of intestine. Excitatory amino acid carrier 1 (EAAC1) is the major transporter of glutamate in the intestine. The amino acids in suckling Huanjiang mini-piglets present study was conducted to measure the effects of body weight with different body weight (BW) on the expression of EAAC1 in jejunum during suckling period using Huanjiang mini-piglets as a model. EAAC1 was cloned from Huansheng Yang, Dezhi Fu, Xiangfeng Kong*, Wence Wang, Huanjiang mini-pig using PCR method, which encoded a deduced and Yulong Yin* 524-AA protein with 8 putative transmembrane domains. The jejunum expression pattern of EAAC1 in suckling Huanjiang mini-piglets 1Research Center for Healthy Breeding of Livestock and Poultry, with large BW (LBW) and small BW (SBW) was tested by real-time Hunan Engineering and Research Center of Animal and Poultry RT-PCR and Western Blotting, respectively. Comparing with the Science and Key Laboratory for Agro-ecological Processes LBW piglets, SBW piglets had lower expression levels in both in Subtropical Region, Institute of Subtropical Agriculture, mRNA and protein during the early suckling period, and the contents the Chinese Academy of Sciences, 410125 Hunan, China of glutamate in liver and muscle changed at this period. These find- 2Graduate University of the Chinese Academy of Sciences, Beijing ings suggested that the dysfunction of intestine will influence the 100039, China metabolism states of other organs and EAAC1 may be a potential 3College of Animal Science and Technology, Southwest target for improving intestine function in SBW piglets. University, Chongqing 400715, China *[email protected] Keywords: Excitatory amino acid carrier 1 (EAAC1), Body or [email protected] weight, Huanjiang mini-pig

Genetic selection strategies towards higher prolificacy resulted in more and more impaired fetal development and increased littler size. Monosodium glutamate intake and body weight The piglets with small body weight (SBW) have long-term alterations in structure, physiology and metabolism, and permanent negative in Vietnamese adults growth performance and sub-optimal carcass quality. The present study was conducted to measure the metabolism and transporter Vu Thi Thu Hien1, Nguyen Thi Lam1, Le Thi Hop1, expression of neutral amino acids in piglets with large body weight and Shigeru Yamamoto2 (LBW) and SBW. Suckling Huanjiang mini-piglets (5 with LBW and 5 with SBW) were selected on days 0, 7, 14 and 21 of age, respec- National Institute of Nutrition, Vietnam tively, and the jejunum was collected for analyzing transporter 2International Nutrition, Department of Food and Nutritional expression by real-time RT-PCR and Western blot. The levels of Sciences, Graduate School of Human Life Sciences, Jumonji neutral amino acids in plasma, liver and skeletal muscle were mea- University, Japan sured by automatic amino acid analyzer. Results showed that, compared with the LBW littermates, SBW piglets had higher levels of Background: Vietnamese have been familiar with umami from tra- some neutral amino acids in liver and skeletal muscle, while lower ditional fermented seasonings, which are rich in the umami amino plasma levels during the suckling period. Consisted with the level acid, Glutamate. There have been controversial reports concerning the alteration in neutral amino acids, the expression profiles of system association between monosodium glutamate (MSG) intake and ASC amino acid transporter-2 (ASCT2) in jejunum were changed, overweight/obesity. with lower expression levels in SBW piglets compared with the LBW Objective: To determine the association between MSG intake and littermates. These findings suggested that the intestine dysfunction overweight in Vietnamese adults. may be one of the reasons in altering physiology and metabolism Method: A cross-sectional survey was carried out in Vietnamese states of other organs which result in lower growth rate and carcass aged from 20 and over. A total of 1,528 participants living in rural and quality. urban areas of Hanoi, Thua Thien Hue and Ho Chi Minh cities were

123 12th International Congress on Amino Acids, Peptides and Proteins S57 randomly selected. The majority of participants prepared their foods at Protective effects of arginine supplementation against home, without using commercially processed foods. Dietary intake was diquat-induced oxidative stress in piglets assessed by 24 h-recalling method in 3 non-consecutive days. MSG intake of a family was assessed by weighing containers of MSG and other seasonings before and after the survey. Individual MSG intake was Ping Zheng, Bing Yu, Gang Tian, Keying Zhang, and Daiwen Chen** estimated based on the energy intake ratio of the family. Physical activity was assessed by Global Physical Activity Questionnaire. Institute of Animal Nutrition, Sichuan Agricultural University, Key Results: Eighty one percent of participants were MSG users. Average Laboratory of Animal Disease-resistant Nutrition, Ministry MSG intake was 2.2 ± 1.8 g/day. Pearson correlation coefficients of Education, Ya’an, Sichuan, 625014, Peoples Republic China were used to determine associations between MSG intake and BMI, and energy intake. There was no significant association between MSG The present study was conducted to test the hypothesis that dietary intake and BMI nor MSG and energy intake. Multiple logistic arginine supplementation may attenuate oxidative stress in piglets. A regression analysis revealed that factors associated with overweight total of 36 PIC postweaning pigs weighted 8.67 ± 0.43 kg BW were were age, occupation, physical activity and intakes of energy, car- individually penned and assigned to three dietary treatments including bohydrate, saturated fatty acids and animal protein. basal diet (BD) and BD + 0.8% Arg or 1.6% Arg. On day 8, pigs in Conclusion: MSG intake was suggested to have no effect on body each treatment were divided into two groups to be intraperitoneally weight. injected with diquat or sterile saline, respectively. At 96 h post- injection, piglets were killed for evaluation of the performance, activities of antioxidant enzymes, total antioxidative capacity and malondialdehyde in plasma and liver, and the gene expressions of Nutritional significance and molecular basis inflammatory cytokines. All piglets fed ad libitum. The experiment lasted for 11 days. The results showed that arginine supplementation of intestinal metabolism of DL-methionine did not affect (P [ 0.05) piglets growth performance but there was a and its hydroxyl analogue tendency that high dose of arginine can decrease ADG and ADFI of piglets under non-oxidative stress conditions. Oxidative stress Zhengfeng Fang*, De Wu, Yan Lin, and Lianqiang Che induced by diquat significantly decreased ADG (P \ 0.001), ADFI (P \ 0.001) and increased F/G of piglets (P \ 0.05). However, sup- Key Laboratory for Animal Disease Resistance Nutrition of the plementation of 1.6% arginine significantly increased ADFI and the Ministry of Education of China, Institute of Animal Nutrition, Sichuan antioxidative capacity in liver of piglets under oxidative stress Agricultural University, Ya’an 625014, Peoples Republic China (P \ 0.05). Supplementing 0.8 or 1.6% arginine to the diet can *To whom correspondence should be addressed. Tel: +86-835- enhance the activities of GPx, SOD in plasma and decrease the TNF-a 2885164 Fax: +86-835-2885065 Email: [email protected] mRNA level in jejunum under oxidative stress condition (P \ 0.05). It is concluded that arginine can alleviate the growth depression and Methionine is a nutritionally indispensable amino acid (AA) for ver- stress response induced by oxidative stress to some degree through tebrates, but is also toxic to animals and humans due to its increasing food intake, increasing the capacity of anti-oxidative transmethylation to produce homocysteine. Furthermore, the extensive stress, and decreasing inflammatory cytokines in piglets. catabolism of dietary methionine by the intestine or by luminal microbes may result in decreased availability of this AA for protein synthesis in extraintestinal tissues. To test whether intestinal metab- Protective effects of N-acetylcysteine on intestinal olism of methionine is regulated by its sources, DL-methionine (DL-MET) and its hydroxyl analogue, DL-2-hydroxy-4-methylthio- functions of piglets challenged with lipopolysaccharide butyrate (DL-HMTB) receive growing attention in recent studies. 1, 1 1 1 Studies in pigs show that although the directly available L-MET in DL- Yongqing Hou *, Lei Wang , Wei Zhang , Zhenguo Yang , MET diet was about 1.2-fold that in diet containing equal molar Binying Ding1, Huiling Zhu1, Yulan Liu1, Yulong Yin2, methionine source, 30% of which was supplied by DL-HMTB, the net and Guoyao Wu3 portal appearance of L-MET was not different between the two diets. Noticeably, compared with pigs fed the DL-MET diet, pigs fed the 1Hubei key Laboratory of Animal Nutrition and Feed Science, Wuhan DL-HMTB diet had higher net portal balance and/or appearance of Polytechnic University, Wuhan 430023, China; leucine, isoleucine, histidine, arginine and alanine, but had lower 2Research Center for Healthy Breeding of Livestock and Poultry, portal appearance of glutamate. Plasma ornithine and taurine con- Hunan Engineering and Research Center of Animal and Poultry centration was higher, but plasma urea concentration was lower in the Science and Key Laboratory for Agro-ecological Processes DL-HMTB fed than in the DL-MET fed pigs. These differences may in Subtropical Region, Institute of Subtropical Agriculture, be explained by the difference in distribution of enzymes for con- the Chinese Academy of Sciences, Hunan 410125, China; version of DL-HMTB and D-MET. The low mRNA abundance and 3Department of Animal Science, Texas A&M University, College low activity of D-2-hydroxy acid dehydrogenase (D-HADH) and L-2- Station, TX, 77843, USA; hydroxy acid oxidase (L-HAOX), but high mRNA abundance and high *Corresponding author: Dr. Yongqing Hou, activity of D-AA oxidase (D-AAOX) in the intestine indicate a rela- E-mail: [email protected] tively higher capacity of the intestine to utilize D-MET than to utilize DL-HMTB for L-MET synthesis and its subsequent metabolism. Both This study was conducted with the lipopolysaccharide (LPS)-chal- D-HADH and L-HAOX activity in the stomach was comparable with lenged piglet model to determine the effects of N-acetylcysteine those in the liver and/or kidney, indicating the substantial capacity of (NAC) on intestinal functions of piglets. Eighteen crossbred piglets the stomach to convert DL-HMTB to L-MET. In addition, studies in (11.58 ± 0.26 kg BW) were randomly allocated into control, LPS ducks and chicks show some superiority of DL-HMTB to DL-MET in and NAC groups. The control and LPS groups were fed a basal diet, decreasing homocysteine production and alleviating stress responses, whereas the NAC group was fed the basal diet + 500 mg/kg NAC. On respectively. These novel findings may have important implications days 10, 13 and 20 of the trial, the LPS and NAC groups received for intestinal nutrition and health in animals and perhaps humans. intraperitoneal administration of LPS (100 lg/kg BW), whereas the

123 S58 12th International Congress on Amino Acids, Peptides and Proteins control group received the same volume of saline. On day 20 of the Regulatory role for L-glutamine in the utilization trial, D-xylose (0.1 g/kg BW) was orally administrated to all pigs at of amino acids by pig small-intestinal bacteria 2 h after LPS or saline injection, and blood samples were collected at

1 h thereafter. On day 21 of the trial, pigs were sacriﬁced to obtain 1,2,3 3 3 1 intestinal mucosa for analysis. Compared with the control group, LPS Zhao-Lai Dai , Xi-Long Li , Peng-Bin Xi , Jing Zhang , Guoyao Wu2,3*, and Wei-Yun Zhu1* challenge reduced (P \ 0.05) D-xylose content of plasma, activities of

DAO in jejunal mucosa, the ratio of villus height to crypt depth in 1 jejunal mucosa, RNA/DNA, TP/DNA in jejunal and ileal mucosa, Laboratory of Gastrointestinal Microbiology, College of Animal while increased (P \ 0.05) the activities of DAO in plasma, and Science and Technology, Nanjing Agricultural University, Nanjing 210095, China caspase-3 expression in intestinal mucosa. These adverse effects of 2 LPS were attenuated (P \ 0.05) by NAC supplementation. Moreover, State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China NAC prevented the LPS-induced decrease of claudin-1 and occludin 3 expression in jejunal and ileal mucosa. Collectively, these novel Department of Animal Science, Texas A&M University, College findings indicate that dietary supplementation with 500 mg/kg NAC Station, TX, 77843, USA alleviates the mucosal damage and improves the absorptive function *Corresponding author: Dr. Wei-Yun Zhu ([email protected]) of the small intestine in LPS-challenged piglets. or Dr. Guoyao Wu ([email protected]) Our previous study indicated that the pig small-intestinal bacteria were active in the utilization and metabolism of glutamine. This study investigated the effect of L-glutamine on the utilization of amino acids Recent advances in amino acid nutrition of animals (AA) by pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the pig small Yulong Yin1*, ChengboYang2, Wenkai Ren1, and Guoyao Wu3 intestine. Anaerobic AA basal media containing 0–5 mmol/L of glutamine were used for the experiment. After 3 h of incubation, samples 1Research Center for Healthy Breeding of Livestock and Poultry, were taken for the determination of AA concentrations for the calcu- Hunan Engineering and Research Center of Animal and Poultry lation of AA utilization. Results showed that the dose-dependent Science and Key Laboratory for Agro-ecological Processes increase in the bacterial utilization of glutamine and altered flux of in Subtropical Region, Institute of Subtropical Agriculture, glutamine into glutamate in the bacteria. The addition of glutamine the Chinese Academy of Sciences, 410125 Hunan, China affected the arginine utilization and the flux of arginine into ornithine in 2Department of Food Science, University of Guelph, Guelph, Ontario, pig small-intestinal bacteria. Complete utilization of asparagine, Canada aspartate and serine were observed in pure bacterial strains after 3 h of 3Department of Animal Science, Texas A&M University, College incubation. The addition of glutamine reduced (P \ 0.05) the net uti- Station, TX, 77843, USA *[email protected] lization of asparagine by both jejunal and ileal mixed bacteria. Net utilization of lysine, leucine, valine, ornithine and serine by jejunal or Protein nutrition of animals is actually amino acid (AA) nutrition. ileal mixed bacteria decreased with the addition of glutamine. Overall, Recent findings indicate that the gastrointestinal tract (GIT) of the addition of glutamine affected the metabolism of the arginine- nonruminants, like ruminants, has a complex micro-ecosystem family of AA and serine- and aspartate-family of AA in small-intestinal consisting of numerous species of bacteria. As an essential ‘‘organ’’, bacteria and reduced the utilization of most AA in jejunal or ileal mixed the gut microbiota plays an important role in AA nutrition of the bacteria. These findings suggest that the beneficial effects of glutamine host. In contrast to enterocytes, small-intestinal bacteria can degrade to the swine nutrition may partially by regulating the microbial utili- all dietary AA, thereby profoundly affecting their first-pass metab- zation and metabolism of AA in the small intestine. olism in the gut. The metabolic fates of AA vary among bacterial species, with the rates of utilization being higher in E. coli and Klebsiella sp., compared with Streptococcus sp. This finding helps Relationship between dietary lysine levels and serum explain the health- and growth-promoting effects of dietary inter- concentration of amino acids and performance ventions by inhibiting the growth of potential pathogenic bacteria in the intestine and enhancing AA absorption by the small intestine. of growing pigs Also, small-intestinal bacteria metabolism results in the losses of endogenous N and AA in ileal digesta and feces, contributing to N Miguel Cervantes, Adriana Morales, Alfonso Araiza, Jose´ Landero, pollution in the livestock industry. Dietary non-starch polysaccha- John K. Htoo, Hećtor Garcia, and Ernesto Avelar rides (NSP) are the main exogenous factors that cause endogenous N secretion in pigs. Available evidence shows that NSP enzymes Typical feed ingredients are first limiting in Lysine (Lys) for pigs, but can improve pig growth performance by stimulating the degradation usually contain excess of arginine and neutral amino acids (AA), of soluble NSP in the small intestine and reducing the losses of which interact with Lys for its intestinal absorption. The availability of intestinal endogenous N and AA. Furthermore, dietary carbohydrates AA significantly affects the performance in pigs. An experiment was affect the digestion and absorption of N and AA in the portal- conducted to analyze the effect of three dietary Lys levels (0.40%- drained visceral tissues via a mechanism that controls glucose Deficient, DEF; 1.18%-Intermediate, INT; and 1.56%-Excess, EXC) release from starch. The most exciting finding is that arginine can on the performance and serum concentration (SC) of indispensable attenuate the damages to the GIT, sudden deaths, and neurologic AA. Twenty-four crossbred pigs (14.4 ± 1.7 kg) with 8 replicates per lesions caused by toxins of the bacterium Escherichia coli. Arginine treatment were used. A wheat based diet was formulated, using ana- supplementation can enhance immune responses and ameliorate the lyzed ingredient amino acid (AA) contents to meet the requirements of reproductive failure in mice caused by the porcine circovirus type 2 AA other than Lys. Treatments (T) were: T1, wheat-based diet, 0.40% infections. Thus, AA plays a central role in health, growth, and Lys (DEF); T2, basal plus 0.78% L-Lys (INT); T3, basal plus 1.56% reproduction of animals. L-Lys (EXC). At the end of a 28-day trial, all pigs were sacrificed and Keywords: Amino acids (AA), Non-ruminants, Small intestine, blood was collected to analyze the SC of AA. Two contrasts were Bacteria constructed to analyze the effect of the Lys levels (DEF vs. INT; and

123 12th International Congress on Amino Acids, Peptides and Proteins S59

INT vs. EXC), Increasing the Lys level from 0.40 to 1.18% improved (Duroc 9 Large White 9 Landrace) with an average initial BW of the ADG, ADFI, and FCR (p \ 0.01). No further response was 70 kg were surgically fitted with chronic catheters in the portal vein, observed with the EXC level. Serum concentration of arginine, thre- ileal vein and carotid artery and were randomly allocated to two groups onine, and valine reduced (p \ 0.01) when Lys increased from DEF to to receive alanine (103 mg/kg body wt, isonitrogenous control) or L- INT; no further response occurred with the EXC diet. Lys SC linearly arginine-HCl (61 mg/kg body wt) by the portal vein. Blood flows were increased (p \ 0.01). Methionine, leucine and phenylalanine tended to measured with infusion of p-aminohipuric acid (PAH) into the ileal reduce with the increase in Lys. Performance and SC of selected AA vein, and simultaneously obtain blood samples very 0.5 h for 4 h for are well correlated each other, in response to dietary Lys levels. determine concentrations of metabolites in carotid arterial blood and oxygen consumption by portal vein-drained organs (PVDO). Compared with alanine treatment, arginine infusion increased PVBF at 30, 60 and Significant reverse correlation between dietary intakes 90 min after infusion but decreased PVDO at 60, 90, 120 and 150 min of three branched essential amino acids (Leu, IIe and Val) after infusion (P \ 0.05). Plasma concentrations of glutamate at infu- and percent body fat in the Newfoundland population sion time of 180 and 240 min and arginine at infusion time of 60, 120, 180 and 240 min in arginine infused pigs were higher than those of alanine infused pigs on the same infusion time (P \ 0.05). However, Daniel Wadden, Farrell Cahill, Feiyu Han, Yagang Xie, arginine infusion decreased (P \ 0.05) plasma cystine concentrations at Wayne Gulliver, Hongwei Zhang, and Guang Sun infusion time of 60, 120, 180 and 240 min and valine at infusion time of 60 and 120 min compared with alanine treatment. Plasma concentra- Recent studies show that the amount of dietary intake of branched tions of insulin and glucagon at infusion time of 30, 60 and 90 min were essential amino acids (BCAA) may affect the short and long term higher and free fatty acid at infusion time of 60, 90 and 120 min were metabolism. Our lab has previously shown that dietary protein intake lower than those of pigs both in alanine treatment on the same infusion is negatively associated with body adiposity. In the present study we time and at arginine pre-infusion time. These results suggest that acute further investigate the role of BCAA in this beneficial effect in the arginine supplementation improves blood flow and reduces the PVDO Newfoundland population in Canada. A total of 2,334 adults (577 oxygen consumption, thereby enhancing the amino acids availabilities men and 1,757 women) from the CODING study participated. Daily for utilization. And insulin and glucagons may play important roles in BCAA intake was computed from the Willett Food Frequency arginine infusion transiently regulating nutrient metabolism. Questionnaire. Body fat percentage was measured using a dual energy Keywords: L-Arginine, Blood flow, Oxygen consumption X-ray absorptiometry. Body mass index and waist circumference were measured as well. Data were analyzed according to gender using partial correlation (controlling for age, physical activity level and total calorie intake). The daily intakes of the three BCAAs based on The endothelial arginine-nitric oxide pathway obesity status (percent body fat, Bray criteria) were compared among normal, overweight and obese groups. All three BCAAs were found in diabetes and obesity reversely correlated with all obesity phenotypes with WC having the strongest correlation coefficient in both women (r varies from -0.27 Cynthia J. Meininger1 and Guoyao Wu1,2 to -0.35 and p \ 0.001) and men (r varies from -0.23 to -0.28 and p \ 0.001). ANOVA analysis revealed that normal weight subjects 1Department of Systems Biology and Translational Medicine, Texas had the highest intake, and the obese had the lowest intake of all three A&M Health Science Center, Temple, TX, USA; and BCAAs with the overweight in the middle. This strong dose effect 2Department of Animal Science, Texas A&M University, College relationship holds based on any obesity criteria used in the study. Our Station, TX, USA findings provide solid evidence of the beneficial effect of BCAA on obesity at the free living population level. Endothelial cells synthesize nitric oxide (NO) from L-arginine and oxygen utilizing the endothelial isoform of NO synthase (eNOS). NO, initially named endothelium-derived relaxing factor, has a central role The dynamic changes of blood flow, oxygen in regulating endothelial cell function in blood vessels. We demon- consumption and metabolites response to acute arginine strated that in diabetes the bioavailability of NO is reduced because of decreased endothelial tetrahydrobiopterin, a critical co-factor for supplementation in growing-finishing pigs eNOS. Dietary L-arginine increases tetrahydrobiopterin—normalizing NO synthesis and vascular function—by stimulating GTP cyclohy- Bie Tan1, Yulong Yin1*, Xinguo Li2, Xiangfeng Kong1, drolase I, the first and rate-controlling enzyme for de novo synthesis of 1 1 1 1,3 Zhiqiang Liu , Tiejun Li , Ruilin Huang , and Guoyao Wu tetrahydrobiopterin. L-arginine also decreases glucosamine production, helping to reverse impaired endothelium-dependent relaxation, oxida- 1Research Center for Healthy Breeding of Livestock and Poultry, tive injury and vascular insulin resistance in diabetic and obese patients. Hunan Engineering and Research Center of Animal and Poultry The arginine-NO pathway has widespread effects on the metabolism of Science and Key Laboratory for Agro-ecological Processes energy substrates. Arginine supplementation increases plasma arginine in Subtropical Region, Institute of Subtropical Agriculture, while decreasing plasma glucose in diabetic rats. L-arginine reduces the Chinese Academy of Sciences, 410125 Hunan, China body weight loss in type 1 diabetic rats but, interestingly, decreases 2Hunan Institute of Animal Husbandry and Veterinary Medicine, adiposity in genetically obese rats, diet-induced obese rats and humans Changsha, Hunan 410131, China with type 2 diabetes. In Zucker diabetic fatty rats, a genetically obese 3Department of Animal Science, Texas A&M University, College animal model of type 2 diabetes, arginine treatment reduces white Station, TX 77843, USA *[email protected] adipose tissue while increasing NO synthesis. This arginine-induced increase in NO stimulates oxidation of energy substrates (including Arginine plays an important role regulating nutrient metabolism. This fatty acids and glucose). L-arginine supplementation stimulates brown study was conducted to determine the dynamic changes of blood adipose tissue development possibly by enhancing synthesis of cell metabolites response to acute arginine supplementation in growing- signaling molecules and increasing expression of genes that promote finishing pigs using blood catheter technique. Eight barrows mitochondrial biogenesis. Modulation of the arginine-NO pathway may

123 S60 12th International Congress on Amino Acids, Peptides and Proteins be beneficial for vascular function, metabolic homeostasis, and weight Many amino acids (AA) regulate key metabolic pathways crucial to management in diabetes and obesity. maintenance, growth, reproduction and immune responses, making Supported by NIH, JDRF and AHA. their dietary supplementation a major focus of fish nutrition research. Lysine is frequently limiting in ingredients used for feeds, affecting growth performance and health. This experiment aimed to test how The metabolic characteristics of the proteins lysine limitation affects growth and muscle proteome expression in juvenile zebrafish (Danio rerio). From 33 to 49 days post-fertilisation in Canadian hulless barley: comparison (dpf), quadruplicate groups of 8 fish were fed adequate (Lys(+)) or of the zero-amylose waxy, waxy, and high-amylose low-lysine (Lys(-)) diets. Fork length was monitored and white trunk cultivars with the normal starch cultivar muscle proteome at 49 dpf screened by 2D-DIGE and MALDI ToF– ToF mass spectrometry. Growth rate was negatively affected by the low-lysine diet. Of the D. Damiran, L. Yang, and P. Yu* 527 ± 11 (mean ± SEM) protein spots observed (10–150 kDa and 4–7 pI values), 30 were over-expressed and 22 under-expressed in College of Agriculture and Bioresources, The University of skeletal muscle of Lys (-) fish (|fold-change| [1.2). Sixteen proteins Saskatchewan 51 Campus Drive, Saskatoon, SK, S7 N 5A8, Canada of the 20 identified spots were involved in the cytoskeletal network *Corresponding author: Peiqiang Yu, Ph.D. SAF Research Chair and contractile apparatus of skeletal muscle, 11 of which were more College of Agriculture and Bioresources University of Saskatchewan, abundant in the Lys (-) fish (isoforms of myosin-binding protein Room 6D10 Agriculture Building, 51 Campus Drive, Saskatoon, SK, H-like, fast skeletal myosin heavy chain (fsMHC), fast skeletal Canada, S7 N 5A8 Tel: +1-306-9664132 Fax: +1-306-9664151 myosin light chains and skeletal muscle actin; alpha tropomyosin) and [email protected] 5 less abundant (isoform of fsMHC, actin fragments and F-actin- capping protein). Proteins involved in energy metabolism (beta eno- The hulless barley (HB) with normal starch structure is considered to be a lase and mitochondrial ATP synthase) were more abundant in Lys (-) less favourable feed grain for ruminant animals in comparison to the fish. Finally, the proteins apolipoprotein A-I precursor (lipid trans- hulled counterpart. This is largely due to the rapid degradation of its starch port) and Pdlim7 (PDZ-LIM protein family) showed increased in the rumen. Recently, several new Canadian hulless barley cultivars abundance in Lys (+) fish. Overall, these results demonstrate the key with altered starch characteristics (waxy and high-amylose), have been role of lysine as regulator of muscle development, metabolism and developed. The variations in the molecular composition and character- growth. istics of starches substantially affect functional properties, therefore the objectives of this study were to: (1) investigate metabolic characteristics of the proteins and the protein supply of zero-amylose waxy (CDC Fibar), waxy (CDC Rattan), and high-amylose (HB08302) HB in comparison to normal starch feed-type (CDC McGwire) hulless barley using the DVE/ Thyroglobulin and iodine nutrition OEB system and the NRC 2001 model, and (2) compare the two models in predicting the amount of protein supplied by the HB. According to both, Radovan Bı´lek, and Vaćlav Zamrazil the DVE/OEB system and NRC 2001 model, zero-amylose waxy HB was superior to the other cultivars in both, truly absorbed protein in the small Institute of Endocrinology, Prague, Czech Republic Na´rodnı´ 8, intestine (DVE; 123 vs. 111 g/kg DM) and the metabolizable protein 116 94 Prague 1, Czech Republic Tel: +420 224905111, (MP; 112 vs. 92 g/kg DM), while the high starch HB had the lowest DVE Fax: +420 224905325, E-mail: [email protected] (103 vs. 117 g/kg DM) and MP (87 vs. 100 g/kg DM). As a single feed ingredient, all four of the HB had negative protein degradation balance, Our results from population studies indicate that serum thyroglobulin indicating the N shortage for the microbial biosynthesis in the rumen. In (Tg) seems to be a valuable indicator of iodine nutrition. Thyro- general, HB with modified starch structure provided a relatively balanced globulin is the major iodoglycoprotein of the thyroid gland and one of source of energy and protein for microbial synthesis in the rumen. Con- the largest proteins in the human body (molecular weight of soluble sequently, the hulless barley with modified starch might be a better feed dimer about 660 kDa). The immunoanalytical determination of serum grain for dairy cattle than the normal starch HB. However, larger feed Tg is difficult because of the inhomogeneity of the Tg molecule, samples with a wider range of chemical compositions needs to be eval- where various isoforms of Tg exist with differences both in primary uated in order to be more conclusive. structure and iodine or carbohydrate content. In addition, the presence Keywords: Hulless barley, Protein evaluation, Starch structure, of circulating autoantibodies against Tg may substantially interfere Protein metabolic characteristic with determination of serum Tg. The main problem is the specifica- tion of Tg cut-off value corresponding to various levels of iodine intake, which is limited by large interassay variability and lack of The muscle proteome of growing zebrafish is affected reference data for Tg in healthy, iodine-sufficient individuals. by dietary lysine Urinary iodine (laboratory spectrophotometric method based on alkaline ashing and Sandell–Kolthoff reaction) and serum Tg (elect- rochemiluminiscence immunoassay, Roche Diagn., Germany) were 1,2 2 Mahaut de Vareilles , Luis E.C. Conceiçaõ , determined in the general healthy population of 3,014 randomly 1 3 2 Pedro Go´mez-Requeni , Katerina Kousoulaki , Pedro M. Rodrigues , selected individuals aged 6-98 years (1,272 males, 1,742 females), or 2 4 2 Nade`ge Richard , Kari E. Fladmark , Odete Cordeiro , in the hospital population (n = 5,516), which consisted of individuals 2 1 Tome´ S. Silva , and Ivar Rønnestad aged 0–91 years (527 males, 4,989 females) attending the Institute of Endocrinology, Prague. The serum Tg concentration was elevated in 1 Department of Biology, University of Bergen, Norway iodine-deficient subjects and serum Tg decreased progressively as the 2 Centro de Cieˆncias do Mar do Algarve (CCMAR), Faro, Portugal urinary iodine concentration rose. We can conclude that thyroglobulin 3 Nofima Ingredients, Fyllingsdalen, Norway is a valuable indicator of iodine nutrition in a population, if thyroid 4 Department of Molecular Biology, University of Bergen, Norway disorders are not frequent.

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Peptides N-homocysteinylated human serum albumin on sulfhydryl group permits to obtain bioconjugate of protein with the peptide-oligonucleotide conjugate by maleimide residues. A fluorine-labeled bioconjugates was prepared for use as an Aryl hydrazine resin as a tool in the synthesis intravascular contrast agent for magnetic resonance imaging. 19F of C-terminal modified peptides: optimization NMR offers a unique quantitative means of imaging infused agents, as there is essentially no 19F background signal in tissue. In addition, of oxidation conditions 19F spectroscopy is a powerful alternative to 1H NMR spectroscopy because of its high sensitivity (83% with respect to 1H) due to a high A. Walewska, I. Małuch, D. Sobolewski, and A. Prahl gyromagnetic ratio, 100% natural abundance of the (spin = 1/2) 19F isotope, narrow lines, and short longitudinal relaxation times (T1) Faculty of Chemistry, University of Gdan´sk, Sobieskiego 18, which permit rapid pulsing with a corresponding improvement in the 80-952 Gdan´sk, Poland signal-to-noise ratio per unit time. The research is supported by Integration Grant of SB RAS 88, Recently, Y. Kwan et. al. presented a novel and straightforward RFBR Grant 09-04-01483-a, Grant of Novosibirsk region strategy to synthesize peptides with p-nitroanilides and other anilide government. analogues using an aryl hydrazine resin. The resin is also a multi- purpose tool for the synthesis of carboxylic acids, esters and thioesters. When the synthesis is completed, the fully protected peptide hydrazide resin is oxidized with either N-bromosuccinimide Cell penetrating peptides: a nanocarrier for delivery (NBS) or copper(II) acetate in pyridine. The resulting acyl diazene of ssDNA and protein complexes in plant cell resin is then cleaved by peptide displacement at the C-terminus with amine. The fully deprotected peptide amide is finally obtained by Francois Eudes treatment with trifluoroacetic acid (TFA). In our approach, we used a 4-Fmoc-hydrazinobenzoyl AM NovaGel resin to synthesize a pep- Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, tide-substituted amide in the C-terminus. First, the oxidative cleavage Lethbridge, AB, T1J 4B1, Canada was carried out with NBS in pyridine and a nucleophile [a protected 4-(aminomethyl)benzimidamide (Amba)]. However, the yield of the Cell-penetrating peptides (CPPs) are a class of short peptides with reaction was very poor. In the next step, we applied copper(II) acetate property to translocate across cell membranes and target subcellular in the presence of pyridine and Amba. Following optimization, the localisation, such as the nucleus. CPPs also have the capacity to non- efficiency of the process was significantly improved. Herein we dis- covalently bind to cargo molecules, such as nucleic acid and protein, cuss the conditions needed to obtain a reasonably high efficiency of and transport them to its final subcellular destination. Despite fun- the oxidative cleavage in the synthesis of the C-terminal modified damental differences between animal cell and plant cell composition, peptides using the aryl hydrazine resin linker. the CPP-cargo uptake pattern between the mammalian system and the plant system is very similar. The dimer Tat49–57 RKKRRQRRR basic domain, one of the shortest known cell penetrating peptide, has been extensively used for wheat and triticale nuclear targeting and delivery Cathepsin B-sensitive peptide-oligonucleotide of dsDNA, ssDNA and proteins. Synthetic or in vivo produced conjugates for cancer therapy nucleic acid, proteins and CPPs are blocks that conjugate to form nano-complexes in a relatively predictable manner. Single stranded L. A. Yarinich1,2, A. S. Chubarov1,2, L. S. Koroleva1,2, DNA binding proteins such as RecA, Rad51 and VirD2 were used to I. Yu. Serpokrylova1, V. N. Silnikov1, and T. S. Godovikova1,2 form various cargo complexes with ssDNA, and increased the integrity of ssDNA integrated in the haploid plant genome of 1Institute of Chemical Biology and Fundamental Medicine, Siberian microspore. This novel process fit very well with cell culture systems, Branch of Russian Academy of Sciences, 8 Lavrentyev Ave., 630090 as demonstrated with isolated microspore, a unique haploid cell Novosibirsk, Russia amenable to the production of haploid and doubled haploid plant 2Novosibirsk State University, 2 Pirogova St., 630090 Novosibirsk, lines. Stable DNA integration has been achieved following CPP Russia mediated delivery of dsDNA and ssDNA in wheat and triticale mi- crospores, and inheritance has been documented in subsequent Induction of apoptosis in endothelial cells is considered an attrac- generations. CPP mediated transfection in plant microspore opened tive strategy to therapeutically interfere with a solid tumor’s blood new possibilities for precision genetic engineering of this unique cell supply. We constructed cytotoxic conjugates that specifically target type and crops of commercial importance. angiogenic endothelial cells, thus preventing typical side effects of apoptosis-inducing drugs. For this purpose, we conjugated the Designing and efficient synthesis of anticancer peptides antisense oligonucleotide via a lysosomal cleavable linker to N- contained novel linkers and investigation of their homocysteinylated human serum albumin and further equipped this drug-albumin conjugate with RGD-peptides for multivalent inter- activities action with integrin. Peptides: substrates for cathepsins taking part in cancer progression has been described in literature. Application Fatemeh Tahoori, Reza Sheikhnejad, and Saeed Balalaie of such peptides as biodegradable linkers let to release the therapeutic in transformed cells. Synthesis of peptides (AlaLeu)2Gly, Peptide Chemistry Research Center, K. N. Toosi University b-Ala(AlaLeu)2 were carried out in solution using Boc-strategy. of Technology, Tehran, Iran After that flexible amino linker with maleimide fragment was coupled to C-terminal and antisense oligonucleotide was connected Peptides can be used for treatment of different diseases. Although to N-terminal of peptides by phosphamide bond. Modification of numerous anticancers drugs with peptide skeletons exist, most of

123 S62 12th International Congress on Amino Acids, Peptides and Proteins them have some drawbacks. The development and designing of new Epigenetic mechanisms of evolution: peptides regulate peptides is a highly demand and considerable challenge for synthetic gene expression and increase resource of the organism chemists and biochemists. We wish to report, designing and efficient synthesis of a novel proline-rich heptapeptides contained new linkers using combination of solid-phase and solution phase peptide synthesis V. Kh. Khavinson, V. V. Malinin, and B.F. Vanyushin* methodologies. Synthesis of desired heptapeptides was done according to the Saint Petersburg Institute of Bioregulation and Gerontology, Russian known SPPS Strategy. Synthesis was carried out using 2-CTC resin Academy of Medical Sci., 3, Dynamo Pr., Saint Petersburg, 197110, according to the standard Fmoc strategy. Before final deprotection Russia; E-mail: [email protected] and cleavage from the surface of resin, new linker is formed via the *Belozersky Institute of Physical and Chemical Biology, Lomonosov reaction of free amine with anhydrides. The purification was done Moscow State University, Moscow, 119991, Russia using preparative HPLC. The structure of the synthesized peptides were confirmed using HR-Mass(ESI). Peptides increase lifespan of rats by about 30–40% and inhibit growth The anti-cancer activity of the peptide was studied. Since the of tumors. Peptides introduced in vivo influence the gene expression lipophilicity is a key factor in determining the rate at which a drug profile. Peptides introduced to transgenic mice suppressed expression crosses the blood–brain barrier (BBB), we inserted alkyne and alkyl of the HER-2/neu mammary gland cancer gene two to fourfold. The linkers. peptides introduced increase transcription of IL-2 and c-fos genes in The desired products are purified and their biological activity are lymphocytes and various structures of hypothalamus. The mecha- studied. We believe that the existence of lipophilic moieties cause nisms of the peptide action associated with chromatin activation in more lipophilicity and their cell permeability. The investigation of blood lymphocytes of aged patients. Human fibroblasts treated with anti-cancer activity of the synthesized peptides are in progress. peptides showed the telomerase activity induction and a 2.5-fold The details will be discussed in the conference. increase in the mean telomere length. Peptides increased the number of cell divisions by 42.5%, i.e. the overcoming Hayfiick’s limit. Treatment of aged and senile people with peptides improved their Design of peptides with nuclear binding and potent physiological functions and decreased the lethality by 44–49% in a broad-spectrum antimicrobial activities period of 8–12-year clinical observation. Peptides bind to double- and single-stranded deoxyribooligonucleotides containing CNG and CG sequences that are target sites for DNA methylation in eukaryotes. Lirong Li, Guowei Le, and Yonghui Shi Peptides modulate specifically the action of eukaryotic endonucleases depending on DNA methylation status. The peptide modulating action Center for Food Nutrition and Function Factor, College of Food, on DNA hydrolysis seems to be due to site-specific peptide binding Jiangnan University, WuXi, 214122, China with DNA that may protect DNA against enzymatic hydrolysis. Peptide binding to CNG or CG sites in gene promoters should prevent Objective: Emergence of multi-resistant strains has prompted search their methylation with respective DNA-methyltransferases and leave for new antimicrobial drugs. Antimicrobial peptides (AMPs) are of promoters to be unmethylated that is crucial for activation of most greatest potential sa a new class of antibiotics. We designed a series genes. Thus, specific peptide-DNA interactions control epigenetically of AMPs that could translocate across bacterial membranes and bind the cell genetic functions and can be responsible for homeostasis to DNA. Aiming to explore their antibacterial mechanism and analyse recovery and life-span prolongation. the influence of charge and hydrophobicity on biological activity. Method: Physicochemical properties were analysed by antimicrobial peptide database and protein sequence analysis softwares. Minimal inhibition concentration (MIC) was determined using the microdilu- tion assay. We investigated the secondary structure of the peptides using circular dichroism (CD) spectroscopy. Using microbial adhe- Metabolic stability of long acting LHRH antagonists sion to solvents (MATS) method measured strains surface hydrophobicity. The ability of permeabilization of inner membrane Jinfeng Yao1, Ning Zhou2, Keliang Liu2, and Ming Xue1,* (IM) was evaluated by cytoplasmic L-galactosidase release. Binding of the peptides to pathogenic bacterium genomic DNA was evaluated 1Department of Pharmacology, School of Basic Medical Sciences, by gel retardation assays. Capital Medical University, Beijing 100069, China Result: These antibacterial peptides containing of 20 amino acids, net 2Beijing Institute of Pharmacology and Toxicology, Beijing 100850, charge from +3 to +7, with 45–70% of hydrophobic residues showed China *Corresponding author Tel: +86-10-83911520; classic amphipathic characteristic and stability. They fold into an Fax: +86-10-83911520. E-mail address: [email protected] amphipathic a-helical conformation and showed random coil stereo structures. Decreasing peptides mean hydrophobicity and maintaining Purpose: Long acting luteinizing hormone-releasing hormone the original charge could enhance antimicrobial activity. Peptides (LHRH) antagonists are a series of novel LHRH antagonist ana- exhibited less than 50% hemolytic activity at 600 lg/ml. The increase logues, which synthesized on the base of the new concept of the in hemolytic activity was correlated with mean hydrophobicity long acting peptides design for protease-resistant. The bioactivities increase. All peptides except P7 had no defined secondary structure in of them were evaluated in rats by the testosterone test model. The aqueous buffer, but in membrane mimetic condition peptides fold into designed peptides showed a long action of inhibiting testosterone an a-helical conformation. Treating with peptides, strains surface secretion comparing with parent peptide. In the present study, a hydrophobicity decreased and cytoplasmic membrane permeabiliza- method was established to assess metabolic stability of the LHRH tion increased. Peptides with net charge from +5 to +7 and low mean antagonists in vitro rapidly. hydrophobicity inhibited migration of DNA above a weight ration of Methods: The cassette dosing (N in one) was used, 12 compounds DNA to peptide of 1:1. were divided into 3 groups. For cassette dosing, 4 compounds were Conclusion: these design peptides may serve as useful templates mixed with leuprorelin (as a control), and added to solution of pan- which had potent. creatin, then the mixture were incubated at 37°C for 4 h. Samples

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(100 ll) were withdrawn at different time intervals and determined by This work presents the development of controlled drug delivery HPLC method. vehicles based on magnetic nanoparticles. The system based on mag- Results: Calibration curves of all compounds were linear in the netoliposomes with incorporated peptide on surface was tested as drug concentration range of 1.0–400 lg/ml, correlation coefficients were delivery device. Iron oxide nanoparticles were synthesized by co-pre- more than 0.9991. The low limits of quantification of these twelve cipitation of ferrous and ferric salts in alkali medium. The black powder compounds were 1.0–10 lg/ml. The inter-day and intra-day preci- obtained was functionalizated with phosphatidylcholine to form bio- sions were within ±10.0% at three concentration levels. The accuracy compatible magnetoliposome. The surface modification of iron oxide percent error was 2.80–6.0%. The half-life of leuprorelin was nanoparticle after functionalization with phospholipid was accompanied 4.2 min, the half-lives of the other compounds were 0.73–5.5 h. with zeta potential measures and DLS. Magnetoliposome exhibited Conclusion: The half-lives of the 12 LHRH antagonists were not values of zeta potential (f = 30 mV) higher than those values obtained different from those determined, respectively (p [ 0.05). Among for suspension of iron oxide nanoparticle in water (f = 2.5 mV) them LY617B had a long half-life. These results showed that this showing a better colloidal stability. The hydrodynamic diameter method had been successfully applied to predict the metabolic sta- obtained by DLS measurements confirmed this behavior. The peptide bility of LHRH antagonists in vitro pancreatin system. was prepared by solid-phase Fmoc strategy. It was immobilized on magnetoliposome surface and characterized by Fourier transform spectroscopy, DLS and controlled release. Peptide release was carried out in an aqueous medium at 37°C and under heating. The amount of Peptides regulate proliferative activity of stem cells released peptide was monitored by measuring the absorbance using UV spectrophotometry. The magnetic heating characteristics were obtained V. Kh. Khavinson, N.S. Linkova, A.V. Dudkov, A.V. Trofimov, to samples in an alternating magnetic field of strength 502 Oe and and S.V. Trofimova frequency 230 kHz. Samples were entered into the centrifuge tubes which were then placed in the center of the induction coil. The alter- Saint Petersburg Institute of Bioregulation and Gerontology, Russian nating magnetic field was applied for a given time 30 s until 5 min, in Academy of Medical Sci., 3, Dynamo Pr., Saint Petersburg, 197110, 30 s intervals. The results suggested that the new system prepared in this Russia; E-mail: [email protected] study have a great potential to biomedical applications.

The objective of the experiment was the evaluation of the proliferative activity of the MSC under the influence of peptides H-Ala-Glu-Asp- Gly-OH, H-Lys-GLu-Asp-OH and H-Lys-Glu-OH. MSC was culti- Preparation and identification of Zn (II) chelating vated (37°C, 5% CO2) in the a-MEM media (Biolot, Russia) with the peptides and their complexes from sesame protein cattle fetal serum (Gibco, USA), 2 mm of glutamine (Gibco, USA) and hydrolysates gentamicin (100 units/ml). 13 samples of cultures were used in the experiment—10 main and 3 control, into which the one of the peptides Chan Wang, and Bo Li* were added (20 ng/ml). To the control samples instead of the peptide phosphate-buffered saline was added. On the 3rd and 5th day of the College of Food Science and Nutritional Engineering, China culture growth the cells were counted in the Gorayev chamber. Agricultural University, Qinghua East Road 17, Haidian District, In the control on the 3rd and 5th day the quantity of the cells was Beijing 100083, People’s Republic of China 50,000 ± 5,600 and 65,000 ± 6,200 correspondingly. Under the *Corresponding author Tel: 86-10-6273-6243; Fax: 86-10-6234-7334 influence of the tetrapeptide on the 3rd and 5th day the quantity of E-mail: [email protected] cells in the culture has risen reliably by 1.7 and twofold in comparison with the control (85,000 ± 9,900 and 125,000 ± 11,500). Under the The sesame protein hydrolysates (SPH) obtained with trypsin, which influence of the dipeptide and tripeptide on the 3rd day the quantity of exhibited high metal-chelating abilities were used for separation of the MSC showed a tendency to reduction and was valuated at metal-chelating peptides. The metal chelating peptides were isolated 50,000 ± 3,200 and 40,000 ± 3,400 cells. On the 5th day of the from the hydrolysates using affinity chromatography with zinc immo- introduction of the dipeptide and tripeptide to the culture of the MSC bilized on solid supports (chitosan). Further, six zinc-chelating peptides the quantity of cells reduced reliably by 1.9- and 1.6-fold and was were identified with reversed phase (RP)-HPLC and electrospray ioni- valuated at 35,000 ± 3,000 and 40,000 ± 2,900 cells. Depending on zation (ESI) -MS/MS, two of these metal-chelating peptides, Ser-Met the structure of the peptide different proliferative ability of the MSC (SM), Asn-Cys-Ser (NCS), were then synthesized and the zinc-chelating was noticed. Tripeptide stimulated growth of the MSC. Di- and tri- abilities were measured by EDTA titration, and radical scavenging peptide inhibited the proliferation of the MSC. activity of peptides and their complexes with zinc were measured by TEAC assay. The zinc-chelating abilities of SM and NCS are 55.4 and 73.7%, respectively. The peptide SM and zinc-SM complex had little Preparation and characterization of peptide conjugated radical scavenging activity, while peptide NCS and zinc-NCS complex had significant radical scavenging activities with 0.514 ± 0.008 (mM magnetoliposomes to biomedical applications Trolox) for peptide and 0.404 ± 0.008 (mM Trolox) for the complex. Studies of zinc complexes of two peptides by pH- potentiometric Roberta V. Ferreira*, Rodrigo M. Verly, Victor Hugo Munhoz, Dorila titration and mass spectrometry (MS) demonstrated that the complex- Pilo´-Veloso and Rosana Z. Domingues ations of two peptides with zinc are both at ratio of 1:1. And zinc-NCS complex is more stable than zinc-SM. According to Infrared ray (IR) Departamento de Quı´mica/ICEx/UFMGl Avenida Antoˆnio Carlos spectrometry of peptides and complexes, the carboxyl of C-terminus 6627, Cidade Universita´ria Pampulha 30270-901, Belo Horizonte, amino acid, peptide bond and side chain such as sulfhydryl group of MG, Brazil *[email protected] Cys, amino and acyl of Asn, play important roles in the complexation between peptide and zinc. From Circular dichroism (CD) spectrometry Keywords: Antimicrobial peptide, Ferrofluids, Magnetic hyperther- of peptide SM, a-helix disappeared from 25.7% and b-sheet was mia, Magnetoliposome increased from 74.3 to 100% after complexation with zinc. However,

123 S64 12th International Congress on Amino Acids, Peptides and Proteins

CD spectrometry of peptide NCS indicated that a-helix increased from Positively charged cell-penetrating peptides (CPPs), which are a 12.9 to 42.5% and b-sheet was increased from 0 to 57.5%. These class of short peptide sequences that can traverse cell membranes structural changes lead to a significant increase in the peptide’s stiffness efficiently, are often used as transporters for various biological and it is just the way the peptides found to adjust its structure to the drugs such as peptides, proteins, and genes, which are attached as presence of the metal ions at minimum energy expense. cargo. The cellular translocation properties of the silica nanopar- Keywords: Sesame protein, Metal-chelating peptides, Hydrolysis, ticles coated with transcription-activating-factor-derived peptide Antioxidant activity, Zinc (TDP), which has 11 key amino acid residues (YGRKKRRQRRR) from the human immunodeficiency virus (HIV-1) Tat protein, has been investigated; to determine the cargo size dependency, Simultaneous determination of long acting LHRH and translocation efficiency into the various living mammalian antagonists and its application to high-throughput cells. We found that there is an optimized cargo size for the efficient cellular uptake, which has to closely related to the pharmacokinetics translocation mechanism due to the presence of CPPs. We will present the size-dependant peptide mediated delivery systems, and Jinfeng Yao1, Ning Zhou2, Keliang Liu2, and Ming Xue1,* plausible mechanism. Based on the efficient Tat-mediated delivery system, we could show that the Tat-associated particles can 1Department of Pharmacology, School of Basic Medical Sciences, be used as a highly cell imaging agent (with fluorescence incor- Capital Medical University, Beijing 100069, China porated cargos), and a gene therapeutic agent (with si-RNA 2Beijing Institute of Pharmacology and Toxicology, Beijing 100850, incorporation). China *Corresponding author. Tel: +86-10-8391 1520; Fax: +86-10-8391 1520. E-mail address: [email protected] Structural and orientational study of peptide Purpose: Long acting luteinizing hormone-releasing hormone (LHRH) antagonists are novel LHRH antagonist analogues. The phenylseptin in micellar environment bioactivities of the long acting peptides were evaluated by the rat testosterone test model. The designed peptides showed a long action V. H. O. Munhoz*1,2, M. T. Q. de Magalha˜es3,4, R. M. Verly1, of inhibiting testosterone secretion compared with the parent peptide. S. F. C. de Paula1, J. M. Resende1, C. Aisenbery2, D. Pilo´-Veloso1, The metabolic stabilities of the peptides were assessed in pancreatin C. Bloch Jr.3, and B. Bechinger2 system. It showed there were good protease-resistant activities for the peptides. In the present study, rapid liquid chromatography-electro- 1Departamento de Quı´mica, Instituto de Cieˆncias Exatas, spray tandem mass spectrometry (LC-ESI–MS/MS) detection was Universidade Federal de Minas Gerais, Av. Pres. Antoˆnio Carlos, established for the simultaneous determination of the five LHRH 6627, 31270090, Belo Horizonte-MG, Brazil (*[email protected]) antagonists in rat plasma for the purpose of high-throughput phar- 2Universite´ de Strasbourg/CNRS UMR7177, Institut de Chimie, 4, macokinetic screening. rue Blaise Pascal, 67070 Strasbourg, France Method: The method was operated under selected reaction moni- 3 Embrapa Recursos Gene´ticos e Biotecnologia, PqEB- Final W5, toring (SRM) mode in the positive ion mode. The analytes were Brası´lia DF, Brazil extracted from 50 ll rat plasma by liquid–liquid extraction with 4 Po´s-graduaçaõ em Biologia Molecular, Instituto de Biologia, acetonitrile. Chromatographic separation of the analytes was suc- Universidade de Brası´lia, Campus Universita´rio Darcy Ribeiro, cessfully achieved on a Hypersil Gold (100 9 2.1 mm, 3 lm) using a Brası´lia DF, Brazil E-mail:[email protected] mobile phase composed of acetonitrile–water (30:70) containing 0.1% (v/v) formic acid. Phenylseptin (Phes) is a new class of antimicrobial peptides isolated Results: The method showed good linearity and no endogenous from the anuran Hypsiboas punctatus. They are known to be material interfered with the marked compounds. The limits of quan- phenylalanine-rich peptides on their N-terminus. There are two tification of five peptides were 10–4,000 ng/ml. The average extract knowing forms belonging to this class which share, to some degree, recoveries of the five compounds in the rat plasma were all over 70%. the same amino acid sequence, except for an enantiomerization at Intra-day and inter-day precisions (R.S.D.%) were all within 15% and one phenylalanine residue. Both peptides show highly a-helical and the method assessed a quite good accuracy (R.E.%). amphipathic structures when in contact micelles and bilayers. Conclusion: This method has been successfully applied in the In vitro assays have shown that both peptides have mild antimi- simultaneous quantification and the pharmacokinetic studies of these crobial activity against Gram-positive and Gram-negative bacteria, five long acting peptides after sublingual intravenous administration being the Phenylseptin containing D-amino considerably more active in rats. than its homologue. Nonetheless, the exact mechanism which describes the antimicrobial activity of these peptides is still not fully determined. Size dependent uptake efficiency of cell penetrating To elucidate some aspects of this mechanism, static cross-polari- 15 2 peptide mediated delivery systems: mechanisms zation N and H solid-echo solid-state NMR were performed in samples with the peptide in contact with mechanically oriented lipid and therapeutic application bilayers. By combining these two experiments, it was possible to determine the orientational constraints which were used on these Heesok Kim, So Ra Kang, Byung Keun Oh, and Kwanwoo Shin* peptides’ structures determined through solution NMR in the presence of DPC micelles, leading to the most probable orientation of the Department of Chemistry and Interdisciplinary program of integrated peptide when it interacts with membrane-like media. Biotechnology, Institute of Biological Interfaces, Sogang University, With these results, it was possible to observe that both peptides, Seoul 121-742, South Korea although similar in amino acid composition, interact very differently *E-mail: [email protected] with membrane-like environments and thus exert different activity.

123 12th International Congress on Amino Acids, Peptides and Proteins S65

The cause of the difference between their activities probably is the contraction induced by bradykinin. These peptides, called bradykinin differential anchoring, showed by the variation on the 2H quadrupolar potentiating peptides (BPPs), have also demonstrated the ability to splitting, which is quite sensitive to strong interactions between the inhibit angiotensin-I converting enzyme (ACE). Typically, BPPs from side chains and the bilayer. Bothrops jararaca contain 5–14 amino acid residues and have similar Keywords: Antimicrobial peptides, Solid-state NMR features, such as a high content of proline residues and the tripeptide Ile-Pro-Pro at the C-terminus. Various therapeutic systems used for targeting drugs to the colon are capable of minimizing the possible drawbacks associated Structural and thermodynamic studies of the dimeric with drug degradation and, moreover, are able to modify the peptide homotarsinin in aqueous and mimetic administered dose. Therefore, cyclodextrins could be used once this membrane environments class of molecules is shown as a potential colon-specific drug carrier system. The heptapeptide BPP7a, p-Glu1Asp2Gly3Pro4Ile5Pro6Pro7, forms Rodrigo M. Verlya*, Jarbas M. Resendea,Fa´bio C. L. Almeidaa an association complex with b-cyclodextrin. The peptide and its Marcelo P. Bemquererb, and Dorila Pilo´-Velosoa complex were characterized by circular dichroism (CD) and isothermal titration calorimetry (ITC), which showed a very weak interaction aDepartamento de Quı´mica/ICEx/UFMG, between b-cyclodextrin and the peptide. Assignments of all hydro- bEmbrapa-Recursos Gene´ticos e Biotecnologia/Brası´lia-DF, gen resonances of the peptide alone and as a complex were made cCNRMN/UFRJ *E-mail: [email protected] using 1H nuclear magnetic resonance (NMR) experiments at 400 and 600 MHz. High resolution diffusion ordered spectroscopy The homodimeric peptide homotarsinin was isolated from Phyllome- (HR-DOSY) experiments were carried out to establish the self- dusa tarsius, a tree-frog that inhabits the Brazilian Amazonian basin. aggregation state of BPP7a. This peptide is composed of two identical linear chains each with 24 In addition, the anti-hypertensive activity of the BPP7a/b-cyclo- amino acid residues carrying a natural amidation at the C-termini and dextrin complex was evaluated in spontaneous hypertensive rats connected by a single disulfide bond. Antimicrobial assays demonstrate (SHR), showing increased activity than pure BPP7a. In vivo experi- that homotarsinin dimmer as well as its monomer shows antimicrobial ments in SHR showed that the complex reduced blood pressure in the activity against both Gram-positive and Gram-negative bacteria. rats, which may be attributed to increased bioavailability of BPP7a From aqueous buffer and DPC micelles NMR data, the ten lowest after its complexation with b-cyclodextrin. energy structures of homotarsinin were calculated, showing strong amphipathic character and also very similar helical contents for both media. In aqueous buffer the inter-chain NOEs observed indicate close contact between the individual chains and the calculated structures showed tertiary coiled-coil arrangement, which is characterized by the hydrophobic residues positioned at the homodimer internal portion. In Synthesis and study of the solvent effect on self- contrast, the absence of inter-chain NOEs when homotarsinin is in assembly in a novel tripeptide molecule contained contact with the membrane suggests the existence of a more open unusual amino acid using molecular dynamics structure. These results imply that hydrophobic peptide–peptide inter- simulations chain interactions stabilize the homotarsinin coiled–coiled structure in water. However, when homotarsinin binds to the membrane, these peptide–peptide interactions are replaced by peptide-membrane inter- Bahareh Talaei, Hengameh Fallah, and Saeed Balalaie* actions in order to allow a better partitioning of the amphipathic helices into the membrane interface. Isothermal titration calorimetric study Peptide Chemistry Research Center, K. N. Toosi University shows a great homotarsinin interaction with both POPC and POPC:- of Technology, Tehran, Iran, POPG (3:1) LUVs. Dynamic light scattering measurements indicated Corresponding author: [email protected] that homotarsinin associates to phospholipid vesicles leading to a supramolecular structure, which is characterized by an increased The diphenylalanine dipeptide is a suitable building block for hydrodynamic diameter, up to certain a point when the disruption occurs. molecular self-assembly. We were encouraged to synthesis some Keywords: Peptide antibiotics, Amphipathic a-helix, Coiled-coil novel peptides which contained c-amino acids in their backbones. structure, nmr Gabapentin and baclofen were the best candidates for this approach. In this article, we focused on the synthesis of some peptides. Between the synthesized peptides, H-Phe-Phe-Baclofen- Study of a bradykinin potentiating peptide, bpp7a, OH was selected due to the existence of aromatic moiety in the structure of baclofen which is responsible for self-assembly via and its b-cyclodextrin complex p–p stacking. The realm of applications of computational chemistry is con- Ivana Lula,1 Frederico B. De Sousa, Aˆ ngelo M. L. Denadai, siderably expanding owing to steady advances in computer power. Danielle Ianzer, Antoˆnio C. M. de Camargo, Robson A. S. Santos, An ab initio quantum mechanical calculation was performed on and Rubeń D. Sinisterra H-Phe-Phe-Baclofen-OH tripeptide using Gaussian 03 package. The gas phase energy of the tripeptide was optimized at the HF/3-21G 1Universidade Federal de Minas Gerais, Instituto de Cieˆncias Exatas- level. Molecular Dynamics Simulations were carried out with Departamento de Quı´mica. Av. Antonio Carlos 6627, Belo Horizonte explicit solvent (water) using Gromacs 4.0.7 package. The details MG 31270-901, Brazil of simulations will be reported. Different possible ways of self- assembly such as H-bonding, p–p stacking and T-stacking can be Serpent venom, especially from Bothrops jararaca, contains small seen in the presentation. The details about the self assembly of molecule peptides which greatly enhance the smooth-muscle these compounds will discuss in the conference.

123 S66 12th International Congress on Amino Acids, Peptides and Proteins

Synthesis of novel bisphosphonate containing peptide esteriﬁcation, methanolysis, Heck and Buchwald. The inﬂuence and unusual amino acids of the different starting materials is evaluated and numerous studies are underway at this time to explore their biological activities. Sorour Ramezanpour, Saeed Balalaie*, Armin Arabanian, and Hamid Reza Bijanzadeh Topological determination of the antimicrobial peptide Peptide Chemistry Research Center, K.N. Toosi University of Technology, Tehran, Iran hylaseptin P2 in oriented bilayers

Biophosphonate (BPs) are an important class of drugs with dif- V. H. O. Munhoz1,2, B. A. Assunçaõ*1, S. F. C. de Paula1, ferent biological activities. They can be used for treatment of J. M. Resende1, D. Pilo´-Veloso1, and B. Bechinger2 diseases characterized by abnormal calcium metabolism; such as osteoprosis and tumor-associated osteolysis. It was shown that the 1Departamento de Quı´mica, Instituto de Cieˆncias Exatas, existence of heterocycle skeleton has an efficient role for the Universidade Federal de Minas Gerais, Av. Pres. Antoˆnio Carlos, biological activity of the bisphosphonate derivatives such as 6627, 31270090, Belo Horizonte MG, Brazil (*[email protected]) imidazole moiety in zoledronic acid as the anti-cancer bisphosph- 2Universite´ de Strasbourg/CNRS UMR7177, Institut de Chimie, 4, rue onate compound. In continuation of our research for the synthesis Blaise Pascal, 67070 Strasbourg, France *[email protected] of novel peptides, some novel peptides containing free carboxylic acid functional group were synthesized via solution phase peptide Keywords: Antimicrobial peptides, Solid-state NMR synthesis (SIS) or solid phase peptide synthesis (SPPS) approaches. The Hylaseptin P2 (HSP-2) is an antimicrobial, a-helical peptide We synthesized novel peptides with histidine in their structure. present on Hypsiboas punctatus anurans, found at the Amazon rain- Meanwhile, some unusual amino acids such as gabapentine and forest. It has activity against both Gram-positive and Gram-negative baclofen, were used as the starting materials. The synthesis of bacteria and fungi. It is believed that its mechanism of action is bisphosphonate moiety was done using reaction of phosphorus guided by its affinity with the bacterial membrane. Although the trichloride and phosphorous acid with free carboxylic acid moiety. outlines of the mechanism are proposed, its full pathway is not yet The details for the synthesis of desired bisphosphonates will be completely unveiled, hence the need to perform studies to elucidate it discussed in the conference. better. One of the techniques that have been useful to study peptide- phospholipid interactions is the solid-state NMR of isotopically labelled peptides in oriented bilayers1. In this work, different experiments were done in order to get complementary information regarding the peptide lipid interaction Synthesis of pseudopeptides contained indole skeletons through 31P, 2H, and 15N cross-polarization NMR experiments. The and their applications in synthesis of lipophilic peptides samples consisted on a mixture containing the peptide and POPC on a 1.5:100 molar rate spread on 18 very thin glass plates, stacked one 15 Sorour Ramezanpour, Nahid Sadeghi Alavijeh, Saeed Balalaie*, over the other, and hydrated at 93% of relative humidity. The N and Hamid Reza Bijanzadeh experiment was performed on Bruker AVANCE AMX400 wide-bore with a commercial double-resonance E-free probe and the 2H experiment, on a Bruker AVANCE 300 with a static commercial tri- Peptide Chemistry Research Center, K.N. Toosi University 15 of Technology, P.O.Box 15875-4416, Tehran, Iran ple-resonance probe. The peptides were synthesized with N label at L18 and 2H label at A16. The resulting 31P spectrum indicates a good orientation of the Pseudopeptides contained indole skeletons, suitable for synthesis of 15 2 lipophilic peptides which can probably affect the ability of these bilayer. The N spectrum shows a peak at d74 ppm and the H bioactive compounds to penetrate blood–brain barrier (BBB) as well spectrum, a quadrupolar splitting of 27 kHz. as transport across cell membranes, can be synthesized by using By calculating the tilt and rotational pitch angle it was possible to suitable indole carboxylic acid derivatives. Indole nucleus is a determine the orientation of the peptide. substructure found in numerous natural products and pharmaceuticals possessing anti-inflammatory, antimalarial, antidepressant, antitumor and various other activities. Therefore, we have been Plant amino acids encouraged to synthesize some new pseudopeptides containing the indole moiety hoping to obtain new compounds with potential biological activities which contained lipophilic moieties and also An omics approach to elucidating amino acid amide bonds. Among the protocols for the synthesis of pseudopeptides and metabolism in plant peptides or amino acids, the important method is the Ugi four component reaction (Ugi-4CR) as it offers significant advantages Kansuporn Sriyudthsak, Eiji Okamura, Yuji Sawada, over stepwise procedures, especially with respect to diversity, and Masami Yokota Hirai complexity, green chemistry, and atom economy. In this approach, we have designed an Ugi-4CR strategy via the reaction RIKEN Plant Science Center/CREST, JST of benzaldehyde derivatives, Indole carboxylic acids, primary amines and isocyanides to construct these valuable pseudopep- These days it is becoming more and more important to understand tides which have a huge potential for further reactions such as plant metabolism, because it is closely related to plant production,

123 12th International Congress on Amino Acids, Peptides and Proteins S67 namely, food and biomass production. However, genes involved in Characterization of type 1 ribosome inactivating metabolism and its regulation are not yet fully identified even in proteins in edible plants Arabidopsis. We are aiming at discovery of novel genes responsible for amino acid metabolism and secondary metabolism by 1 2 3 means of transcriptomics and metabolomics. Based on coexpres- Munish Puri *, Inderdeep Kaur and C. J. Barrow sion analysis using a large-scale transcriptome dataset of 1,3 Arabidopsis, we have identified/predicted novel gene functions in Centre for Biotechnology and Interdisciplinary Biosciences, glucosinolate (secondary metabolite specific to Capparales) and Institute for Technology Research and Innovation (ITRI), Geelong amino acid metabolism. In order to obtain a large-scale metabo- Technology Precinct (GTP), Deakin University, Victoria 3217, Australia, lome dataset, we have established an ultra high-throughput 2 methodology named ‘widely targeted metabolomics’ (Sawada Fermentation and Protein Biotechnology Laboratory, Department et al. 2009, Plant Cell Physiol. 50:37–47). We obtained time- of Biotechnology, Punjabi University, India series metabolome data after a certain metabolic perturbation. Furthermore, we constructed a mathematical model of amino acid The ribosome inactivating proteins (RIPs) from plants possess RNA metabolism by our novel simulation algorithm. The simulation N-glycosidase activity that depurinates the major rRNA, thus damaging results agreed well with the experimental data (time-series me- ribosome in an irreversible manner and arresting protein synthesis. RIPs tabolome data), showing the algorithm is useful to occur in fungi, bacteria and plants and are abundant in angiosperms, topologically predict dynamic behaviors of metabolite levels in the where they appear to have defensive role. RIPs are presently classified as metabolic system. In this presentation, our recent progress will be rRNA N-glycosidase in the enzyme nomenclature (EC 3.2.2.22) and do introduced. exhibit other enzymatic activities such as ribonuclease and deoxy- ribonuclease activities. RIPs are classified into two groups based on their difference in their primary structure. Type I RIPs consist of a single polypeptide chain of approximately 26–35 kDa that possess an RNA N-glycosidase activity. These proteins have attracted a great deal of Bio-available diamino amino acids in soil of differently attention because of their anti-viral, anti-tumor, and anti-microbial managed mountain meadows and forests activities, which is useful in medical research and development. Here, we describe isolation of a novel protein from Momordica sp, a high- climbing vine from family Cucurbitaceae which is native to the tropical Valerie Vranova1, Dalibor Janous3, Klement Rejsek1, 1 2 1 regions of Africa, Asia, Arabia and Caribbean. The purified protein has Jindrich Kynicky , Martin Brtnicky and Pavel Formanek been verified by SDS-PAGE and mass spectrometry to contain only

1 single chain Type-1 ribosome inactivating proteins (RIPs). With present Mendel University in Brno, Faculty of Forestry and Wood experiments, we determined the presence of RIPs in edible plant mate- Technology, Department of Geology and Soil Science, rials, including some that are eaten raw by human beings. The novel Zemedelska 3, 613 00, the Czech Republic, protein is further characterized to validate its therapeutic potential. e-mail: [email protected], 2Mendel University in Brno, Faculty of Agriculture, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Contributions of individual amino acids to CLV3 Zemedelska 1, 613 00, The Czech Republic, 3CzechGlobe, Global Change Research Centre AS CR, v.v.i., peptide in shoot apical meristem maintenance Porici 3b, 603 00 Brno, The Czech Republic in Arabidopsis

Bio-available or so-called ‘‘free’’ amino acids occurring either in Xiufen Song, Dali Yu, Peng Guo, Tingting Xu, Shichao Ren, soil solution or exchangeably bound on soil colloids may be directly and Chun-Ming Liu* taken up by plant roots without previous mineralization. This may be especially relevant in alpine, arctic and boreal areas, where the Center for Signal Transduction and Metabolomics, Institute availability of mineral nitrogen is low, and thus, amino acids are of Botany, Chinese Academy of Sciences, Nanxincun 20, considered to be important for plant nutrition. In this work we have Fragrance Hill, Beijing 100093, China concentrated on selected bio-available diamino amino acids including (1) nitrogen rich and more strongly exchangeably bound Compared to traditional phytohormones, peptide hormones in theory arginine as well as strongly exchangeably bound lysine, and (2) have more flexibility in changing their primary structures and sub- cystine. This work has shown that in the top 7 cm of Ap horizon of sequently specificities in evolution through amino acid (AA) differently managed mountain meadows, arginine, lysine and cystine substitutions. Previous genetic and biochemical studies reveal that formed (on average) 14, 2.5 and 11, respectively, of the totally CLV3 acts as a small peptide to interact with the CLV1/CLV2/SOL2 seventeen amino acids (aspartic acid, glutamic acid, serine, histi- receptor complexes for shoot apical meristem (SAM) maintenance. dine, glycine, threonine, alanine, tyrosine, valine, methionine, To elucidate how peptide specificities are established in AA sequence, phenylalanine, isoleucine, leucine, proline plus the three studied we introduced point mutations to the peptide-coding and its flanking diamino amino acids) extracted from soil using 0.5 M ammonium regions of CLV3 to substitute AAs one by one by alanine and acetate. In Oe horizon of differently managed mountain Norway introduced these constructs to clv3-2 for in vivo complementation. spruce stands, arginine, lysine and cystine formed (on average) 2.7, This study yielded a complete contribution map of individual AAs for 3.1 and 7.1, respectively, of the seventeen extracted amino acids. the CLV3 function, showed that glycine-6, aspartate-8 and histidine- Each of the three studied diamino amino acids formed ca. 7–8% of 11 are among the most important residues, while previously assigned the total bio-available nitrogen including nitrogen of all seventeen modification residues of proline-7 and -9 and flanking sequences amino acids, ammonium and nitrates in soil of all studied showed very little contributions. Further, in vitro SAM treatments ecosystems. using chemically synthesized alanine-substituted CLV3 peptides Acknowledgments: This work was financially supported by IGA revealed an evidently different contribution pattern as the in vivo MENDELU 2010-2012 and NAAR - QI91C054. assay and the previously reported root assay, suggesting different

123 S68 12th International Congress on Amino Acids, Peptides and Proteins perception machineries are involved. The contribution of individual well as their ratio within soil profiles in young spruce stands (soil type AA in CLV3 established in this work may help to understand peptide Typic Haplohumod) and meadows (soil type Oxyaquic Hapludalf) of hormones in general. mountain regions, and young oak stands in lower altitudes (soil type Oxyaquic Haplustept). Results of this work showed that the L-toD- glutamic acid respiration ratio ranged from 1.2 to 5.1 in different Effect of alkali-extracted rice protein on the renal horizons of soil profiles, being the lowest in organic horizons and function in type 2 diabetic rats decreasing with depth. Acknowledgments: This work was financially supported by IGA MENDELU 2010-2012 and NAAR, QI91C054. Reiko Watanabe1, Masatoshi Kubota2, Akihiko Saito3, Takehisa Kumagai4 and Motoni Kadowaki2 Mineralization of c-aminobutyric acid, citrulline, 1Department of Health and Nutrition, University of Niigata Prefecture, Japan, ornithine and b-alanine in soil 2Graduate School of Science and Technology, Niigata University, Japan, Peter Dundek1, Valerie Vranova1,KlementRejsek1, Martin Brtnicky2, 3Graduate School of Medical and Dental Sciences, Niigata Marian Pavelka3,JindrichKynicky1 and Pavel Formanek1 University, Japan, 4Kameda Seika Co., Ltd., Japan 1Mendel University in Brno, Faculty of Forestry and Wood Technology, Department of Geology and Soil Science, Zemedelska 3, Backgrounds and objectives: Type 2 diabetes mellitus (T2DM) is 613 00, The Czech Republic; e-mail: [email protected], rapidly increasing and is becoming a serious problem all over the 2Mendel University in Brno, Faculty of Agriculture, Department world. T2DM caused by life style is associated with several long-term of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, complications, including diabetic nephropathy, and the important role Zemedelska 1, 613 00, The Czech Republic, of dietary protein in kidney function has been recognized. Rice is a 3CzechGlobe, Global Change Research Centre AS CR, v.v.i., stable food and important as energy and protein sources in many Porici 3b, 603 00 Brno, The Czech Republic countries. We had clearly proved that alkali-extracted rice protein (AE-RP) lowered total cholesterol and triglycerides in the plasma and Gamma-aminobutyric acid (GABA), citrulline, ornithine and b-ala- liver. Therefore, we focused on effects of AE-RP on T2DM, espe- nine play different roles in plants including protection against stress. cially diabetic nephropathy, as new beneficial functions. These amino acids were also found in soils of various ecosystems Materials and methods: AE-RP used included high CP content including subtropical heathlands, tundra communities, forests, coastal ([90%) which have high digestibility and bioavailability. Male non- salt marshes, arable land and grasslands. In soil, these amino acids obese type 2 diabetic Goto-Kakizaki (GK) rats were fed high sucrose may occur incorporated in soil organic matter, as ‘‘free’’ in soil diets for 10 weeks with 20% casein (C) or AE-RP. Fasting blood glucose solution or exchangeably bound to soil colloids. GABA was found in levels were measured every other week. Three-day urine collection was the highest concentrations mainly in waterlogged soils forming up to performed to determine albuminuria at the end of experiment. Kidneys 50% of the total ‘‘free’’ amino acid-N pool. Ornithine was found to be are removed and the tissue sections obtained for histological analysis. among dominant amino acids in leachates from some forest soils. Results: AE-RP had no effects on fasting blood glucose levels. Besides the ‘‘free’’ amino acid pool, occurrence of b-alanine was However, the level of urinary albumin in GK rats fed AE-RP was reported in hydrolysates of soils or humic substances. Data on kinetics significantly lower (36%) than that of C group. Degrees of renal of GABA, citrulline, ornithine and b-alanine mineralization in soils of glomerular damages were significantly higher in C group, but those in different properties are presented and discussed in this work. AE-RP group were similar to those in Wistar rats fed high sucrose C Acknowledgments: This work was financially supported by IGA diets. These results suggest that AE-RP has a suppressive effect on MENDELU 2011. progression of diabetic nephropathy.

Nascent peptide-mediated control of gene expression Mineralization of enantiomers of glutamic acid in soil in Arabidopsis: feedback regulation of methionine biosynthesis and beyond Peter Dundek1, Valerie Vranova1, Klement Rejsek1, Marian Pavelka3, Jindrich Kynicky1, Martin Brtnicky2 and Pavel Formanek1 Hitoshi Onouchi1,2,IsaoEbina3,MarikoTakemoto1, Shun Watanabe3, 3 3 4 1 1Mendel University in Brno, Faculty of Forestry and Wood Noriyuki Onoue , Yui Yamashita , Yayoi Endo , Hiroaki Koyama , 1 5 3 Technology, Department of Geology and Soil Science, Zemedelska 3, Yoko Yamashita-Nagami ,HiroTakahashi, and Satoshi Naito 613 00, the Czech Republic, e-mail: [email protected], 1 2Mendel University in Brno, Faculty of Agriculture, Department Division of Applied Bioscience, Graduate School of Agriculture, of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Hokkaido University, Sapporo 060-8589, Japan, 2 Zemedelska 1, 613 00, the Czech Republic Core Research for Evolutional Science and Technology, Japan 3CzechGlobe Research Center, Porici 3 b, Brno, the Czech Republic Science and Technology Agency, Kawaguchi 332-0012, Japan, 3Division of Life Science, Graduate School of Life Science, Hokkaido D-Amino acids artiﬁcially added to soils are mineralized at lower rates University, Sapporo 060-8589, Japan, 4 compared to their corresponding L-enantiomers; nevertheless, the Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan, 5 mechanism of this effect is not known. The L-toD-amino acid res- College of Bioscience and Biotechnology, Chubu University, piration ratio is commonly reported in the range from 1.0 to 8.7. The Matsumoto-cho 1200, Kasugai, Aichi 487-8501, Japan differences between L- and D-amino acid respiration were found to be affected by stress conditions (acidity, heavy metals). In this work we Expression of the Arabidopsis CGS1 gene, which encodes the ﬁrst have attempted to determine respiration of L- and D-glutamic acid as committed enzyme of methionine biosynthesis, is feedback

123 12th International Congress on Amino Acids, Peptides and Proteins S69 regulated at posttranscriptional level. A 14-residue stretch of amino Proteome and free amino acid of Polygonum minus leaf acid sequence, termed the MTO1 region, encoded within the first exon of CGS1 itself is involved in this regulation. Our in vitro Roohaida Othman1,2, Hana-Marlin Mahfodz2, Chang Li Yen3, studies have revealed that, during translation of CGS1 mRNA, the Nur Afiqah Sukiran1, Syarul Nataqain Baharum1, MTO1 region within the nascent CGS1 polypeptide acts inside of and Normah Mohd Noor1 the ribosome before emerging from it to cause translation elon- gation arrest in response to S-adenosyl-L-methionine (AdoMet). 1Institute of Systems Biology, Universiti Kebangsaan Malaysia, The translation arrest then triggers CGS1 mRNA degradation. In Malaysia, addition, we recently found that AdoMet induces compaction of the 2School of Biosciences and Biotechnology, Faculty of Science nascent peptide chain inside the ribosomal exit tunnel upon the and Technology, Universiti Kebangsaan Malaysia, Malaysia, translation arrest. Apart from the CGS1 system, most of eukaryotic 3Department of Medical Microbiology, Faculty of Medicine, examples of nascent peptide-mediated ribosomal regulation involve University of Malaya, Malaysia, peptides encoded by upstream open reading frames (uORFs) Correspondence: [email protected] located in 50 untranslated regions. In order to further identify regulatory nascent peptides, we searched for Arabidopsis uORFs The aromatic plant, Polygonum minus, produces essential oil of high whose amino acid sequences are conserved in a wide range of economic value with high demands in the food, flavor and fragrance dicot plants by using a comparative genomic approach, and iden- industry. The essential oil contains a variety of secondary metabolites, tified 14 novel conserved uORFs. Mutational analyses revealed including aliphatic aldehydes and terpenes that have been associated that 4 of the conserved uORFs control the expression of the with this plant unique smell. To understand the secondary metabolite main coding sequence in an amino acid sequence-dependent biosynthesis, a proteomic approach was undertaken to identify the manner. proteins and amino acids produced in this plant. P. minus leaf proteins were resolved into 1869 polypeptides with pI values ranged between 4 and 7 and relative molecular masses from 10 to 100 kDa. A master Proline accumulation is inhibitory to Arabidopsis leaf polypeptide profile was generated based on the consistently seedlings during heat stress expressed protein pattern. Proteins present in 97 high quality spots were identified using Mascot software and Viridiplantae database (NCBI) and by comparison with in-house EST database of this plant. Wei-Tao Lv, Bin Lin, Min Zhang, and Xue-Jun Hua* Enzymes involved in carbohydrate metabolism and photosynthesis were among the main proteins identified. Subsequent amino acid Research Center for Molecular and Developmental Biology, analysis using LC–TOF–MS showed that from 19 amino acids Key Laboratory of Photosynthesis and Environmental detected, leucine was found to be the most abundant amino acid. The Molecular Physiology, Institute of Botany, Chinese Academy enzyme involved in the precursor biosynthesis of leucine was identi- of Sciences, Beijing 100093, People’s Republic of China, fied from the proteomic study. The proteome map obtained provides Graduate School of Chinese Academy of Sciences, Beijing 100049, the basis for further study on P. minus physiology and will contribute China to improve our understanding of plant metabolism in aromatic plant thus aiding in crop improvement effort. The effect of proline (Pro) accumulation on heat sensitivity was investigated using transgenic Arabidopsis plants ectopically expressing Delta(1)-pyrroline-5-carboxylate synthetase 1 gene (AtP5CS1) under the control of a heat shock protein (HSP) 17.6II The defense function and secondary structure analysis gene promoter. During heat stress, the heat-inducible expression of AtP5CS1 transgene was capable of enhancing Pro biosynthesis. of HpaXm from Xanthomonas citri subsp. malvacearum 12-day-old seedlings were first treated with heat at 37°C for 24 h to induce Pro, and then were stressed at 50°C for 4 h. After Weiguo Miao1*, Congfeng Song2, Fucong Zheng1, recovery at 22°C for 96 h, the growth of Pro-overproducing plants and Jinsheng Wang2* was significantly more inhibited than were control plants that do not accumulate Pro, manifested by lower survival rate, higher ion 1College of Environment and Plant Protection, Hainan University, leakage, higher ROS and MDA levels and increased activity of Haikou 570228 China, Pro/P5C cycle. The activity of antioxidant enzyme SOD, GPX, and 2Department of Plant Pathology, Nanjing Agricultural University, CAT, but not that of GR and APX, increased in all lines after heat Nanjing 210095 China, treatment, but the increase was more significant in Pro-overpro- *Corresponding author Weiguo Miao, e-mail: ducing seedlings. Staining with MitoSox-Red, reported for being [email protected], Jinsheng Wang, - able to specifically detect O2 formed in mitochondria, showed that e-mail: [email protected] Pro accumulation during heat stress resulted in elevated level of ROS in mitochondria. Interestingly, exogenous abscisic acid and HpaXm from, HpaXm (harpin) is a type III secreted protein of the ethylene were found to partially rescue the heat sensitive pheno- cotton blight bacterial pathogen Xanthomonas citri subsp. malva- type of Pro-overproducing seedlings. Measurement of ethylene and cearum that elicits a hypersensitive response (HR) in nonhost ABA levels further confirmed that these two hormones are nega- tobacco. Two a-helical domains were predicted in HpaXm by the HNN tively affected in Pro-overproducing seedlings during heat stress. program; a hydrophobic h-region was found between the N- and Our results indicated that Pro accumulation under heat stress C-terminal flanking regions. The former contains the 1st to 18th decreases the thermotolerance, probably by increased ROS pro- residues, in which ab-strand resides. The latter contains the 101st to duction via Pro/P5C cycle and inhibition of ABA and ethylene 133rd residues. H-regions were also found in other harpin-like pro- biosynthesis. teins. A signal peptide was predicted to exist in the 15 leading amino

123 S70 12th International Congress on Amino Acids, Peptides and Proteins acids of the N-terminus. The identical heptads, 39-LDQLLTQ– The effect of amino acids on activity of extracellular LIMALLQ-52, were found in the N-terminal a-helix region of HpaXm. acid phosphomonoesterase in soil Italicized letters indicate different residues in HpaXm. Three DNA- binding residues were found in N and C-terminal regions in HpaXm, Valerie Vranova1, Silvia Chersich2, Dalibor Janous4, Klement especially C-terminal flanking regions. HpaXm is predicted to contain 1 1 3 1 two potential coiled-coil (CC) regions, one at the N-terminus with a Rejsek , Jindrich Kynicky , Martin Brtnicky and Pavel Formanek high probability of formation, and one at the C-terminus with a lower 1 probability of formation. To investigate the role of the CC regions in Mendel University in Brno, Faculty of Forestry and Wood Technology, Department of Geology and Soil Science, HpaXm function, two peptides and two mutated peptides containing 14 residues of the N- and C-terminal CC regions were designed and Zemedelska 3, 613 00, The Czech Republic, e-mail: [email protected], synthesized. Peptide purities of the synthetic peptides were all higher 2 than 84%. Tobacco leaves infiltrated with aqueous solutions of these Pavia University, Department of Earth Sciences, Via Ferrata, 1 27100, Pavia, Italy, peptides showed an HR within 24 h. The result indicated that the 3 peptide, containing 14 amino acids from the N-terminal CC region of Mendel University in Brno, Faculty of Agriculture, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, HpaXm, was sufficient to cause an HR after infiltrating the intercel- Zemedelska 1, 613 00, The Czech Republic, lular spaces of tobacco leaves. Specifically, the CC domain of the 4 N-terminal a-helix contributed to HR activity. CzechGlobe, Global Change Research Centre AS CR, v.v.i., Acknowledgments: This work was supported by grants from Porici 3b, 603 00 Brno, The Czech Republic the NKBRPC (2003CB114204 and 2006CB101902), NKPC (2004BA901A36), RFDP(20104601110004), CARS-34-GW8 and Extracellular acid phosphomonoesterase (orthophosphoric monoes- KYQD1006(China). ter phosphohydrolase, E.C. 3.1.3.2) catalyzes hydrolysis of a variety of organic phosphomonoesters and is therefore important in soil organic phosphorus mineralization and plant nutrition. Activity of acid phosphomonoesterase extracted from e.g. larvae of cod- The effect of abandonment of mountain meadows worm (Phocanema decipiens) or muscle of codfish (Gadus morhua) on uptake of 14C-labelled glutamic acid and alanine was found to be inhibited by different compounds including amino by soil microbial community acids. We have adopted this knowledge to find out if amino acids (L-cysteine, L-glutamic acid, L-arginine and D-alanine), naturally occurring in soil, may affect activity of extracellular acid phos- Klement Rejsek1, Valerie Vranova1, Dalibor Janous3, phomonoesterase in soil and thus participate in regulation of Michael Po¨schl4, Jindrich Kynicky1, Martin Brtnicky2 phosphorus cycling in terrestrial ecosystems. Amino acids were and Pavel Formanek1 applied to soil in concentration of 5 lgg-1 dry soil. Results of this work showed that L-arginine has no effect, whereas D-alanine, L- 1Mendel University in Brno, Faculty of Forestry and Wood glutamic acid and L-cysteine tend to slightly increase (by up to Technology, Department of Geology and Soil Science, Zemedelska 3, 10%) activity of extracellular acid phosphomonoesterase in tested 613 00, the Czech Republic; e-mail: [email protected], soils. 2Mendel University in Brno, Faculty of Agriculture, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemedelska 1, 613 00, The Czech Republic, 3CzechGlobe Research Center, Porici 3 b, Brno, The Czech Republic, Polyamines 4Mendel University in Brno, Faculty of Agriculture, Department of Molecular Biology and Radiobiology, Zemedelska 1, 613 00, The Czech Republic Characterization of the hypusine biosynthetic pathway The effect of a 13-year abandonment of previously long-term mown from Leishmania donovani 14 14 mountain meadows on uptake of L-glutamic acid ( CO2H[ CH2]2 14 14 14 14 [NH2] CO2H) and L-alanine ( CH3 CH[NH2] CO2H) by microbial Rentala Madhubala community of Ap horizon (3–13 cm) (soil type Oxyaquic Hapludalf) was determined in this work. Soil of Ap horizon of mown and School of Life Sciences Jawaharlal Nehru University, New Delhi, abandoned meadows showed the same total carbon content and India similar total nitrogen content (0.29 vs. 0.33%). One hundred fifty microlitres of alanine solution (15.7–717 lM) or glutamic acid Hypusine (N€-(4-amino-2-hydroxybutyl) lysine), an unusual amino solution (9.2–588.3 lM) was briefly mixed with 0.3 g wet soil in acid derived from the polyamine spermidine, is present in all the 15-ml polypropylene tubes. One ml of 1 M NaOH was added to each eukaryotes. It is synthesized as a result of post-translational modi- 14 sample tube to capture the respired CO2 and the sample tubes were fication occurring exclusively on one cellular protein, eukaryotic sealed with a gas-tight stopper and incubated at 14.5°C for 3 h. initiation factor 5A (eIF5A). It is formed in two enzymatic steps. Subsequently, the soils were extracted with 0.5 M K2SO4 to remove The first step is catalyzed by the enzyme deoxyhypusine synthase the not-utilized amino acids (10 min, 200 rpm) followed by centri- (DHS) (EC 2.5.1.46) which catalyses the NAD+ dependent transfer fugation (3,550 rpm, 20 min) and counting of the 14C label remaining of the 4-aminobutyl moiety of spermidine to a specific lysine resi- in the supernatant solution. The abandonment of the meadow had no due of the eIF5A precursor protein to form an intermediate, significant (P [ 0.05) effect on L-alanine or L-glutamic acid uptake. deoxyhypusine. This intermediate is subsequently hydroxylated by -1 Uptake rate of L-alanine at 717 lM was 135-159 nmol g dry soil the enzyme deoxyhypusine hydroxylase (DOHH) (EC 1.14.99.29) h-1; glutamic acid uptake rate at 588.3 lM was 83-85 nmol g-1 dry which completes the synthesis of hypusine and maturation of eIF5A. soil h-1. Leishmania has two genes containing DHS domains viz., DHS-like Acknowledgments: This work was financially supported by IGA gene (DHSL20) and DHS34 gene, of which only DHS34 protein was MENDELU 2010-2012 and NAAR, QI91C054. found to contain functional activity in vitro. DHS34 is much longer

123 12th International Congress on Amino Acids, Peptides and Proteins S71 with 601 amino acids as compared to the human enzyme (369 Establishment of growth arrest by polyamine depletion amino acids) and contains several unique insertions. Gene replace- ment studies for DHS34 showed that the enzyme deoxyhypusine Guy Landau, Ester Feldmesser, Zippy Bercovich, Avichai Ran, synthase and eIF5A modification play an essential role in cell via- and Chaim Kahana bility of L. donovani. The second enzyme of the hypusine biosynthetic pathway, DOHH, is a HEAT-repeat protein with eight Department of Molecular Genetics, the Weizmann Institute tandem repeats of a-helical pair. Four conserved histidine-glutamate of Science, Rehovot 76100, Israel, sequences have been identified which may act as metal coordination e-mail: [email protected] sites. A *42 kDa recombinant protein was obtained by heterolo- Escherichia coli gous expression of DOHH in . Circular dichroism The polyamines spermidine, spermine and their diamine precursor L. donovani spectroscopy revealed that the purified recombinant putrescine are naturally occurring polycations that are essential for L. donovani DOHH is largely a-helical. DOHH showed low cellular growth and proliferation. Depletion of cellular polyamines sequence identity (40.6%) with the human homolog. Metal chelators results in growth cessation that will be resumed upon re-addition of like ciclopirox olamine (CPX) and mimosine significantly inhibit the polyamines to the growth medium of the arrested cells. The tight L. donovani growth of and DOHH activity in vitro. These inhibitors association between polyamines and cellular proliferation made the were found to be less effective against the human enzyme. The polyamine metabolic pathway an appealing therapeutic target for L. donovani alignment of DOHH with the human homolog shows treating hyperproliferative pathologies including cancer. However, that there are two significant insertions in the former, corresponding the mechanism by which polyamine depletion inhibits cellular pro- to the alignment positions 159–162 (four amino acid residues) and liferation is mostly unknown. 174–183 (ten amino acid residues), the latter being present near the We set out to investigate the molecular mechanism by which L. substrate binding site. The structural differences between the polyamine depletion causes growth inhibition by investigating chan- donovani enzymes and the corresponding human homologs may be ges in translational and transcriptional activity in the depleted cells. exploited for structure based design of selective inhibitors against 35S-methionine incorporation experiments demonstrated that both the parasite. DFMO and GC7 treatments inhibit protein synthesis activity. Po- lysomal profile analysis demonstrated that both treatments arrested the translation process at the initiation step, with GC7 being more potent. We therefore inferred that the more profound effect of GC7’s Effect of L-arginine on the catabolism of polyamine is a result of its immediate competition with the physiological poly- and oxidative stress in testes of rats treated by ethanol amines on cellular sites of action. On the mechanistic side we have demonstrated that DFMO and GC7 treatment affected the phos- Pavlovic D1, Stojanovic I1. Nikolic J1, Kocic G1, Cvetkovic T1, phorylation state of two translation factors that regulate the initiation Jevtovic-Stoimenov T1, and Visˇnjic´ M2 step. Both treatments increased phosphorylation of eIF2a and decreased phosphorylation of 4E-BP1, two changes that each by 1Institute of Biochemistry, Medical Faculty University of Nis, Serbia, itself can provoke the initiation block. Kinetic studies have demon- 2Surgery Clinic of the Clinical Center, Medical Faculty University strated that the phosphorylation of eIF2a that might be part of the of Nis, Serbia, [email protected] establishment of stress response preceded all other changes. Charac- terization of transcriptional changes occurring during polyamine Despite the low oxygen tensions characterizing the testicular micro- depletion further supported the notion that the growth of polyamine- environment, this tissue remains vulnerable to oxidative stress. depleted cells is inhibited due to establishments of stress response Previous studies have reported that excessive alcohol consumption program in the treated cells. has a negative effect of testicular function through the induction of oxidative stress and concomitant disruption of testicular antioxidant status. Polyamines are ubiquitous organic cations essential for Monitoring cancer by urinary polyamine analyses testosterone synthesis, DNA and RNA, being necessary for normal mammalian spermatogenesis, cellular proliferation and tissue Uriel Bachrach integrity. On the other hand degradation products of polyamines exert cytotoxic effects both in vitro and in vivo. Since L-arginine is Department of Molecular Biology, Hebrew University-Hadassah the precursor in the synthesis of polyamines, the aim of this study Medical School, Jerusalem, Israel, e-mail: [email protected] was to determine the parameters of oxidative stress and the activity of polyamine oxidase (PAO) and diamine oxidase (DAO)— The naturally occurring polyamines, spermine, spermidine and the enzymes responsible for polyamine catabolism in the testes of rats diamine putrescine are widespread in nature. Due to their cationic treated by ethanol, and the effect of L-arginine administration. The nature, they interact with negatively charged macromolecules, such as animals were allocated into four experimental groups: ethanol DNA and transfer RNA and stabilize them. Therefore, during growth treated (15% solution in drinking water), arginine treated (0.5% processes polyamines are synthesized along with nucleic acids rep- solution of arginine in drinking water), ethanol plus arginine, and lications. Over 75,000 research papers have been written on this controls. Animals were treated for 3 weeks. The obtained results subject since 1900 and more than half (54%) were published between demonstrated that ethanol administration inhibit the antioxidant 1990 and 2009. As cancer cells grow rapidly, polyamines accumulate enzymes in the testes—catalase, SOD, GST, diminishes the tes- in cancerous tissues and their concentration is elevated in body fluids ticular content of GSH, while MDA concentration, as well as PAO of cancer patients. Various analytical methods were used to determine and DAO activity are increased. L-arginine ameliorates ethanol the amounts of polyamines in biological fluids. We developed an induced oxidative stress in the testes, suppressing lipid peroxidation enzymatic-based chemiluminescence method to determine urinary and restoring catalase, SOD, GST, PAO, DAO and GSH to normal polyamines and analyzed more than 2,000 urine samples from breast, levels. These results raise the possibility of novel nutrition strategy colon, lung and rectal cancer patients. Polyamine levels are high in for preventing and treating ethanol-dependent testicular the urine of cancer patients. Upon successful treatment, urinary dysfunction. polyamines return to their normal values. When therapy fails,

123 S72 12th International Congress on Amino Acids, Peptides and Proteins polyamine levels remain high. Longitudinal studies of polyamine They are involved in cell growth, developmental processes as well as levels can permit the assessment of the relapse or remission of the responses to environmental stress. The effect of osmotic stress disease. Elevated urinary polyamines, indicate the presence of can- imposed by sorbitol on total polyamine contents and polyamine cerous cells, sometimes even before the appearance of clinical oxidases were investigated in unicellular cyanobacterium Synecho- symptoms. Polyamines are present also in red blood cells and in the cystis sp. PCC 6803. The growth rate of the cells decreased when hair of cancer patients. It may be concluded that the determination of BG11 medium contained 300 mM sorbitol. The cells could not grow polyamines in biological ﬂuids, may serve as a useful tool for cancer in the presence of 700 mM sorbitol. The changes in polyamine oxi- diagnosis and treatment. dase activity correlated with total polyamine content under long term osmotic stress. The putative amine oxidase was expressed in E.coli BL21 (DE3). The enzyme could oxidize spermidine and spermine but showed no activity towards putrescine. On the other hand, Synecho- Polyamine and nitric oxide metabolism interaction cystis crude extracts were found to oxidize putrescine, spermidine and markers in colorectal cancer, surrounding and healthy spermine. Overall results suggest that at least two amine oxidases are present in Synechocystis cells. tissue samples: possible prognostic value

1 1 2 1 I. Stojanovic , A. Veljkovic , B. Brankovic , D. Pavlovic , Spermine enzymatic oxidation products in cancer G. Stanojevic2, G. Kocic1, D. Sokolovic1, P. Janosevic3, M. Nestorovic2, D. Petrovic2, and M. Visnjic2 therapy: Lysosomotropic compounds and Docetaxel potentiate their cytotoxicity 1Institute of Biochemistry, University of Nis, Faculty of Medicine, Serbia, Enzo Agostinellia, Giampiero Temperaa, Nikenza Vicecontea, 2 Clinic for Surgery, Clinical Centre Nis, University of Nis, Faculty Stefania Saccoccioa, Eris Bidollaria, Maria Condellob, of Medicine Serbia, Giuseppina Bozzutob, Giuseppe Aranciab and Agnese Molinarib 3Clinic for Stomatology, University of Nis, Faculty of Medicine, Serbia aDepartment of Biochemical Sciences, SAPIENZA University of Rome and CNR, Institute of Molecular Biology and Pathology, Although L-arginine-derived polyamines and nitric oxide are known P.le A. Moro 5, 00185 Rome as the promoters of tumorigenesis, the contradictory literature data bDepartment of Technology and Health, Istituto Superiore di Sanita` point out anti-proliferative activity of nitric oxide, opposed to Viale Regina Elena 299, 00161 Rome, Italy, polyamine role in cancer cell proliferation and differentiation. [email protected] Considering the literature data that dietary arginine can enhance the risk for colorectal cancer development by a mechanism including The in situ formation of cytotoxic metabolites by an enzyme-cata- NOS2 and polyamine synthesis, we have examined the markers of lyzed reaction is a recent approach in cancer chemotherapy. It was L-arginine metabolism in the samples of cancer tissue, as well as demonstrated that bovine serum amine-oxidase (BSAO) and spermine adjacent surrounding and healthy tissue at the incision margin in (SPM) addition to human cancer cells induces cell growth inhibition 50 patients with colorectal cancer. The obtained results suggest that and over-run the multidrug resistance (MDR) phenotype through the L L the present -arginine seems to be competent for -ornithine pro- oxidative stress caused by polyamine metabolites H2O2 and aldehydes duction in context of high putrescine production and, consequently, produced by the oxidative reaction. The results demonstrate that spermidine and spermine synthesis. Together with diminished cytotoxicity induced by SPM metabolites was greater in multidrug putrescine catabolism through diamine oxidase pathway, this could resistant cells (MDR) than in the corresponding wildtype ones (WT), be the way to estimate the proliferative potential of surrounding due to an increased mitochondrial activity. adjacent and healthy colon tissue in these patients. Increased nitric It was observed that the combination of BSAO/SPM enzymatic oxide level in relation to enhanced polyamine synthesis and high system with docetaxel (DTX) had a synergistic effect on cell growth arginase activity in cancer tissue points out the possibility of L- inhibition through apoptosis in both human epidermoid KB and breast arginine synthesis from citrulline, rising arginine bioavailability for cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX com- both polyamine and nitric oxide synthesis, leading to tumor pro- bination on apoptosis were caspase 3 and 9-dependent. The results gression. The differences in polyamine and nitric oxide content, as suggest that DTX could sensitize tumour cells to the oxidative stress well as other markers of L-arginine metabolism, in healthy and and apoptosis induced by BSAO/SPM. Preliminary studies also show adjacent tissue compared to cancer one offer a possibility to esti- that the cytotoxicity of BSAO/SPM was enhanced on human mela- mate the risk of tumor recidivity and the survival of these patients noma M14 cells by pre-treatment of tumour cells with the anti- at the time of surgical intervention. malarial drug chloroquine (CQ), a lysosomotropic compound. Chloroquine sensitized WT and MDR cells to the subsequent exposure to SPM metabolites. Cytotoxicity was greater by the combined Polyamine oxidases in cyanobacterium Synechocystis treatment than by BSAO/SPM alone and was higher in MDR cells than their wild-type counterparts. The release of lysosomal enzymes sp. PCC 6803 under osmotic stress into the cytoplasm by CQ, as shown by confocal microscopy, is the major reason for its sensitizing effect. Aran Incharoensakdi, and Khanittha Samasil The ﬁndings suggest that the association DTX with BSAO/SPM, the lysosomotropic effects caused by CQ and mitochondrial altera- Chulalongkorn University, Bangkok, Thailand tions induced by SPM oxidation products, allow the design of a new therapeutic strategy based on the use of these combinations in human Polyamines namely, putrescine, spermidine and spermine, are poly- neoplasms, making this approach mainly attractive in treating MDR cationic compounds found in both prokaryotic and eukaryotic cells. cancer patients.

123 12th International Congress on Amino Acids, Peptides and Proteins S73

Tracing the evolutionary origin of polyamine glucose uptake in the obese. Muscle biopsies showed increased pos- interconversion pathway by characterizing zebraﬁsh tabsorptive inhibitory IRS-1 serine phosphorylation in T2D and lesser Hyper3 increases in T2D and obese, vs lean. Signaling via Akt and spermidine/spermine N1-acetyltransferase 1 isoenzymes downstream of mTORC1 was also less than lean. Thus, in insulin- resistant states hyperaminoacidemia: (1) can overcome resistance of Yi-Chin Lien, Ting-Yu Ou, Po-Chih Kuo, Yu-Tzu Lin protein anabolism and (2) does not exacerbate impaired glucose and Han-Jia Lin uptake. Protein restriction in obesity and T2D thus appears unjustiﬁed.

Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan, e-mail: [email protected] Analysis of amino acids in plant extracts using thin Spermidine/spermine N1-acetyltransferase 1 (SSAT1) catalyzes the layer chromatography, amino acid analyzer and matrix rate-determining step in polyamine interconversion pathway, which maintains the homeostasis of polyamines. In addition, mammalian free material enhanced laser desorption ionization mass SSAT1 is also involved in many physiological and pathological spectrometry events, such as hypoxia, cell migration and carcinogenesis. By using cross-genomic bioinformatic analysis, we found that bony fish of Muhammad Nasimullah Qureshi1,2, Guenther Stecher2,3, vertebrates seems to be the origin of polyamine interconversion Gudrun Abel4, and Guenther K. Bonn2 pathway. It might be helpful to further clarify the physiological functions of human SSAT1 by comparing it with the original SSAT1, 1Medicinal Botanic Centre, PCSIR Laboratories Complex Peshawar, such as zebrafish SSAT1. There are three potential SSAT1 isozymes, Pakistan, tentatively denoted as zSSAT1a (NM_001093748), zSSAT1b 2Institute of Analytical Chemistry and Radiochemistry, Leopold- (NM_001030199) and zSSAT1c (NM_001002169) in the zebrafish Franzens-University Innsbruck, Innrain 52 a, 6020 Innsbruck, Austria, genome. The RNA messengers of the three zSSAT1 isogenes were all 3Bionorica Research GmbH, Mitterweg 24, 6020 Innsbruck, Austria, detectable in the zebrafish tissue that suggests none of these isogenes 4Bionorica AG, Kerschensteinerstr. 11-15, 92318 Neumarkt, are psudogenes. The recombinant enzymes of zSSAT1 isogenes were Germany prepared to characterize their enzymatic kinetic properties. Moreover, the regulation and protein–protein interaction relationship of these The standardization of natural product drugs needs reliable methods isoenzymes were also studied, and such properties were compared for the analysis of raw materials and final products. In this study, with that of human SSAT1. different analytical techniques were evaluated in order to recognize the most suitable method for qualitative and quantitative analysis of amino acids in herbal extracts. The specific focus was on thin layer chro- Proteomics matography (TLC), amino acid analyzer, and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption ionization time of flight mass spectrometry (mf-MELDI- MS). Samples employed in the study were standards and water extracts Amino acids can overcome the insulin resistance from Althaea officinalis, Taraxacum officinale, and Matricaria of protein metabolism in humans chamomilla. TLC analysis not only proved the presence of different amino acids in the biological sample, but also hinted at the existence of Re´jeanne Gougeon, Maya Bassil, Ste´phanie Chevalier, Sergio Burgos, other unknown compounds. The application of mf-MELDI-MS further Jose´ A. Morais, and Errol B. Marliss confirmed the presence of different amino acids. The quantification of amino acids in the plant extracts was performed using an automatic McGill Nutrition and Food Science Centre, McGill University, amino acid analyzer. Evaluation of all three techniques employed Montreal, QC, Canada clearly proved the adequate performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as targets do not need Amino acids (AA) stimulate protein synthesis both independently and modification and analysis requests only a few minutes. Furthermore, interactively with insulin. Insulin resistance of protein accompanied amino acid analyzer is suitable for quantitative analysis. that of glucose metabolism in our studies of whole-body protein anabolism (13C-leucine) in obese and diabetic persons. The protocol ‘‘clamped’’ hyperinsulinemia at postprandial levels, with euglycemia Global analysis of synaptic protein complexes and isoaminoacidemia maintained by variable infusions of glucose and by interaction proteomics AA solutions. However, hyperaminoacidemia (HyperAA) may impede insulinstimulated glucose uptake. Hence, we quantified rates of protein anabolism and glucose uptake (3-3H-glucose) during a simulated fed Ning Chen, August B. Smit, and Ka Wan Li steady-state, with serum insulin, glucose and AA at peak postprandial levels (‘‘Hyper3’’). Marked increases in anabolism, and attenuation of Department of Molecular and Cellular Neurobiology, Center insulin-stimulated glucose uptake occurred in lean men. Next, severely for Neurogenomics and Cognitive Research, Neuroscience Campus insulin-resistant men with Type 2 diabetes (T2D) underwent Hyper3 Amsterdam, VU University, Amsterdam, The Netherlands clamps. Unexpectedly, their protein synthetic and net anabolic responses (/kg FFM) matched those of the lean subjects, despite lesser Glutamatergic synapses represent the primary fast excitatory con- hyperinsulinemia. Furthermore, their resistance of glucose uptake was nections that link principal neurons in all brain areas into circuits. Pre- not worsened. We then tested less insulin-resistant obese men. Iden- synaptic terminals are responsible for converting electrical signals tical hyperinsulinemia in obese and lean required pancreatic clamps from axons into released chemicals packaged in synaptic vesicles (octreotide, glucagon, and hGH). HyperAA again induced equal net (SV). The exocytosis and endocytosis of SV involve coordinated protein anabolism in both groups, without worsening the attenuated dynamic molecular events within networks of proteins in the pre-

123 S74 12th International Congress on Amino Acids, Peptides and Proteins synaptic active zone. In the postsynaptic spine, AMPA-type glutamate have implications on bryozoan anti-fouling methods. Despite many receptors (GluA/AMPAR) function as primary carrier of synaptic studies on metamorphosis of this species, little is known about the current, and mediate the majority of excitatory synaptic transmission. molecular mechanism of these processes. Here, we report a compar- Both synaptic transmission and plasticity are governed by changes in ative study of swimming larvae and metamorphosing individuals at spatio-temporal patterns of AMPAR complexes via differential sub- 4 h and 24 h post-attachment using label-free quantitative proteomics. unit expression and dynamic protein interactions. While a few selected We identified more than 1,100 proteins at each stage, 61 of which were synaptic proteins have been extensively examined, the global synaptic differentially expressed. Specifically, proteins involved in energy protein interactome and their dynamics that underlie synapse function metabolism and structural molecules were generally down-regulated, and plasticity remain largely unknown. In this study we use a pro- whereas proteins involved in transcription and translation, the extra- teomics approach to characterize the synaptic protein interactome. We cellular matrix, and calcification were strongly up-regulated during have characterized [50 synaptic protein complexes including those metamorphosis. Many tightly regulated novel proteins were also involved in (1) SV docking, priming, vesicle fusion, endocytosis and identified. Subsequent analysis of the temporal and spatial expressions vesicle recycling, (2) AMPAR accessory proteins, (3) proteins of some of the proteins, and assay of their functions indicated that they involved in actin dynamic, and (4) synaptic cell adhesion molecules. In may have key roles in metamorphosis of B. neritina. a typical immunoprecipitation experiment we characterize around 150 proteins. A draft of this synaptic protein network will be presented, showing the complexity of the synaptic protein interactome. The role of O-linked GlcNAc modification on the glucose response of ChREBP

Proteomic analysis of the anticancer mechanism Haruhiko Sakiyama, Noriko Fujiwara, Takahiro Noguchi, of resveratrol against neuroblastoma Hironobu Eguchi, Daisaku Yoshihara, and Keiichiro Suzuki

Xiaoqing Ren, Qingsong Wang, Xuyang Zhao, Mingrui An Department of Biochemistry, Hyogo College of Medicine, and Jianguo Ji* 1-1 Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan

State Key Laboratory of Protein and Plant Gene Research, College The carbohydrate response element-binding protein (ChREBP) func- of Life Sciences, Peking University, Beijing 100871, China tions as a transcription factor in mediating the glucose-activated gene expression of multiple liver enzymes, which are responsible for con- Resveratrol (Res), a dietary polyphenolic compound, has strong verting excess carbohydrate to storage fat. ChREBP is translocated into anticancer properties on various tumors. However, the mechanism of the nucleus in response to high glucose levels, and then up-regulates the chemopreventive effect of Res against neuroblastoma remains transcriptional activity. Although this glucose activation of ChREBP is unclear. Treatment of neuroblastoma cell SH-SY5Y with Res resulted generally observed only in liver cells, overexpression of wild type max- in a decrease in cell viability and an induction of apoptosis in a dose- like protein X (Mlx), but not an inactive mutant Mlx, resulted in the dependent manner. Comparative proteomic approaches were applied exhibition of the ChREBP functions also in a human kidney cell line. to explore the profile of protein expression changes. Applying two Because high glucose conditions induce the glycosylation of cellular dimensional gel electrophoresis and MALDI TOF/TOF mass spec- proteins, the effect of O-linked GlcNAc modification on ChREBP trometry techniques, we analyzed the proteome of 20 lM Res treated functions was examined. Treatment with an O-GlcNAcase inhibitor SH-SY5Y and untreated, 20 proteins were found significantly chan- (PUGNAc), which increases the O-linked GlcNAc modification of ged. Seven proteins were up-regulated and four down-regulated. cellular proteins, caused an increase in the glucose response of These proteins are involved in oxidation reduction, microtubule ChREBP. In contrast, treatment with a glutamine fructose amido- polymerization, cell cycle, signal transduction, electron transport transferase inhibitor (DON), which decreases O-GlcNAcylation by chain. Further experiments demonstrate that Res can suppress the inhibiting the hexosamine biosynthetic pathway, completely blocked proliferation of SH-SY5Y through reducing oxidation, inhibiting the glucose response of ChREBP. These results suggest that the microtubule polymerization, participating protein signal transduction. O-linked glycosylation of ChREBP itself or other proteins that regulate The results suggest that Res has good potential as effective chemo- ChREBP is essential for the production of functional ChREBP. preventive agent against neuroblastoma. Redox Quantitative proteomics identify molecular targets that are crucial in metamorphosis of the marine Characterization of antioxidative peptide in free radical bryozoan Bugula neritina system: electronic properties in key positions C4,C1,N1 Tim Yue Him Wong for ORAC and superoxide databases; C2 for TEAC database The Hong Kong University of Science and Technology, Division of Life Science Yao-Wang Li, and Bo Li*

Metamorphosis of the marine bryozoan Bugula neritina transforms a College of Food Science and Nutritional Engineering, China ball-shaped swimming larva into a tubular sessile juvenile within 48 h. Agricultural University, Qinghua East Road 17, Haidian District, The metamorphosis consists of various complex processes such as the Beijing 100083, People’s Republic of China morphogenetic rearrangement of larval tissues and the development of juvenile tissues from primordial cells. Understanding metamorphosis Antioxidative peptide attracts more and more attention of researchers of B. neritina can provide insights into their colonization, as well as since it possesses special functions. However, the relationship

123 12th International Congress on Amino Acids, Peptides and Proteins S75 between the structure and activity is not clear, especially the anti- Redox reactions of hydroperoxides formed on amino oxidative peptide in the free radical system. Therefore, many acids and proteins: key intermediates in oxidative antioxidative peptide which come from different sources measured with different measuring methods are formed three databases with our damage to proteins effort: TEAC, ORAC and superoxide databases respectively. Before these three databases are dealed with PLSR method with amino acid Michael J. Davies descriptors to build QSAR models, they have been processed by TTPN methods since the length of peptide are different. Then each of The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, databases have found suitable descriptors for describing their struc- NSW 2042, Australia, e-mail: [email protected] tures (R2 [ 0.7, Q2 [ 0.5 for TEAC database, R2 [ 0.9, Q2 [ 0.5 for ORAC database, R2 [ 0.9 for superoxide database), and there are Proteins are major targets for oxidative damage due to their high some signiﬁcant positions in the sequence for antioxidant activity, abundance and rapid rates of reaction with many radicals and 1 they are C4,C2,C1 and N1. Additionally, low electronic properties excited states (e.g. O2). Exposure of proteins to radicals generated which is the most important properties for activity and high hydro- by radiation, metal ion/hydroperoxide systems, peroxyl radicals, phobic properties for these position are suitable, especially position peroxynitrite, and activated white cells, results in the formation of C4,N2 need acidic or basic amino acid and position C1 welcome protein-derived radicals. Subsequent reaction with O2 gives rise to amino acid which possess electronic, hydrophobic properties and new reactive groups including hydroperoxides and 3,4-dihydroxy- steric or bulky property for obtaining high activity. This research phenylalanine. These species are long-lived, can diffuse from their shows the features and key positions of antioxidative peptide in the site of generation due to poor removal by enzymes, and can be free radical system which will beneﬁt the further research and the detected in intact cells. Reaction of these hydroperoxides with thiol mechanism. (Cys) groups is rapid. As a result, we hypothesized that protein hydroperoxide formation might inactivate thiol-dependent enzymes and result in altered cell function, metabolism, signalling, redox maintenance and apoptosis. We have shown that GAPDH, gluta- Oxidation of Cys111 residue in loop VI of human thione reductase, caspases, cathepsins, Ca2+-ATPases, and protein copper/zinc superoxide dismutase tyrosine phosphatases are inactivated by amino acid-, peptide- and protein hydroperoxides. Inactivation occurs in a concentration-, time- and structure-dependent manner. These reactions result in 1 2 3 Noriko Fujiwara , Kentaro Ihara , Shinsuke Kato , concomitant hydroperoxide consumption and thiol oxidation; in 1 1 1 Daisaku Yoshihara , Hironobu Eguchi , Haruhiko Sakiyama , some cases sulfenic acid intermediates are detected. Some protein Soichi Wakatsuki2, Naoyuki Taniguchi4 and Keiichiro Suzuki1 hydroperoxides are more effective than H2O2, probably as a result of the longer lifetime of protein hydroperoxides in cells. Overall, 1 Department of Biochemistry, Hyogo College of Medicine, Japan, these data support the hypothesis that hydroperoxides formed on 2 Structural Biology Research Center, Institute of Material Structure oxidized proteins may contribute to cellular dysfunction and altered Science, High Energy Accelerator Research Organization, Japan, redox signalling in systems subject to oxidative stress by inducing 3 Department of Neuropathology, Institute of Neurological Sciences, strand breaks and mutagenic lesions in DNA, inhibiting key cellular Faculty of Medicine, Tottori University, Japan, enzymes, altering cellular redox status and signalling, and depleting 4 Systems Glycobiology Group, Disease Glycomics Team, Advanced antioxidants. Science Institute, RIKEN, Japan

Modifications of cysteine residues in proteins involve two types of oxidation. One is reversible oxidation such as disulphide bonding, Study on P16 damage induced by peroxynitrite sulfenation (Cys-SO) and S-nitrosation (Cys-SNO), and the other is irreversible oxidation such as sulfination (Cys-SO2H) and sulfonation Yunjing Luo*, Jingjing Li, Yang Ding, and Rugang Zhong (Cys-SO3H). Copper/zinc superoxide dismutase (SOD1) catalyzes the conversion of toxic superoxide anion radical into molecular oxygen College of Life Science and Bioengineering, Beijing University and hydrogen peroxide, thereby protecting cells against oxidative of Technology, 100124 People’s Republic of China, *Corresponding stress. In contrast, mutations in SOD1 have been found from patients Author: e-mail: [email protected] of familial amyotrophic lateral sclerosis (FALS). Only human and great ape SOD1 s among mammals have two free cysteine residues, P16, as a CDK4 inhibitor, is redox regulated in the course of cell cycle Cys6 and Cys111. We have shown that Cys111 located in the loop VI control. Over-expression of CDK4 induced by the deactivation and at the surface of the SOD1 molecule is selectively sulfonated even by abnormality of p16 can cause uncontrollable cell proliferation and divi- air oxidation. The polyclonal antibody raised against a synthesized sion, increasing the possibility of cell carcinogenesis. Oxidation injury of peptide containing Cys111-SO3H reacted with oxidized SOD1 but not p16 has been involved in the pathogenesis of cancer. In this paper, with reduced-form SOD1 by Western blot analysis and immunostained the aim to determine nitration sites, we used liquid chromatography-mass inclusions in the spinal cord of FALS patients and ALS model mice. spectrometry technology. Tyr129 and Tyr44, which is more vulnerable to On the other hand, the recombinant human SOD1 oxidized with ONOO-, were detected to be two nitration sites of p16. Since they are 2-mercaptoethanol (2-ME) only at Cys111 by disulphide bonding (2- both essential for CDK4 binding, we focus on the need for the nitration ME-SOD1, developed by Ube Industries Ltd.) was resistant to oxi- damage of ONOO- on the combination of p16 and CDK4 by SDS-PAGE dation. We will present a crystal structure of the dimer of 2-ME- of the reaction compound. The result revealed that under the conditions of SOD1 showing some interactions between 2-ME molecules in the low accumulated doses of peroxynitrite, the gradual decrease of P16 both subunits. These results indicate that Cys111 is a primary target compound is proportional to the increase of monomers over time, along for oxidative modification with oxygen or other thiols and plays an with small molecular weight polymers over time, suggesting the potent important role in oxidative damage to human SOD1 including FALS nitration modification by ONOO-on P16 has played a critical role in mutants. activity of CDK4, even implicating the cell progression.

123 S76 12th International Congress on Amino Acids, Peptides and Proteins

Acknowledgements: This work was supported by National Natural in the interstitial space. In a contralateral limb design (Rest vs. Science Foundation of China (No. 20875006) and Beijing Natural Exercise), this study used in vivo microdialysis to study the change in Science Foundation (No. 2102005). skeletal muscle amino acid concentration following protein feeding. Four male subjects undertook unilateral, concentric lower limb knee extensor resistance exercise on two occasions. A microdialysis Sports & Exercise catheter (CMA 63) was inserted into m. vastus lateralis of both the exercise and resting limb and perfused at 0.3 ll min-1 and serially sampled over a period of 7 h. Following a 2 h equilibration period subjects consumed either a 9% w:v whey protein isolate (WPI) Chronic effects of leucine supplementation of whey solution or placebo (flavoured water). Amino acid concentration was protein on mTOR activation in the skeletal muscle determined using reverse phase HPLC. of sedentary and exercised Wistar rats Compared to PLA, ingestion of WPI induced a significant increase in plasma amino acids to a peak concentration at 60 min post ingestion; BCAA (894 vs. 19 lM), Leu (355 vs. 17 lM) and GLU 1 2 2 P. C. B. Lollo ; T. M. Batista ; E. M. Carneiro ; (192 vs. -17 lM). Analysed over the same time point analysis of the 1 and J. Amaya-Farfan. * amino acids recovered in the lD revealed a 30% greater increase in the interstitial amino acid concentration in the exercise compared to 1 School of Food Engineering and the resting muscle; BCAA (912 vs. 545 lM), Leu (362 vs. 226 lM) 2 Institute of Biology, University of Campinas, SP, Brazil. and GLU (176 vs. -154 lM). *[email protected] Nutrient uptake into tissues from the circulation is influenced both by concentration and flow. The observed difference in the interstitial The milk-whey proteins are rich sources of L-leucine and L-leucine concentration of amino acids in the exercised muscle is probably can promote animal growth by activating m-TOR-mediated skeletal related to an exercise-induced increase in nutritive blood flow. muscle protein synthesis. We wished to investigate the dose–response effect of supplementing the L-leucine-rich milk-whey proteins (MWP) with L-leucine on protein synthesis and whether physical exercise Human skeletal muscle interstitial cytokine response would reduce the hyperinsulinemic effect of L-leucine, as ascertained by m-TOR activation and the determination of common blood bio- to protein feeding measured by in vivo microdialysis markers. Twenty-four sedentary weanling male Wistar rats were divided into four diet groups for 30 days as follows: (a) Control (AIN Philip M Jakeman 93-G); (b) 3% (AIN93-G +3% L-leucine); (c) 4.5% (AIN93-G +4.5% L-leucine); (d) 6% (AIN93-G +6% L-leucine). Another four like Faculty of Education and Health Sciences, University of Limerick, groups were submitted to physical exercise. Serum insulin, uric acid, Limerick, Ireland glucose, AST, ALT, CK, LD and gastrocnemius mass, total and body mass-adjusted protein were determined by standard methods, and In vivo microdialysis is a minimally invasive, membrane based mTOR by Westernblot analysis. The data were analyzed by ANOVA sampling technique, capable of determining the solute concentration and pos-hoc Duncan. mTOR concentration increased 3.91-fold at 6% in the interstitial space of a target tissue. Insertion of a microdialysis L-leucine in the sedentary, against 6.34-fold in the exercised animals. probe leads to a sterile inflammatory response characterised by an Exercise damped the hyperinsulinemic effect of higher serum L-leu- innate immune reaction and cytokine signalling. Whey proteins are cine. Other blood parameters and muscle mass/protein and markers of promoted as anti-inflammatory. This study investigated the effect of muscle damage did not change with supplementation. mTOR whey protein feeding upon the cytokine response to in vivo expression in the gastrocnemius of Wistar rats was increased by microdialysis. supplementing MWP with L-leucine, up to the 6% level, with a Microdialysis catheters (CMA 70) with a 100 kDa molecular mass reduction of hyperinsulinemia and no other apparent detrimental cut-off were inserted into the vastus lateralis of subjects and perfused effects. at a flow rate of 1.0 ll min-1 and serial sampled for 7 h. Following a Keywords: L-Leucine, Milk whey proteins, Dose–response; 2 h equilibration period subjects consumed either a 9% w:v whey mTOR, Skeletal muscle tissue protein isolate (WPI) solution or a flavoured water placebo (PLA). Pro-inflammatory cytokine), IL-6, IL-8 and TNFa, concentration of the microdialysate (lD) was measured by multiplexed immunoassay Effect of exercise and protein feeding on the interstitial (SectorÒ, Meso Scale Discovery). amino acid profile of human skeletal muscle sampled Following the 2 h equilibration the mean concentration of all cytokines increased in both the PLA and WPI to a peak 6 h after by microdialysis insertion of the cannula. Compared to PLA, feeding of WPI showed no effect on the increase in the peak mean concentration of IL-6 [690 William McCormack (285) vs. 786 (150) pg/ml; p [ 0.05] and a 56% decrease in the peak mean concentration IL-8 [2582 (1166) vs. 1130 (776) pg/ml Faculty of Education and Health Sciences, University of Limerick, p = 0.15]. TNFa was below the LLOD in the majority of lD samples Limerick, Ireland and was therefore omitted from the analysis. Though the sample size is small, the reduction in the recovery of In vivo microdialysis is a minimally invasive, membrane based the pro-inflammatory cytokine IL-8 is indicative of a potential sampling technique, capable of determining the solute concentration immunomodulatory function of whey protein isolates.

123 12th International Congress on Amino Acids, Peptides and Proteins S77

Inﬂuence of increased blood ammonia by long duration Protein S-guanylation and its unique regulation running on the performance of calculation task mechanisms involving cysteine metabolism

Hajime Ohmori1, Daisuke Aoki2, Keisuke Ishikura3, Song-Gyu Ra1, Takaaki Akaike Takafumi Suzuki1, Shoichi Komine1, Michio Okido1, and Teruo Miyazaki4 Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan 1Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan We recently clarified the physiological formation of 8-nitroguanosine 2Master’s Program in Health and Physical Education, University 30,50-cyclic monophosphate (8-nitro-cGMP) in cells in culture, which of Tsukuba, Tsukuba, Japan is the first demonstration of a new second messenger derived from 3Sports Research and Development Core, University of Tsukuba, cGMP in mammals since the discovery of cGMP more than 40 years Tsukuba, Japan ago. 8-Nitro-cGMP is formed via nitric oxide (NO) and has a unique 4Department of Development for Community Medicine, Tokyo electrophilic property. It can thus react with nucleophilic sulfhydryls Medical University, Ami, Japan of cysteine and protein, causing protein S-guanylation. However, regulation mechanisms of formation of 8-nitro-cGMP and S-guany- The purpose of this study was (1) to investigate the influence of the lation are still unclear. Here, we precisely quantified NO-dependent interval exercise simulated for soccer game (INT) and the steady rate formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatog- exercise with same average running speed as the interval exercise (SR) raphy-tandem mass spectrometry. More than micromolar in calculation task performance, and (2) to investigate the relationship concentration of 8-nitro-cGMP was evident in C6 cells that had been between blood ammonia level and calculation task performance. stimulated to express inducible NO synthase with excessive NO pro- Subjects were 5 soccer players (age 21.5 ± 1.6 years, VO2max duction. These unexpectedly large amounts of 8-nitro-cGMP suggest 56.7 ± 4.3 ml/kg/min).They were required to perform the three con- that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, ditions (INT, SR and REST). Three times of calculation tasks were may undergo guanine nitration. 8-Nitro-cGMP caused S-guanylation required in each condition, and they were evaluated in two different of Keap1 in cells, which led to Nrf2 activation and subsequent ways, the work speed expressed as work rate, and the work accuracy induction of antioxidant enzymes including heme oxygenase-1, and expressed as error rate. For the error rate, there was significant dif- thus 8-nitro-cGMP protected cells against cytotoxic effects of hydro- ference between INT and REST in 3rd calculation task. Significant gen peroxide. We revealed that 8-nitro-cGMP S-guanylated the correlation between error rate and blood ammonia level (r = 0.65). Cys434 of Keap1 in cells. Protein S-guanylation induced by 8-nitro- This study suggests that the rise in blood ammonia induced by long cGMP may thus have important implications in NO-related physiology duration running would influence the decrease of work accuracy. and pathology, pharmaceutical chemistry, and development of therapeutics for many diseases. Moreover, our recent investigation clarified a new regulatory molecule derived from cysteine metabolism, which is Sulfur containing amino acids critically involved not only in the 8-nitro-cGMP signaling but also in other electrophilic cellular signaling in general.

Mercaptopyruvate sulfurtransferase knockout mouse Structure and function of selenocysteine lyase production: what is the physiological role of the enzyme? and cysteine desulfurase

1 2 Noriyuki Nagahara , and Takaaki Ito Hisaaki Mihara

1 Dept. of Environ. Med., Nippon Med. Sch., Department of Biotechnology, Institute of Science and Engineering, 2 Dept. Pathol. Exp. Med., Grad. Sch. Med., Kumamoto Univ College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan In cysteine catabolism, mercaptopyruvate sulfurtransferase (MST: EC 2.8.1.2) catalyzes the reaction from mercaptopyruvate to pyruvate. Selenocysteine lyase (SCL) is a pyridoxal 50-phosphate (PLP)- MST is localized in the cytoplasm and mitochondria in animal cells. dependent enzyme that specifically acts on L-selenocysteine to yield We found higher MST expression during mouse embryonic develop- L-alanine and selenium. The enzyme is proposed to function in the ment than in the postnatal period although the MST gene is a recycling of the micronutrient selenium from degraded selenoproteins housekeeping gene. Our studies of the structure–function relationship containing selenocysteine residue as an essential component. We have of MST revealed that (1) a disulfide bond between the dimeric MST and revealed that the catalytic reaction of SCL proceeds via the formation a catalytic site cysteine contributes to redox-dependent regulation of of an enzyme-bound selenopersulfide intermediate on Cys375. MST activity; (2) the disulfide bond serves as a redox-sensing molec- Cys375 on the flexible loop directed L-selenocysteine, but not L- ular switch, and (3) a catalytic site cysteine is oxidized to form low cysteine, to the correct position and orientation in the active site to redox potential sulfenate, which is reduced not by glutathione but by initiate the catalytic reaction. This provides the basis for under- thioredoxin. These findings suggest that MST contributes to maintain standing how trace amounts of a selenium-containing substrate is cellular redox homeostasis. To confirm the physiological function and distinguished from excessive amounts of its cognate sulfur-containing tissue specificity of a gene repression of MST, we generated MST compound in a biological system. knockout mice (C57BL/6) by gene targeting in embryonic stem cells L-Cysteine is the sulfur source for the biosynthesis of a variety of using the Cre/loxP system. Functional expression of a Cre/LoxP site- cofactors such as thiamin, iron-sulfur clusters, molybdopterin (MPT), specific recombination system is applied in generating tissue-specific and tRNA thionucleosides. Cysteine desulfurase, a PLP-containing knockout mice. A comprehensive analysis of pathologic and morpho- homodimer, decomposes L-cysteine to L-alanine and sulfane sulfur via logic changes caused by MST deficiency is in progress. the formation of an enzyme-bound persulfide intermediate. The

123 S78 12th International Congress on Amino Acids, Peptides and Proteins persulfide sulfur is subsequently incorporated into the biosynthetic most MCR chemistry performed with isocyanides relates to the pathways of several sulfur-containing biofactors, which provides an classical reactions of Passerini and Ugi. Passerini reactions involve an elegant mechanism for making sulfur atoms available without releasing oxo component, an isocyanide and a carboxylic acid. The success of them in solution. In molybdenum cofactor biosynthesis, MoeB acti- multicomponent condensations in organic synthesis during the last vates the C terminus of the MoaD subunit of MPT synthase to form few years has increased the interest for doing novel reactions or MoaD-adenylate, which is subsequently converted to a thiocarboxylate modifying the old ones. Such modifications include the use of poly- for the generation of the dithiolene group of MPT. We revealed that functional building blocks, the employment of non-classical starting IscS, among three cysteine desulfurases present in E. coli, is the pri- units, or new solvents according to green chemistry. mary physiological sulfur-donating enzyme in MPT biosynthesis. As part of our continuing interest in isocyanide-based multicomponent reactions, herein we describe an efficient synthesis of a-acyloxythioamides 4 from N-protected a-amino acids 1 as an acid Sulfur metabolism in the cysteine dioxygenase knockout component in the Passerini reaction in 1-butyl-3-methylimidazoli- nium bromide ([bmim]Br) as ionic liquid (IL) at room temperature. mouse: impairment in taurine synthesis and increased formation of acid labile sulfur

Stipanuk, Martha H, Krista Fieselmann, Lawrence L. Hirschberger, A solvent-free synthesis of highly functionalized Ueki, Iori, Jimmy Lam, Rachel Peters, Heather B. Roman, benzothiazolediamides (mimic natural peptide) and Alessandro Valli via Ugi four component reaction

Division of Nutritional Sciences, Cornell University, Ithaca, Fatemeh Sheikholeslami Farahani*1, and Ashraf S. Shahvelayati2 NY, 14853, USA 1Department of Chemistry, Firoozkooh Branch, Islamic Azad Cysteine homeostasis is dependent on the regulation of cysteine University, Firoozkooh, Iran, dioxygenase (CDO) in response to changes in sulfur amino acid 2Department of Chemistry, Islamic Azad University Shahr-e Rey intake. Cysteine dioxygenase oxidizes cysteine to cysteinesulfinate, Branch, Tehran, Iran which is further metabolized to either taurine or to pyruvate + sulfate. To gain insight into the physiological function of CDO and the Multicomponent reactions (MCRs) are one-pot processes that com- consequence of a loss of CDO activity, mice carrying a null CDO +/- -/- +/- bine three or more substrates simultaneously. Such processes are of allele (CDO mice) were crossed to generate CDO , CDO , +/+ -/- great interest in diversity-oriented synthesis, especially to generate and CDO mice. CDO mice exhibited postnatal mortality, -/- compound libraries for screening purposes. The Ugi four-component growth deficit, and connective tissue pathology. CDO mice had reaction (Ugi 4CR) is one of the milestones in this field and great extremely low taurine levels and somewhat elevated cysteine levels, efforts have been devoted to the exploration of the potential of this consistent with the lack of flux through CDO-dependent catabolic transformation. A primary amine, a carbonyl compound, a carboxylic pathways. However, plasma sulfate levels were slightly higher in -/- +/- +/+ acid, and an isocyanide react together to give a-amidoamides in this CDO mice than in CDO or CDO mice and tissue levels of remarkable reaction. In recent years several modifications of the acid-labile sulfide were elevated, indicating an increase in cysteine classical Ugi 4CR have been described; these include variations of catabolism by cysteine desulfhydration pathways. Null mice had one of the components or the introduction of a linkage between two of lower hepatic cytochrome c oxidase levels, suggesting impaired them. In particular, the groups of Zhu and Do¨mling have contributed electron transport capacity. H S has been identified as an important 2 significantly to the advancement of this transformations. gaseous signaling molecule as well as a toxicant, and pathology may As part of our continuing interest in multicomponent reactions, be due to dysregulation of H S production. Control of cysteine levels 2 herein we describe an efficient synthesis of unsaturated thioamid- by regulation of CDO may be necessary to maintain low H S levels 2 odipeptides (pseudopeptides) 4 from 4-benzothiazol-2-ylamino-4- and facilitate the use of H S as a signaling molecule. 2 oxo-2-butenoic acid 1, prepared from 2-aminobenzothiazol and furan- Acknowledgments: Supported by grant DK056649 from the National 2,5-dione, as a new acid component in the Ugi reaction under solvent- Institute of Diabetes and Digestive and Kidney Diseases. free condition.

Synthesis Comparative syntheses of peptide thioesters derived from mouse and human prion proteins A one-pot synthesis of functionalized a-acyloxythioamides ˇ ˇ from N-protected a-amino acids as an acid component Jaroslav Sebestı´k, Zbigniew Zawada, Martin Safarˇ´ık, and Jan Hlavaćˇek in the Passerini reaction in an ionic liquid Institute of Organic Chemistry and Biochemistry, AS CR, 16610 Prague, Czech Republic Ashraf S. Shahvelayati*1, Issa Yavari2, and Maryam Ghazvini Prions are suspected as causative agents of several neuropathogenic 1Department Chemistry, Islamic Azad University Shahr-e Rey diseases. However, they mode of action is still not clear. Combination Branch, Tehran, Iran, of chemical and recombinant synthesis can provide suitable probes 2Department Chemistry, Islamic Azad University Science for determination of prion role in pathogenesis of related diseases. and Research Branch, Ponak, Tehran, Iran Peptide thioesters are key building blocks for chemical syntheses of proteins by native chemical ligation. Prions are sources of difficult Multicomponent reactions (MCRs) are generally defined as reactions sequences for synthesis by Fmoc/tBu approach. Therefore, a scan of where more than two starting materials react to form a product. Today the prion domain 93–231 was carried out in order to discover which

123 12th International Congress on Amino Acids, Peptides and Proteins S79 thioesters are easily available as suitable building blocks for total non-neuronal cells suggesting that they could be implicated in chemical synthesis of prion protein based probes. First, the synthesis carcinogenesis. on chlorotritylchloride resin was employed and after deprotection of mGlu receptors are G-protein-coupled receptors and eight sub- small quantity the segments were purified and characterized. If the types (mGluR 1–8) have been identified and classified into three difficulties were observed during synthesis, the segment was re-syn- groups (I–III) based upon sequence homology, transduction mecha- thesized with either special dipeptides or by splitting to 2 or 3 smaller nism and pharmacological profile. segments. When the sequences were synthesized correctly without Because of their modulating properties, mGlu receptors are rec- main impurities, the protected segments were coupled with EtSH ognized as promising therapeutic targets and many ligands (agonists using DIC/DMAP activation. If the technique did not provide a and antagonists) have been prepared to better understand the phar- suitable peptide thioester, the protected segment was coupled using macology of mGlu receptors in order to selectively activate the PyBOP/p-Ac-NH–Ph-SH. In some special cases, the other techniques different groups and subtypes of receptors. of thioester formation were carried out. Peptide thioesters from An a-amino acid moiety can be found in all mGlu receptors C-domain of prion protein were synthesized and characterized. competitive ligands and most of the side chains hold an acidic func- Acknowledgments: This work was supported by grant of Czech tion. Examination of the glutamate binding site in the mGlu receptors Science Foundation (GA CR) No. 203/07/1517 and Research Project and pharmacological data of some ligands shows that sterically con- Z40550506. strained structures with an optimal distance between functional groups could lead to potent and selective new ligands. It is known that introducing an unsaturation in a biologically active structure could modify the conformation of the molecule and EAAC1-mediated neuronal glutathione synthesis thus the biological activity. In this respect, the synthesis of new acetylenic analogues of glutamate will be described. Koji Aoyama

Department of Pharmacology, Teikyo University School of Medicine, Tokyo, Japan Novel efficient and stereoselective synthesis of the b- amino-a-hydroxy acid units in bestatin and amastatin Glutathione (GSH) is a tripeptide comprised of glutamate, cysteine and glycine. Cysteine is the rate-limiting substrate for GSH synthesis Youngran Seo, Hyeonjeong Kim, Young Gyu Kim* in neurons. Most neuronal cysteine uptake is mediated by a sodium dependent excitatory amino acid transporter, known as excitatory School of Chemical and Biological Engineering, Seoul National amino acid carrier 1 (EAAC1). EAAC1-null mice have reduced University, Seoul, 151-744, Republic of Korea neuronal GSH levels and showed increased susceptibility to oxidant injury. GSH depletion in the brain has been considered to be an early Development of efficient synthetic methods for b-amino-a-hydroxy event in some neurodegenerative diseases. Endogenous mechanisms acids has been of interest because the b-amino-a-hydroxy acid units are to increase the neuronal GSH level in the brain might be a potential frequently found in many biologically active natural products such as strategy to protect against neurodegeneration. Our ongoing studies bestatin and amastatin as well as paclitaxel. Bestatin is an immune focus on the mechanisms to regulate neuronal GSH synthesis via response modifier and a dipeptide containing (2S,3R)-3-amino-2- EAAC1. hydroxy-4-phenylbutanoic acid. Amastatin, an aminopeptidase inhibitor with (2S,3R)-3-amino-2-hydroxy-5-methylhexanoic acid as a key component, has shown antitumor, immunoregulatory, and antibacterial New developments in the synthesis of acetylenic activities. Both efficient and stereoselective construction of the same analogues of glutamate functional motif in the above aminopeptidases, the b-amino-a-hydroxy acid unit, will be reported by a facile intramolecular conjugate addition with an N-hydroxylmethyl group of the stable lactols derived from 1,2 1,2 1,2 1 P. Meffre* , Z. Benfodda , D. Benime´lis , V. Rolland commercially available a -amino acids, phenylalanine and leucine. and F. C. Acher3

1 ´ Institut des Biomolecules Max Mousseron-UMR 5247 -CNRS- Novel synthetic methodologies to prepare unnatural Universite´s Montpellier 1 et 2, Place E. Bataillon, 34095 Montpellier Cedex 5, France, acids, aminoacids and peptidomimetics 2UNIVERSITE DE NIMES, Laboratoire de ChimieBioOrganique- LCBO, Site des Carmes, Place Gabriel Pe´ri, 30000 Nıˆmes, Mauro F. A. Adamo 3Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601, CNRS Universite´ Paris Descartes, We have developed an original synthon, 3-methyl-4-nitro-5-styr- 45 rue des saints Pe`res, 75006 Paris, France ylisoxazole 1 (Scheme 1), 1 and novel organocatalyses (Schemes 1, 2). Compounds 1 are stable crystalline compounds which could be Glutamate ((S)-Glu) is the major excitatory amino acid in the central generated in multigram scale from isoxazole 2 and aldehyde 3. nervous system. It acts by stimulating ionotropic and metabotropic Compounds 1 contain two electrophilic centres: (1) soft E1 which glutamate receptors (iGluR and mGluR, respectively). Glutamate has reacted selectively with stabilised nucleophiles; (2) hard E2 which been shown to be involved not only in many neuropathologies such as reacted selectively with hard nucleophiles such as –OH. The addition anxiety, pain, ischemia, Parkinson’s disease, epilepsy and schizo- of –OH to E2 is the bases of a synthetically useful reaction which phrenia. More recently, mGlu receptors have also been detected in transformed the 4-nitroisoxazol-5-yl core into a carboxylate,

123 S80 12th International Congress on Amino Acids, Peptides and Proteins

demonstrating compounds 1 as synthetic equivalent to cinnamates 4. One-pot efficient synthesis of a-aminophosphonates The 4-nitroisoxazole-5-yl core also activated the alkene as the derivatives via multicomponent reactions of triphenyl resulting anion, delocalised across a large number of conjugated atoms, was more stable: compared to cinnamic esters and aldehydes, phosphite, aldehydes and amines in ionic liquid isoxazoles 1 reacted faster and more efficiently. Compounds 1 were intermediate in multi component processes (over 20 peer-reviewed Rahimeh Hajinasiri* papers published 2005–2010) and more recently substrates for enantioselective phase transfer catalyses (Scheme 1). Chemistry Department, Qaemshahr Branch, Islamic Azad University, c-Aminoacids 8,2 derived from 5, a-aminoacids 6 and their P. O. Box 163, Qaemshahr, Iran derivatives 9, heavily substituted cyclopropanes 10, and chiral iso- nitriles 11 were obtained from 1 in high yields and ees. My group has Ionic liquids (ILs) have gained great attention in recent years. Ionic also developed a unique sulfonylation process which allowed liquids have a high polarity and low vapor pressure. These charac- obtaining multigram amounts of enantiopure sulfonic acids 13 teristics combined with immiscibility with most less polar organic (Scheme 2). 3 Sulfonic acids are extensively employed as resolving solvents led to their use as solvent or co-solvent in catalysis. a-am- agent and possess several interesting biological properties, exerting inophosphonic acids are probably the most important substitutes for key metabolic roles in the central nervous system (CNS). The sulfonic the corresponding amino acids in biological systems. A number of acid functionality is also present in natural products, for example in potent antibiotics, enzyme inhibitors, and pharmacological agents are 6-gingesulfonic acid, extracted from ginger (Zingiberis Rhizoma) and a-aminophosphonic acids as well as their derivatives, particularly used as an anti-ulcer drug in Chinese and Japanese medicine. My peptides. independent work resulted in 40 peer reviewed publications, five In this study, we describe an efficient synthesis of a-aminophos- patents and three application pending. The paper describing the phonates through the reaction of aldehydes 1, amines 2 and synthesis of five was selected as Hot paper in Angewandte Chemie phosphites 3 in ethyl methyl imidazolium bromide. For optimizing the Int. Ed., and was highlighted twice in Synfacts for the novelty of one reaction conditions, a sample reaction between benzaldehyde, benzyl being synthetic equivalent to cinnamates and for the use of five in the amine and triphenylphosphite was carried out in different ILs as the preparation of Baclophen. The synthesis of compounds 6–7 is still solvent. The results indicated that ionic liquid ethyl methyl imi- unpublished. The patent describing the sulfonylation of alkenes dazolium bromide is excellent solvent for these reactions. (Scheme 2) won the IDEA price 2009 hosted by Enterprise-Ireland.

123 12th International Congress on Amino Acids, Peptides and Proteins S81

Selective synthesis of fluorinated cyclic b-amino acids Kahalalide A is one of a family of peptide natural products isolated from the marine mollusk Elysia rufescens and its algal diet Bryopsis Lorańd Kiss1, Eniko} Forro´1, Santos Fustero2, and Ferenc Fu¨lo¨p1 sp. Among these, the structurally simpler kahalalide A is one of the few marine-derived cyclic peptides with antimycobacterial activity, 1Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 and it is not cytotoxic to various tumor cell lines. Recently, Line Szeged, Eo¨tvo¨s u. 6, Hungary, Bourel et al. has reported a chemical route to generate kahalalide A 2Departamento de Quı´mica Orgańica, Facultad de Farmacia, and its analogues. Besides the motivation of discovering biologically Universidad de Valencia, Valencia, Spain, E-mail: active compounds, there was a purely chemical impetus for the total [email protected] synthesis. Based on our previous enormous work on synthesis of cyclic peptides, a parallel synthesis of kahalalide A derivatives were b-Amino acids are key components of natural products and precursors designed and carried out in our laboratory by a total solid-phase route of bioactive b-lactams. A number of cyclic b-amino acids possess with Fmoc chemistry. Later, we will investigate their biological antifungal or antibacterial activities. The alicyclic and O-orN-con- activities in detail. taining heterocyclic, conformationally rigid b-amino acids are building blocks for the synthesis of biologically active novel peptides. Among the large family of bioactive fluorinated compounds, Syntheses and selective functionalisations of carbocyclic fluorinated amino acids and peptides present a high importance in b-amino acids medicinal chemistry as enzyme inhibitors, antitumoural agents and antibiotics. Due to the special characteristics of fluorine, the chemistry of fluorinated cyclic amino acids is a very interesting area. In Ferenc Fu¨lo¨p spite of the great potential of cyclic amino acids, only few cyclic fluorinated derivatives have been reported so far, among either a- Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 amino acids or c-amino acids. Furthermore, although cyclic b-amino Szeged, Eo¨tvo¨s utca 6, Hungary, [email protected] acids have recently generated increasing interest, an extremely few fluorinated cyclic analogues have been synthetized so far. The alicyclic b-amino acids have acquired great interest in recent The regio- and stereoselective approach to fluorinated b-amino- years in view of their pharmacological potential. Cispentacin, an cyclohexane or cyclohexane esters has been developed, from a antifungal antibiotic with a cyclopentane skeleton, is one of the most bicyclic b-lactam. The procedure involves six or seven steps, and important derivatives. (1R,2S)-2-amino-4-methylenecyclo-pentane- consists in regio- and stereoselective iodolactonization, lactone carboxylic acid (Icofungipen) is a known antifungal agent. Cyclic, opening and hydroxy-fluorine exchange. The method has been conformationally rigid b-amino acids such as cis and trans-2-ami- extended to the synthesis of fluorinated amino ester enantiomers. nocyclopentanecarboxylic acid have been used as building blocks in the synthesis of peptides1. The present lecture will demonstrate different strategies that result in functionalized b-amino acids from 2-aminocycloalkenecarboxylic One-pot synthesis of functionalized 2,3-dihydrothiazoles acids. The Scheme illustrates one example where conformationally Maryam Ghazvini, Issa Yavari, and Nasir Iravani restricted, orthogonally protected 2,4-diaminocarboxylates with a cyclopentane skeleton were efficiently synthetized. The syntheses Payam noor university, Iran involve strategies of diastereoselective epoxidation of the b-lactam and the corresponding protected amino esters with opposite selec- Thiazoles and their derivatives exhibit various biological activities tivities, followed by regioselective opening of the oxirane ring with such as antimicrobial, anti-inflammatory, antiviral, antituberculosis sodium azide. This new class of compounds can be regarded not only and cytotoxic activities. For example, the thiazolium ring present in as conformationally constrained b,c-diamino acid derivatives, but also vitamin B1 serves as an electron sink, and its coenzyme form is as potential functionalized carbocyclic nucleoside precursors. important for the decarboxylation of a-keto-acids. Thiazolines show interesting anti-HIV or anticancer activities and can inhibit cell division. We wish to report a convenient and facile synthesis of Synthesis of constrained carbocyclic and heterocyclic functionalized dihydrothiazole derivatives in good yields. A simple one-pot synthesis of ethyl 3-(alkyl)-4-methyl-2-(phe- b-amino acid derivatives nylimino)-2,3-dihydrothiazole-5-carboxylate or 1-(3-cycloexyl-4- methyl-2-(phenylimino)-2,3-dihydrothiazole-5-yl)ethanone from the Sven Mangelinckx1, Karen Mollet1, Tamara Meiresonne1, reaction of phenylisothiocyanate, primary amines and ethyl 2-chlo- Lorand Kiss2, Matthias D’hooghe1, Ferenc Fu¨lo¨p2, roacetoacetate or 3-chloroacetylaceton is described. and Norbert De Kimpe1 Keywords: 2,3-Dihydro-chloroacetylaceton drothiazole; Phenyli- sothiocyanate; Primary alkylamines, Ethyl 2-chloroacetoacetate 1Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium, Parallel synthesis of kahalalide A analogues through 2Institute of Pharmaceutical Chemistry, University of Szeged, H-6701 on-resin cyclization Szeged,PO Box 427, Hungary The synthesis of conformationally constrained carbocyclic and het- Tingting Wei, Jin Ren, and Chuanguang Qin* erocyclic b-amino acids is becoming increasingly important in synthetic, agricultural and pharmaceutical chemistry. This attention Faculty of Life Sciences, Northernwestern Polytechnical University, results from the specific properties of these non-proteinogenic con- Xi’an 710072, China strained amino acids with respect to biological activity (antifungal,

123 S82 12th International Congress on Amino Acids, Peptides and Proteins gametocidal, peptidase-inhibition) and structural features (building is a very abundant amino acid, whereas its concentration is signifi- blocks for structurally and functionally unique b-peptides). cantly lower in the adult brain. It also was the most prominent amino In this presentation, the synthesis of new conformationally con- acid released in 7-day-old mice in ischemia from all brain regions strained b-amino acid derivatives via elaboration of different strategies studied, cerebral cortex, striatum, cerebellum, hippocampus and brain starting from electrophilic allylic halides and halogenated imines is dis- stem. In 3-month-old mice glutamate, aspartate and GABA were closed. Diverse targeted carbocycles and heterocycles, such as released at relatively high amounts. Significantly less taurine was cyclopropanes, cyclobutanes, aziridines, azetidines and tetrahydrofurans, released in adult than in developing mice. However, ischemia caused all with the b-amino acid (or ester) motif have been successfully prepared. a markedly larger increase in taurine release than in the release of The presence of a constrained ring and/or functional groups in the syn- other neuroactive amino acids even in the adult brain. In all brain thesized b-amino acid derivatives allowed further study of ring opening regions taurine was the amino acid whose release was increased most reactions, ring transformations and functional group transformations. by the ischemic conditions. It is assumed that the release of inhibitory amino acids could counteract the effects of glutamate released in excess in ischemia. Only taurine is released in such amounts, in the developing brain in particular, that it may counteract the harmful Taurine effects of excitatory amino acids in ischemia. Supported by the competitive research funding of Pirkanmaa hospital district. Alteration of gene expression profile by taurine in human intestinal Caco-2 cells Effects of lotus and taurine supplementation Hideo Satsu, Yusuke Gondo, and Makoto Shimizu in diet-induced obesity rat model

Department of Applied Biological Chemistry, Graduate School Kyung Ja Chang, Jeong Soon You, and Zhao Xu of Agricultural and Life Sciences, The University of Tokyo, Japan Department of Food and Nutrition, College of Human Ecology, Inha Taurine, present in high concentration in several mammalian tissues, University, Incheon 402-751, Korea plays an important role in essential biological processes such as anti- oxidation, anti-inflammation, and osmoregulation. However, the Taurine is the most abundant free amino acid in the body and may serve as mechanisms involved in these physiological roles at the genetic level an anti-obesity agent. Lotus (Nelumbo nucifera) has been used to treat remain to be elucidated. In the present study, we investigated the effect obesity. The adipokines secreted by adipose tissue including leptin and of taurine on gene expression profile in human intestinal Caco-2 cells adiponectin are known to play an important role in the pathogenesis of to analyze which genes or signaling pathways are involved in the obesity. In order to investigate the effect of lotus and taurine on obesity, function of taurine. Caco-2 cells were treated without or with 50 mM 5-week-old male Sprague-Dawley rats were randomly divided into five taurine for 24 h. Then global analysis of gene expression change was groups for a period of 8 weeks (normal diet, N group; high fat diet, HF performed by DNA microarray. The mRNA and protein expression group; high fat diet + taurine, HFT group; high fat diet + lotus leaf, HFL level of selected genes was measured by real-time PCR and by western group; high fat diet + lotus root, HFR group). Taurine was supplemented blotting, respectively. As for the transcriptional activity, the reporter by dissolving in feed water (3% w/v). Lotus leaf and root hot water extract vector including its promoter region was constructed and the promoter was orally administrated (400 mg/kg/day) to HFL and HFR groups and activity was measured by luciferase assay. The result of DNA the same amount of distilled water was orally administrated to N and HF microarray and real-time PCR analysis showed that taurine increased groups. Final body weight and relative weights of retroperitoneal adipose the expression level of thioredoxin interacting protein (TXNIP) tissue were significantly lower in HFR groups compared to HF group. In mRNA in a time- and dose-dependent manner. On the other hand, serum, total cholesterol concentration was lower in HFT, HFL and HFR taurine decreased glucose transporter 1 (GLUT1) and GLUT3 mRNA groups compared to HF group. Serum triglyceride concentration was levels. The increase in TXNIP mRNA was not observed by b-alanine lower in HFL and HFR groups compared to HF group. Serum adiponectin or GABA treatment, suggesting that this induction was specific for concentration was higher in HFT and HFR groups compared to HF group. taurine. We further revealed that taurine increased the protein level These results suggest that lotus root hot water extract has the effect of and also the promoter activity of TXNIP. These findings demonstrated reducing the body weight and adipose tissue weight, and taurine and lotus the novel insight of taurine at the molecular level. root have preferable effects in adiponectin levels.

Effect of ischemia on the release of taurine and other Effects of taurine and complex magnesium taurate neuroactive amino acids from the brain on calcium oxalate crystallization and antioxidant activities in vitro Simo S. Oja1 and Pirjo Saransaari

1,2 1,2 1 1 Department of Paediatrics, Tampere University Hospital, Tampere, Zhang Chaoyan , Wu Wenhui , Shu Yunying , and Wang Jue Finland 1College of Food Science and Technology, Shanghai Ocean University, Shanghai, People’s Republic of China, Ischemia is a condition which dramatically hampers the functions of 2 brain cells which derived most of their energy from oxidative Institutes of Marine Sciences, Shanghai Ocean University, Shanghai, metabolism. We studied under ischemic conditions the release of People’s Republic of China endogenous taurine and other neuroactive amino acids from slices prepared from different brain regions from developing 7-day-old and The purpose of this study was to evaluate the inhibitory effect of young adult 3-month-old mice. In the developing mouse brain taurine taurine and complex magnesium taurate on the crystallization of

123 12th International Congress on Amino Acids, Peptides and Proteins S83

calcium oxalate (CaC2O4) with measurement of electrical conduc- Mechanism underlying the antioxidant activity tivity and its morphology. The inhibition effects of taurine and of taurine complex magnesium taurate with different concentrations on

CaC2O4 crystallization were measured by electrical conductivity; in 1 2 1 addition, crystals generated in the mixing solutions were harvested Chian Ju Jong , Junichi Azuma and Stephen W. Schaffer and analyzed by inverted microscope. The antioxidant activities of 1 taurine and complex magnesium taurate were evaluated for scav- University of South Alabama College of Medicine Department of Pharmacology, Mobile, AL 36688; enging of 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) 2 activities in vitro. The results of electrical conductivity research Hyogo University of Health Sciences School of Pharmacy, Kobe, Japan suggest that both taurine and complex magnesium taurate can inhibit the growth of calcium oxalate crystals, but the latter has much Taurine is a potent antioxidant, however, the mechanism underly- stronger capability to inhibit that growth. The morphological of ing its antioxidant activity is unclear. Because taurine is neither a calcium oxalate crystals were traced with inverted microscope, to free radical scavenger nor a promoter of the antioxidant defenses, induce calcium oxalate dehydrate (COD) formation than did taurine the possibility that taurine might be a determinant of mitochondrial under same condition. With the increasing of concentrations, the superoxide production was examined. To test this hypothesis, scavenging activity of taurine and complex magnesium taurate intracellular taurine levels were reduced *50% by exposing iso- against DPPH radical increased; furthermore, the antioxidant ability lated neonatal cardiomyocytes to medium containing the taurine of complex magnesium taurate better than that of taurine. So it transport inhibitor, b-alanine. Associated with the decline in concludes that the inhibition of complex magnesium taurate is intracellular taurine levels was a decrease in the expression of clearly superior to that of taurine. specific mitochondria encoded proteins, such as ND5 and ND6, a Acknowledgments: Financial support: National 863 plans projects pattern indicative of inadequate decoding of UUG. The decline in 2011AA09070109. ND5 and ND6 content in turn led to a decrease in both the activity of respiratory chain complex I and oxygen consumption. It is widely accepted that impaired electron transport chain activity can lead to the diversion of electrons from the respiratory chain to Functional consequences of taurine interaction oxygen, forming in the process superoxide. Therefore, three measures of oxidative stress (elevations in the glutathione redox state, with the GABAergic system MitoSox fluorescence and aconitase activity) were examined in the b-alanine treated cells. All three measures revealed that taurine Abdeslem El Idrissi deficiency is associated with an elevation in oxidative stress. However, co-administration of taurine with b-alanine abolished the City University of New York/College of Staten Island rise in oxidative stress, suggesting that b-alanine-mediated taurine depletion promotes the generation of superoxide by the mito- The goal of this study is to characterize the functional conse- chondria. Hence, taurine is required for normal mitochondrial quences of taurine interaction with the GABAA receptors. Taurine function; severe taurine depletion reduces mitochondrial function is a sulfur-containing, conditionally-essential amino acid amino and enhances the generation of reactive oxygen species by the acid, found in high concentrations in the brain and act as a electron transport chain. GABAA receptor. We found that acute injection of taurine to mice alters a variety of neurobehaviors that are mediated by GABAA Metabolic changes mediated by taurine depletion: role receptors. These include suppression of locomotor activity, anxiety and heightened fear potentiated freezing responses. Outside the in mitochondrial function CNS-mediated behaviors, acute taurine injection resulted in a hypoglycemia and hypotension. All these affects (both central and Stephen Schaffer1, Chian Ju Jong1 and Junichi Azuma2 peripheral) are mediated through activation of the GABAA recep- 1 tors and could be blocked by specific GABAA receptors University of South Alabama College of Medicine Department antagonists. On the other hand, taurine supplementation in drinking of Pharmacology, Mobile, AL 36688; water induced a state of brain excitability characterized by 2Hyogo University of Health Sciences School of Pharmacy, Kobe, Japan increased susceptibility to kainic acid-induced seizures. Taurine-fed mice had elevated brain levels of glutamate and GABA. This Rats treated with the taurine transport inhibitors, guanidinoethylsulfo- increase in neurotransmitter levels was accompanied by an increase nate or b-alanine, lose about 50% of their myocardial taurine content. in the expression of GABA synthesizing enzyme, glutamic acid Associated with the decline in taurine levels is a modest change in decarboxylase. Furthermore, taurine-fed mice have reduced myocardial relaxation (-dP/dt) but no apparent change in other mea- expression of GABAA receptors. The down-regulation of GABAA sures of myocardial function. However, the taurine depleted hearts receptors is due to the sustained interaction of taurine with GABAA experienced significant changes in energy metabolism, which are receptors which decreases the efficacy of the inhibitory synapses at characterized by a shift away from aerobic metabolism in favor of postsynaptic membrane. As a compensatory mechanism to this anaerobic metabolism. The taurine deficient hearts experienced a two- increased excitability, there is increased GAD expression as dem- fold increase in glucose utilization, a twofold increase in pyruvate onstrated biochemically and pharmacologically. Peripheral effects production and a three- to fourfold increase in lactate production. These of taurine supplementation included glucose tolerance and hyper- effects were exaggerated in hearts perfused with buffer containing tension. Thus, chronic supplementation of taurine induces several insulin. Crossover plots revealed that the stimulation in glycolysis by the biochemical changes in the GABAergic system. taurine depleted heart was largely caused by the activation of

123 S84 12th International Congress on Amino Acids, Peptides and Proteins phosphofructokinase, in part because of a decrease in citrate levels. The confined to the S3 segment of renal proximal tubules—the primary size of the creatine phosphate and adenine nucleotide pools of the site for renal adaptive regulation of the taurine transporter, we taurine deficient heart fell by 26 and 6%, respectively. These data are hypothesized that TauT is a target of p53 and c-Jun during cisplatin- consistent with evidence in isolated cardiomyocytes that b-alanine- induced nephrotoxicity. A recent study showed that oxidative stress- mediated taurine depletion slows flux through the electron transport induced JNK signaling pathway together with c-Jun and AP1 activity chain secondary to impaired assembly of electron transport chain contribute to drug resistance. In the present study, we have shown that complexes. TauT is a direct target of p53 and c-Jun. Expression of TauT is down- regulated by p53 and up-regulated by c-Jun in human embryonic 293 renal cells as determined by promoter analysis, reporter assay, DNA Perinatal taurine exposure alters neural control binding, and western blot analysis. Forced overexpression of TauT of arterial pressure via the renin-angiotensin system protects cisplatin-induced apoptosis. Inhibition of TauT by RNA interference resulted in a significant reduction of 293 cell growth and but not estrogen in rats enhanced the sensitivity of 293 cells to cisplatin in dose- and time- dependent manners. Furthermore, we have demonstrated that over- Sanya Roysommuti1, Atcharaporn Thaeomor1, Wichaporn expression of TauT prevented the progression of cisplatin-induced Lerdweeraphon1, Sawita Khimsuksri1, Dusit Jirakulsomchok1, AKI in TauT transgenic mice, as measured by the levels of BUN and and Stephen W. Schaffer2 serum creatinine, and TUNEL assay. Cisplatin activated p53 and PUMA (a p53-responsive proapoptotic Bcl-2 family protein) in the Department of Physiology, Faculty of Medicine, Khon Kaen kidneys of both wild-type and TauT transgenic mice. However, AKI University, Khon Kaen 40002, Thailand and was only clearly observed in the wild-type animals, suggesting that 2Department of Pharmacology, College of Medicine, University functional TauT plays a critical role in protecting against cisplatin- of South Alabama, Mobile, Alabama 36688, USA induced AKI, possibly through attenuating a p53-dependent pathway. In conclusions, functional TauT plays an important role in cis- Perinatal taurine exposure alters neural and renal controls of arterial platin-induced renal injury. Expression of TauT is regulated by p53 pressure in adult rats. This study tests the hypothesis that perinatal and c-Jun and the balance of such regulation will determine the levels taurine status influences arterial pressure control by altering the renin- of TauT that may decide the fate of the renal cells. angiotensin system in adult female rats. Female Sprague-Dawley (SD) rats were fed normal rat chow with 3% beta-alanine (TD), 3% taurine (TS) or water alone (C) from conception to weaning. Their female offspring were fed with the normal rat chow with either 5% glucose in Taurine and Calculus Bovis: benefits tap water (TDG, TSG, CG) or tap water alone (TDW, TSW, CW) of ethnopharmacological knowledge throughout the experiment. Acute inhibition of the renin-angiotensin system with captopril (CW + Cap, CG + Cap, TDW + Cap, 1,2 2 3 TDG + Cap, TSW + Cap, TSG + Cap) or estrogen receptors with Kyoko Takahashi , Yuko Azuma , Junichi Azuma 4 tamoxifen (CW + Tam, CG + Tam, TDW + Tam, TDG + Tam, and Stephen W. Schaffer TSW + Tam, TSG + Tam) were studied. At 7–8 weeks of age, mean 1 arterial pressures (MAP) and heart rates were not significantly dif- The museum of Osaka University; 2 ferent among control treated groups and only MAP decreased in all Graduate School of Pharmaceutical Sciences, Osaka University, rats with the captopril but not tamoxifen. Baroreflex sensitivity con- Osaka, Japan; 3 trols of heart rate and renal nerve activity significantly decreased only School of Pharmacy, Hyogo University of Health Sciences, Hyogo, in TDG and these were restored to CW groups by the captopril but not Japan; 4 tamoxifen. In addition, sympathetic nerve activity significantly and School of Medicine, University of South Alabama, Mobile, AL, USA markedly elevated and parasympathetic nerve activity decreased only in TDG and these were also restored to the CW groups by the captopril The use of animal products in healing is an ancient and widespread but not tamoxifen treatment. Altogether, the present data suggest that cross-cultural practice. Calculus Bovis (the gallstone of Bos Taurus high sugar intake alters the autonomic nervous system control of domesticus Gmmelin) is one of the most precious and commonly-used arterial pressure via the renin-angiotensin system in perinatal taurine medicinal materials in Japan and China. Taurine, discovered from ox depleted adult female rats and this effect is not attenuated by estrogen. gall in 1827, is one of the major active components of C. Bovis. Although C. Bovis has medicinal benefits in improving cardiac contraction, promoting sedation, relieving fever, diminishing Role of taurine and taurine transporter (TauT) inflammation and/or normalizing function of the gallbladder, its detailed mechanism remains largely unknown. To ensure sustainable in cell death use of traditional medicines derived from C. Bovis, we felt that several issues needed to be addressed: (1) the source of the C. Bovis materials Xiaobin Han and quality control; (2) the role of taurine in the efficacy of C. Bovis. The present work provided some references for the quality control and University of Tennessee Health Science Center, Department the efficacy of C. Bovis by using ICP-MS and cultured cardiac cells, of Pediatrics, 50 N. Dunlap St. CRFC 336, Memphis, TN, USA, respectively. First, principal component analysis (PCA) and multi- Email: [email protected] elemental focus were effective in discriminating C. Bovis samples derive from different habitats. Second, C. Bovis was found to exert Cisplatin is a commonly used chemotherapeutic agent that has a cardioprotection against antiarrhythmias by: (1) diminishing arrhyth- major limitation because of its nephrotoxicity. We have demonstrated mias produced by low and high medium Ca2+; (2) antagonizing the that cisplatin down-regulates the expression of taurine transporter pro-arrhythmogenic actions of beta-alanine (an inhibitor of taurine gene (TauT) in LLC-PK1 proximal tubular renal cells and forced transport); (3) attenuating the harmful actions of bile acids. It is overexpression of TauT protects against cisplatin-induced apoptosis plausible that the relationship between taurine, Ca2+ and the bile acids in renal cells in vitro. Since cisplatin-induced renal injury is mainly contribute to the therapeutic effect of C. Bovis.

123 12th International Congress on Amino Acids, Peptides and Proteins S85

Taurine and metabolic syndrome In our pilot clinical study both treatments produced comparable beneficial results. Therefore, the results from clinical studies are in Masato Imae1 and Shigeru Murakami2 agreement with previous in vitro data and strongly suggest that TauBr could be considered a new therapeutic agent in acne vulgaris. Finally, 1R&D Laboratories and we speculate on a therapeutic potential of taurine/TauCl in a pre- 2R&D Headquarters of Taisho pharmaceutical Co. Ltd., Saitama-shi, vention of the induction of autoimmunity. This idea is supported by Saitama 331-9530 and Toshima-ku, Tokyo 170-8633, Japan our recent studies showing that scavenging of HOCl by taurine (formation of TauCl) neutralizes oxidative modification of self pro- Metabolic syndrome is a cluster of cardiovascular risk factors teins by HOCl. Therefore, this reaction protects proteins against including abnormal obesity, hyperglycemia, dyslipidemia and ele- enhancement of their immunogenicity and reduces probability of vated blood pressure. The prevalence of metabolic syndrome is autoantibodies production. increasing in modern societies and is becoming a significant problem around the world. Many studies in animal models and humans have The combined effect of taurine with BCAA revealed that taurine prevents these components of metabolic syn- on the delayed-onset-muscle-soreness and damages drome. The beneficial effects of taurine are thought to be attributable to its basic physiological actions, including osmoregulation, antioxi- after eccentric exercise—a double-blind study dation, immunomodulation, Ca2+ modulation, angiotensin II antagonism, and bile acid conjugation. Taurine stimulates bile acid Song-Gyu Ra1, Teruo Miyazaki2, Keisuke Ishikura3, synthesis from cholesterol in the liver, and thereby decreases the Takafumi Suzuki1, Seiji Maeda1, Shumpei Miyakawa1, cholesterol levels in the plasma and liver, thus leading to improve- Yasushi Matsuzaki2, and Hajime Ohmori1 ments in hyperlipidemia, atherosclerosis and fatty liver. The anti- diabetic effect of taurine is related to its improvement of insulin 1Graduate School of Comprehensive Human Sciences, University resistance through amelioration of lipid and glucose metabolism, and of Tsukuba, Tsukuba, Japan; islet b-cell protection. Taurine has also been shown to enhance the 2Department of Development for Community Medicine, Tokyo energy production in adipose tissue. Anti-hypertensive effects of Medical University, Ami, Japan; taurine have also been demonstrated in hypertensive rats and humans. 3Sports Research and Development Core, University of Tsukuba The suppression of the sympathetic nerve activity may be responsible for the effects of taurine on blood pressure. In addition, the protective Aim: A high-intensity-exercise causes delayed-onset-muscle-soreness effect on endothelial cells may be important for the prevention of (DOMS) and damages that disturb continuous and practice of exer- hypertension as well as atherosclerosis. Worldwide epidemiological cise. Although previous studies have evaluated the effectiveness of studies have showed that urinary taurine excretion as a marker of BCAA on the DOMS, consistent finding has not still convinced. taurine intake is inversely related to the body mass index, blood Therefore, the present study investigated the combined effect of pressure, serum total cholesterol and coronary heart disease mortality. taurine that has many physiological and pharmacological roles with As a result, taurine may therefore prevent metabolic syndrome via BCAA on the DOMS and damages. multiple mechanisms. Method: Untrained volunteers (22.5 ± 3.8 years of age) were assigned to four groups: Control (Placebo; n = 5), BCAA-supplementation (9.6 g/day; n = 6), taurine-supplementation (6 g/day; n = 7), Combination (taurine + BCAA; n = 7). The subjects inges- Taurine haloamines as anti-microbial, ted these supplementations before 2 weeks, on the day, and after 4 days of exercise. Muscle damages were caused by repeating of anti-inflammatory and anti-oxidant agents: forced eccentric elbow flexors. All experiments were carried out new perspectives for clinical use under double-blind method. Result: In the period by 4 days after exercise, the Combination sig- Janusz Marcinkiewicz nificantly improved the DOMS evaluated by VAS, upper arm circumferences, range of elbow’s motion, and serum markers of Department of Immunology, Jagiellonian University Medical muscle damages, although there were some effects in both the taurine College, Krakow, Poland and BCAA alone compared to those in the Control. Conclusion: Combination of taurine with BCAA would be useful Taurine haloamines, taurine bromamine (TauBr) and taurine chlora- way to attenuate DOMS and muscle damages after high-intensity- mine (TauCl), the physiological products of peroxidase halide system, exercise. are generated by eosinophils and neutrophils at a site of inflammation. TauBr and TauCl show anti-inflammatory and anti-microbial properties. Moreover, both haloamines are components of ‘‘the antioxidant network’’ by their ability to induce the expression of heme The effect of taurine treatment on the differentiation oxygenase-1 (HO-1). On the other hand, only TauBr was found to be of cultured skeletal muscle cell highly membrane-permeable, showing stronger microbicidal activity than TauCl. In addition, TauBr shows in vitro ability to reduce for- Teruo Miyazaki1, Akira Honda1, Tadashi Ikegami1, Mutsumi Shirai1, mation of bacterial biofilm. Therefore, TauBr is promising candidate Shumpei Miyakawa2, and Yasushi Matsuzaki1 for a local treatment of chronic infectious/inflammatory disorders associated with the presence of biofilm, namely in acne vulgaris and 1Tokyo Medical University Ibaraki Medical Center, and chronic rhinosinusitis. 2University of Tsukuba, Ibaraki, Japan To supported the idea to use TauBr for topical anti-acne therapy we have compared the effectiveness of TauBr to that of Clindamycin, Background and aim: Taurine has been considered as one of one of the most common topical agent used in the treatment of acne. essential factors on the differentiation/growth of skeletal muscles

123 S86 12th International Congress on Amino Acids, Peptides and Proteins because deficiency of taurine causes incomplete muscular develop- cells. Meanwhile it is well known that taurine deficiency associates ment and exercise abilities. During development and growth periods, with some pathology- or aging-impaired tissue function. However, the abundant taurine is endogenously supplied through placenta and roles of taurine deficiency in the tissue dysfunction have not been milk due to no biosynthesis capacity. The present study examined fully clarified. Recently, we have generated taurine transporter- the role of taurine treatment on the differentiation of mouse myo- knockout (TauTKO) mice and demonstrated that these mice exhibited blast to myotube. Method: Confluent C2C12 cell was cultured with a deficiency in tissue taurine level, especially in heart and skeletal *20 mM taurine in a differentiation medium for up to a week with/ muscle. TauTKO mice also exhibited loss of body weight, cardio- without silencing of taurine transporter gene (taut), transport com- myopathy and the reduced exercise capacity. Furthermore, we petitor; b-alanine, Ca2? chelator; nifedipine, or calcineurin inhibitor; recently found that life span is reduced in TauTKO mice compared to FK506. The expressions of differentiation markers were evaluated wild-type mice. Therefore, taurine deficiency increases the suscepti- by RT-qPCR, fluorescence immunohistochemical stain, or Western bility against physiological stress. Moreover, mitochondrial defects, blot. such as ultra-structural abnormality and a reduction in mitochondrial Result: The differentiation to myotube was significantly and dose- complex I activity, were observed in TauTKO mice. These data imply dependently enhanced by taurine treatment, in particular sixfold in not only the involvement of mitochondrial dysfunction in taurine 20 mM taurine, estimated by fusion index and maximal diameter in depletion-related impairment of various physiological functions but the MHC-positive myotubes. The phosphorylations of Akt and also the cellular mechanism responsible for aging. p38MAPK were increased by taurine. The enhanced differentiations by taurine were significantly cancelled by the taut silencing, b-alanine, nifedipine, or FK506. Conclusion: Exogenous taurine might play as an essential role for the mature differentiation/growth on the skeletal muscle through calcium signaling.

The role of taurine deﬁciency in physiological stress: a study in taurine transporter knockout mice

Takashi Ito, Stephen W. Schaffer, and Junichi Azuma

Hyogo University of Health Science

Taurine has a variety of biological actions, such as anti-oxidation, osmoregulation and modulation of calcium handling, in mammalian

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