Abstracts presented at the 12th International Congress ... - Springer Link

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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Analysis A hydrophobic interaction chromatographic media prepared from poly (N-isopropylacrylamide-co-methyl methacrylate) Junmin Yuan, Jing Li, Chaozhan Wang* 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, 229 Tai Bai North Road, Xi’an 710069, People’s Republic of China, Tel: +86-29-88302604, [email protected] 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 to prepare hydrophobic interaction chromatographic media. The separation efficiency of the prepared media was tested by using a standard protein mixture, it was found that the media has quite good performance for protein separation. Effects of temperature on protein separation and retention were investigated, the results showed that the hydrophobicity of the media increased gently with increasing the ratio of methyl methacrylate in the grafted polymer. For a given prepared media, the retention of proteins increased when the column temperature was elevated, and the resolution of proteins changed dramatically with varying temperature. The prepared chromatographic media was used for purification of myoglobin from pig heart as well as lysozyme and ovalbumin from hen egg white. This work was supported by the National Natural Science Foundation in China (No. 20705028).

Allergens presence in peanut proteins extracted by enzymatic aqueous extraction: analysis by mass spectrometry S. Latif1*, J. Pfannstiel2, H. P. S. Makkar1 and K. Becker1 1 Institute for Animal Production in the Tropics and Subtropics (480), University of Hohenheim, 70593, Stuttgart, Germany 2 Life Science Center Core Facility, University of Hohenheim, 70593 Stuttgart, Germany

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 proteomic-based approach was used to evaluate the allergens presence in the protein extracted through EAAE. Recoveries of 92% oil 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 EAAE was subjected to matrix-assisted laser desorption/ionizationtime of flight mass spectra (MALDI-TOF/MS) which revealed it as a rich source of peptides. Peptides (107 in number) with molecular masses ranging from m/z 700.39–2,369.08 Da were identified. In order to get detailed information on peptide sequences, the protein fraction was further analyzed by nano-LC–ESI-MS/MS on a high resolution mass spectrometer (LTQ-Orbitrap XL). The ESI-MS/MS

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12th International Congress on Amino Acids, Peptides and Proteins data were used to perform a database search using the Mascot 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 the APMs DLAFPGSGEQVEKL, SARQQWELQGDRRCQS, RSVNELDLPIL and KRELMNLPQ for Ara h 1, Ara h 2, Ara h 3 and Ara h 6, respectively. These data suggest that enzymatic treatment during EAAE did not eliminate the presence of peanut allergens, however, as a rich source of peptides the protein extracted through EAAE may have potential for nutritional applications (e.g. in sports nutrition, enteral and infant formulas).

Analysis, characterization and separation of N-acyl derivatives of 2,6-diaminopimelic acid by capillary zone electrophoresis and micellar electrokinetic chromatography Miloslava Vı´tovcova´, Jan Hlavaćˇek, Jan Pıćha, Vaćlav Vaneˇk, Jirˇ´ı Jiraćˇek, and Vaćlav Kasˇicˇka Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague, Czech Republic Recently, the mono-N-protected derivatives of 2,6-diaminopimelic acid (DAP) were prepared to function as competitive inhibitors of the mono-N-succinyl-DAP hydrolysis catalyzed by dapE enzyme, with aim to interrupt the pathways leading to development of bacteria. In this work, capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (CMEKC) were employed for determination of the electrophoretic purity degree, limit of detection (LOD) and limit of quantification (LOQ) of twelve monoN-acylated derivatives of DAP, using an HPCE analyzer equipped 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 classical and isoelectric buffer-based acidic and alkaline background electrolytes within a pH range 2.18–8.64. A new procedure has been developed for correction of effective mobilities to the reference temperature of 25°C. The best separation of the mixtures of DAP derivatives was achieved by CMEKC in acidic background electrolyte (500 mmol/L acetic acid, pH 2.54) using anionic surfactant 60 mmol/L SDS as a constituent of the micellar pseudostationary phase. Acknowledgments: Supported by the Czech Science Foundation (203/08/1428; 203/09/0675) and the Academy of Sciences of the Czech Republic (GAV A400550614; Research Project AV040550506).

Chemical ligation: an effective tool to characterize the interaction between Nedd4L WW domains and the Smad3 linker region by NMR Nina Goerner Smad proteins are the mediators of the TGF-b signalling pathway that controls cell growth and specific cell differentiations. Defects in this pathway can lead to rapid cell growth resulting in cancer diseases. Smad proteins interact with other proteins through their linker region that connects a DNA binding domain named MH1 and a TGF-b-receptor-binding domain known as MH2 domain. The

12th International Congress on Amino Acids, Peptides and Proteins Smad2/3 linker region consists of four phosphorylation sites and a PPxY (PY) motif that is recognized by WW domains. WW domains are small binding domains present in around 170 proteins of variable function, which are involved in many cellular processes, such as transcription, splicing or protein degradation to mention a few. We have characterized in detail the interaction of Nedd4L and a fragment of Smad3 linker including the PY site and also combinations of the phosphorylated residues. To obtain the structural information we have used protein constructs containing pairs of domains and a segmental labelling approach that requires the use of a chemical ligation strategy.

Chiral discrimination of amino acids using achiral electrophoresis separation hyphenated with mass spectrometry Vaćlav Ranc1, Radim Knob1, Jan Petr2, Vitezslav Maier2, Eva Tesarova3, and Juraj Sevcik1 1 Department of Analytical Chemistry, Faculty of Science, Palacky´ University in Olomouc, 17. Listopadu 12, 77146 Olomouc, Czech Republic 2 Regional Centre of Advanced and Technologies, Department of Analytical Chemistry, Faculty of Science, Palacky´ University in Olomouc, 17. Listopadu 12, 77146 Olomouc, Czech Republic 3 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 128 40 Prague 2, Czech Republic,

The study of chiral body constituents like amino acids (AAs) represents one of the frequent tasks of modern analytical chemistry. This increasing demand tension for a development of chiral methods is given by a large number of physiological processes involving studied compounds. As analytical tools, chromatographic or electrophoretic approaches are usually employed. Mass spectrometry could present an interesting alternative. This work deals with a mass spectrometric behavioral study of 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 D,L-Val, where L-Trp was chosen as the chiral host. Firstly, the target amino acids were separated by foregoing electrophoresis using 0.75 M formic acid as a background electrolyte. After the separation step, they AAs were injected to the MS part using electrospray ion source. Sheath liquid (water– methanol 1:1, v/v) contained host compound (total concentration = 1 9 10-3 M). Chiral discrimination was calculated using relative intensities of protonated host molecule and cluster containing host and guest molecule. It was shown that the ratio of these two relative intensities is proportional to the chiral composition of initial sample. Quantification of chiral composition (for a determination of optical purity) is based on five point calibration curves containing 0, 25, 50, 75 and 100% of corresponding D-enantiomer for a determination of optical purity. Represented correlation coefficients are 0.986 for Ala, 0.995 for Phe and 0.997 for Val, respectively. This work was supported by the Research Project of the Ministry of Education of the Czech Republic No. MSM6198959216, by a grant agency of the Palacky University PRF_2011_025 and by Operational Program Research and Development for Innovations–European Social Fund (project CZ.1.05/2.1. 00/03.0058).

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Enantioselective separation of branched amino acids using various separation systems/modes in HPLC Kveˇta Kalı´kova´1, Pavel Repko2, Marta Kowalczyk4, Zuzana Bosa´kova´2, Juraj Sˇevcˇ´ık3, and Eva Tesarˇova´1 1

Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 128 43 Prague 2, Czech Republic 2 Department of Analytical Chemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 128 43 Prague 2, Czech Republic 3 Department of Analytical Chemistry, Faculty of Science, Palacky University in Olomouc, Czech Republic 4 Department of Analytical Chemistry, Faculty of Science, University of Warsaw, Poland, [email protected] Enantiomers of branched amino acids (BAAs), namely alanine, valine, leucine and isoleucine, are present in fresh food. Their 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 addition BAAs are important components of food supplements, in which just L-enantiomers should be found. Separation and determination of enantiomers of amino acids can be achieved in various separation systems. In HPLC mostly chiral stationary phases are employed. A problem in the branched amino acids determination can cause low sensitivity of UV detection. Therefore, derivatization of these amino acids is usually required. In this work, enantioseparation of BAAs was performed in various separation systems employing different chiral stationary phases in various separation modes. The best enantioseparation was achieved on a teicoplanin based column in reversed phase mode or in hydrophilic interaction liquid chromatography (HILIC). In order to improve sensitivity of detection two derivatization procedures, in which dansyl chloride and fluorenylmethyl chloroformate were used as derivatization agents, were tested and compared. In both cases derivatization of BAAs resulted in decreasing LOD and LOQ values, approximately two orders of a magnitude, as compared to underivatized BAAs.

EXAFS and XANES analysis of the Fe and Zn environment in tissue samples at different growth stages Sunil Dehipawala, Todd Holden, E. Cheung, Robert Regan, P. Schneider, G. Tremberger Jr, D. Lieberman, and T. Cheung City University of New York, Queensborough Community College, 222-05 56th Ave, Bayside NY 11364, USA The zinc and iron environments in different growth stages have been studied with EXAFS and XANES with Brookhaven Synchrotron Light Source. Tissue samples included meat, organ, vegetable, bean, leaf, and yeast. The EXAFS data was background subtracted and standard Fourier component analysis was used. The XANES spectra were analyzed with standard methods including the use of a linear combination of XANES standard spectra. The Zn nearest neighbor bond length deduced from EXAFS and XANES are found to be associated with aging in bean, leaf, yeast samples. Zn signal in chayote pith is higher than those in the seed and skin. Edible chicken tissues show that the liver has the smallest Zn signature while thigh tissue has the highest as compared to drumstick, neck, breast, and wing tissues. Duck embryo

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S4 tissues from an edible source (balut) show that the heart has the smallest Zn signature as compared to liver, brain, stomach, intestine, and thigh. The accompanying yolk and white show negligible zinc and would be consistent with the early utilization of zinc during embryo growth. The Fe nearest neighbor bond length analysis show similar results, but is less striking as a bio-marker when compared to Zn. Nevertheless the Fe environment changes in calf liver after cooking as was detected in XANES data. The results suggest an effective synchrotron probe for the study of cellular changes in tissue samples as well as the effect of microbe/ virus in diverse environments such as bio-energy production. Extension to cobalt in vitamin B12 will be discussed.

Non-destructive analysis of coffee by NMR spectroscopy Feifei Wei, Kazuo Furihata, Fangyu Hu, Takuya Miyakawa, Masanori Koda and Masaru Tanokura Modern nuclear magnetic resonance (NMR) spectroscopy with dramatically improved resolution and sensitivity has been widely applied in food science to achieve a direct and comprehensive observation of foods in a non-destructive way, as it is capable in a rapid, single experiment of simultaneously detecting multiple components without destruction of the food. By identifying the different spin systems from appropriate two-dimensional NMR spectra, comprehensive analysis with NMR makes it possible to identify and quantify the components of foods as complex mixtures without physically separating them. Coffee is one of the most consumed beverages in the world. The analyses on the chemical compositions of coffee are still necessary since it is closely related to the quality, security and nutrition of coffee. A complex mixture analysis by one- and two-dimensional NMR spectroscopy was carried out for the identification and quantification of organic compounds in green and roasted coffee without any separation. A detailed NMR signal assignment, as well as quantification, coupled with multivariate statistical chemometric methods offers a powerful new approach for coffee bean quality control and assessment. As an important result of this study, amino acids can be used as significant marker compounds to distinguish the species of green coffee beans.

Nonchiral and chiral gas chromatographic: mass spectrometric analysis of amino acids in aqueous solutions. Application of a novel heptafluorobutyl chloroformate sample preparation approach to investigations in amino acid and peptide chemistry Petr Sˇimek*, Helena Zahradnıćˇkova´, and Petr Husˇek Department of Analytical Biochemistry, Biology Centre, Academy ˇ eske´ of Sciences of the Czech Republic, Branisˇovska´ 31, 37005 C Budeˇjovice, Czech Republic, [email protected] Nonchiral and chiral gas chromatographic (GC) amino acid analysis has been an important research tool in the field of amino acid,

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12th International Congress on Amino Acids, Peptides and Proteins peptide and protein chemistry. However, the polar analytes occur mostly in aqueous media and need to be converted into volatile, thermally stable derivatives prior to GC. Two decades of extensive research have proved alkyl chloroformates highly attractive for this purpose. The reagents react immediately with most amino acid functional groups in aqueous matrices and the process can easily be coupled with liquid–liquid extraction of the resulting less polar derivatives into immiscible organic phase. Here a novel, simple protocol for in situ derivatization of amino acids with heptafluorobutyl chloroformate (HFBCF) is described followed by subsequent nonchiral as well as chiral GC/MS (mass spectrometric) 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 investigations of more than 25 resolved enantiomeric antipodes in peptide hydrolysates, human serum and environmental samples. Acknowledgments: The Czech Science Foundation, projects No. 203/09/2014 and P206/10/2401.

Phylloseptin-2 conformational analysis by solution NMR at different pH values Dorila Pilo´-Veloso*, Bruno Aldrin Assunçaõ, Naira de Oliveira Toˆrres, Rodrigo Moreira Verly, and Jarbas Magalha˜es Resende Departamento de Quı´mica/ICEx/UFMG/Brazil [email protected] Keywords: Peptide antibiotics, Peptide structure, Amphipathic a-helix, Solution NMR, Conformational analysis The cationic peptide Phylloseptin-2 (PS-2) has been isolated from the skin secretion of the tree-frog Phyllomedusa hypochondriasis that inhabits Brazilian tropical forests.2 It is naturally amidated at C-terminus, is composed of nineteen amino acid residues, of which three possibly charged histidines are at positions 7, 17 and 18. Antimicrobial assays demonstrate that it exhibits activity against both Grampositive and Gram-negative bacteria.1,2 We have previously described the NMR structure of PS-2 in TFEd2:H2O (60:40, v/v) at pH 7.0 (phosphate buffer).2 In this work it is 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 1 H-15N HMQC experiments were acquired at 20°C on a Bruker AVANCE-III 800 MHz spectrometer equipped with a triple resonance (1H/13C/15N) 5 mm gradient probe. The assignment of backbone atoms was performed by the sequential assignment methodology.3 The NMR spectra were processed with NMRPipe and then analysed using NMRVIEW. The obtained NOE intensities were converted into semi-quantitative distance restrains and then 200 structures were calculated using XplorNIH. The quality of the lowest energy structures was validated by PROCHECK-NMR. The display, analysis, and manipulation of the 3D structures were performed with MOLMOL. These structures were compared with the published results.2 As a result the most stable structures indicate that PS-2 maintains an amphipathic distribution of its residue side chains within the helical segment, albeit the presence or absence of a positive charge on the His residues leads to important structural differences.

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Preparation and characterization of a hydrophobic interaction chromatographic media with a polymeric ligand

Quantitative and qualitative profiling of mitochondrial 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 and Materials Science, Northwest University, Xi’an 710069, China *College of Chemistry and Materials Science, Northwest University, 229 Tai Bai North Road, Xi’an 710069, People’s Republic of China. Tel: +86-29-88302604, Email: [email protected] A series of temperature responsive polymer with different lower critical solution temperatures were synthesized by using chain transfer free radical polymerization. And a novel hydrophobic interaction chromatographic media, with one of the synthesized polymers as ligand, was prepared for protein separation. The prepared media was used to separate a protein mixture containing seven proteins, and was compared with two normal hydrophobic interaction chromatographic media, with phenyl and PEG 600 as ligands, respectively, under various temperatures and solution compositions. It was found that the newly prepared chromatographic media has much better performance for protein separation than media with phenyl and PEG 600 as ligands. Moreover, it is more sensitive to temperature, and selectivity for proteins can be easily modulated by varying temperature. With temperature increasing, its hydrophobicity is increasing correspondingly. Therefore, a series of hydrophobicity could be obtained on one column packed with the prepared media, which is very important for separation of very complex protein samples. This work was supported by the National Natural Science Foundation in China (No. 20705028), National Fund for Talent Training in Basic Science (No. J0830417).

Purification of cytochrome c from pig heart by using frontal hydrophobic interaction chromatography Rong Chang, Xiangqin Chen, and Chaozhan Wang* 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, 229 Tai Bai North Road, Xi’an 710069, People’s Republic of China. Tel: +86-29-88302604, [email protected] Protein separation and purification is one of the most important steps during production of protein products, especially protein drugs. Cytochrome c is a small heme protein found loosely associated with the inner membrane of the mitochondrion, it is an essential component of the electron transport chain, where it carries one electron. In this work, cytochrome c was extracted from pig heart, and ammonium sulfate precipitation was used as the first step for cytochrome c purification, in which cytochrome c was precipitated, and frontal hydrophobic interaction chromatography (FHIC) was employed to further purify cytochrome c to homogenous. Various concentrations of ammonium sulfate in the mobile phase of FHIC were investigated, and a suitable concentration under which cytochrome c was not retained on the column but impurities retarded was selected. The cytochrome c precipitated by ammonium sulfate was solubilized in this concentration of ammonium sulfate and was pumped onto the column to be purified by using FHIC. Cytochrome c with a purity of 99% was obtained finally, almost all cytochrome c was recovered during FHIC. This work was supported by the National Natural Science Foundation in China (No. 20705028).

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Aging Research Center, Korea Institute of Oriental Medicine, Daejeon 305-811, Korea, Tel: +82 42 868 9508, Fax: +82 42 868 9301, [email protected], b Department of Biological Sciences, Konkuk University, Seoul 143-701, Korea, [email protected] The analysis of mitochondrial DNA sequence variation is of great interest in medical, forensic, and population genetics. Quantitative and qualitative analysis of mitochondrial DNA length for performed using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination by sequence-specific based antlers. Specific primers and fluorescence tags were designed by using mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus. When primers that differentiated Cervinae and Rangifer antlers were used to amplify mtDNA, a 466 bp fragment that was observed for both Cervinae and Rangifer antlers served as a positive control, while a 270 bp fragment was specific for Rangifer antlers. Allele discrimination was used to differentiate Cervinae and Rangifer antlers on the basis of the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for quantitative and quantitative analyses of Cervinae and Rangifer antlers in a single step without any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and especially in this study for the identification of deer products Cervinae from Rangifer antlers.

Specific modification of amino acids to induce separation Yinglai Teng, Elinor L. Scott and Johan P. M. Sanders Amino acids (AA’s) are interesting raw material as the feedstocks for the chemical industry due to the functionalities they contained. This makes them similar to conventional petrochemicals and provides the possibility to circumvent process procedures, additional reagents and energy. They could be obtained as a mixture by hydrolysis of potentially inexpensive protein sources such as agriculture waste or the side stream of biofuel productions. Still, separation is required in order to carry out further transformation into the desired products. Theoretically it could be achieved by electrodialysis (ED). To increase the efficiency of separating AA’s with similar charge behavior, specific modification, preferably to the products with industrial interest, is required. This could be achieved by enzymatic reaction such as decarboxylation. Here we focus on the specific modification for the separation of basic AA mixture containing L-arginine (Arg) and L-lysine (Lys). L-lysine decarboxylase (LDC) was applied to convert Lys to 1,5-pentanediamine (PDA) in the presence of Arg to obtain products with different charge so as to be separated by ED.1 Immobilization of LDC was performed to enhance the operational stability. Though product inhibition was seen, the enzyme is highly specific and at 30°C the presence of Arg has little effect. Based on these, a potential process using a mixture of Arg and Lys was designed and the cost was estimated based on the volumetric productivity, raw material price and transformation costs.

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Structure determination at molecular resolution using atomic force microscopy: focusing on artifacts and reproducibility Yang Gan School of Chemical Engineering and Technology, Harbin Institute of Technology, Harbin, Heilongjiang 150001, People’s Republic of China, [email protected] Despite the enormous success and wide application of Atomic Force Microscope (AFM) in various areas including biology and chemistry, 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 molecular resolution AFM topographs. In the last 15 years, reports of true atomic and molecular resolution in ambient conditions were by no means scarce; nevertheless, some have been overlooked and are rarely known, even to an AFM specialist! The AFM community could hardly benefit from this overlook because an AFM user, trained with this biased philosophy, might be frustrated and regard the challenge of achieving trustworthy high resolution as an impossible task. Instead, one needs to appreciate the power of AFM as an atomic and molecular scale analytical technique that is capable of producing reliable and reproducible topographs with various operation modes; at the same time, one needs to learn that AFM, as a local probe technique, is particularly vulnerable to artifacts. An experienced AFM user should be able to identify artifacts, avoid artifacts, and live skeptically with artifacts if necessary. This presentation, based on my review article, will review critically and timely the achievements molecular resolution in ambient conditions by AFM. The concept of resolution is discussed after a brief introduction of various AFM operation modes. Various types of tipsurface forces, particularly the forces prominent in liquid and in air, are introduced. Different viewpoints on the conditions for achieving molecular resolution are discussed. The important issues of reproducibility and artifacts are discussed in depth, with some examples taken from the latest biology literature. Finally, the challenges of AFM as a trustworthy high resolution technique are discussed.

Study of spectrophotometric characteristics of the charge transfer reaction of taurine with 7,7,8,8-tetracyanoquinodimethane Shengyun Li Department of Chemistry, Taiyuan Normal University, Taiyuan, Shanxi, People’s Republic of China Charge transfer (CT) complex of taurine drug as electron donor with 7,7,8,8-tetracyanoquinodimethane (TCNQ) as electron acceptor has been studied in Britton-Robinson buffer solution. The spectrum obtained for taurine/TCNQ system shows the maximum absorption band at wavelength of 420 nm, which is not due to the absorption of any of the reactants. This band is characteristic of an intermolecular charge transfer involving the overlap between the lowest unoccupied molecular orbital (LUMO) of the acceptor with the highest occupied molecular orbital (HOMO) of the donor. The results reveal that the interaction between the donor and acceptor is due to n–p* transitions by the formation of radical ion pairs. The formation of such complex was confirmed by both infrared and 1H NMR measurements. The stoichiometry of the complex was also

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12th International Congress on Amino Acids, Peptides and Proteins found to be 1:1 ratio by the Job’s and straight line methods between donors and acceptor. The observed data were discussed in terms of equilibrium constant (KCT), molar extinction coefficient (CT), thermodynamic standard reaction quantities (DG°, DH° and DS°). Different variables affecting the reaction were studied and optimized. At the optimum reaction conditions Beer’s law was obeyed in a concentration limit of 1–18 lg ml-1 for taurine. The developed method was applied successfully for the determination of taurine in drug preparation, and relative standard deviation of the method was 0.58–2.63%. Percentage recoveries ranged from 98.5 to 102.8%. Keywords: Taurine, 7,7,8,8-Tetracyanoquinodimethane (TCNQ), Charge transfer complex, Spectrophotometry

Study on enzyme kinetics of glutamic pyruvic transaminase and amino acids conversion by microchip electrophoresis Xiaoyu Mu1,2, Li Qi1,*, Juan Qiao1, Haizhi Zhang1,2 1 Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, 100190 Beijing, China 2 Graduate School, Chinese Academy of Sciences, 100049 Beijing, China, Tel: +86-10-82627290, Fax: +86-10-62559373, [email protected]

In this work, we firstly presented an effective microchip electrophoresis-laser induced fluorescence (MCE-LIF) technique to analyze amino acids (including L-Glu and L-Ala) with ethylene oxide/propylene oxide block copolymer as the surfactant by using the micellar electrokinetic chromatography (MEKC). Then a new method based on the determination of the variation of the concentrations of L-Glu and L-Ala was developed to assay the glutamate pyruvate transaminase (GPT) activity directly. Both the forward catalyzed reaction and the reverse catalyzed reaction were investigated to study the kinetics of GPT by monitoring the concentration changes of L-Glu and L-Ala, and the Km and Vmax were 3.6 mM, 0.2 mM/min for L-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 gripping hepatic diseases, but also showed the potential of exploring other transaminase activities simply by the new method.

The advantages of performing interaction assays in complex matrices, particularly native membranes Darryl J. Bornhop1, Amanda Kussrow1, Michael Baksh2, M. G. Finn2, Paige E. Selvy3, H. Alex Brown3, and Craig W. Lindsley3 1

Department of Chemistry and The Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville TN, 37235, USA 2 Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA, 92037, USA 3 Department of Pharmacology, Chemistry and The Vanderbilt Institute for Chemical Biology, Vanderbilt University Medical Center, Nashville, TN, 37232, USA Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general

12th International Congress on Amino Acids, Peptides and Proteins method for the direct, label-free measurement of membrane-bound 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 a solution to equilibrium binding constants over a range of several orders of magnitude. Values obtained by BSI for small- and largemolecule interactions with receptors in vesicles derived from synthetic lipids and from cell extracts were found to be in good agreement with those previously obtained by indirect methods. The possibility of using BSI as a generally applicable methodology for quantifying membrane-associated protein–ligand interactions of biochemical and pharmacological interest, over a wide range of binding affinities, will be discussed. Further, we show that is has been possible to measure changes in interfacial and kinetic parameters on in vitro reconstituted extruded lipid vesicles and purified protein with BSI. These studies characterized the molecular mechanism of inhibition for a recently identified class of potent and isoform-selective small molecules that block phospholipase D (PLD) activity and compared activity to other known small molecule inhibitors of mammalian PLD. Our preliminary results confirm that BSI is a useful nanoscale technique to measure Ki and Kd, indicates these new compounds disrupt interfacial binding and bulk lipid binding disruption (Ks) can be quantified with BSI.

Using SR-IMS technique with synchrotron for imaging molecular chemistry of protein Peiqiang Yu College of Agriculture and Bioresources, The University of Saskatchewan 51 Campus Drive, Saskatoon, SK, S7 N 5A8, Canada, [email protected] The cutting-edge synchrotron-radiation infrared microspectroscopy (SR-IMS) is able to study cell or living cell biochemistry at cellular and molecular levels. The objective of this synchrotron-based research program was to use the advanced synchrotron-based SRIMS technique as a novel approach to image the molecular chemistry of protein and image cell architecture in plant-based feed and food tissues. The experiments were conducted on beamline U2B at the National Synchrotron Light Source, Brookhaven 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 systematically carried out from various structural layers under various chemical functional groups which were related to protein structures. The results showed that with the advanced SR-IMS plus multivariate molecular spectral analyses, unique cell architecture, the molecular spatial distribution, intensity and nutrient make-up and conformation were revealed at an ultraspatial resolution. This imaging technique provides a high potential that could be used to develop a specific plant/feed/food with targeted nutrition qualify. Keywords: Synchrotron, SR-IMS, Protein image, Biological tissue

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Biochemistry Analysis of skin secretions from the Lithobates pustulosus frog using high-resolution mass spectrometry Emmanuel Rıós, Erika Patricia Meneses, Lorena Hernańdez and Cesar V. F. Batista Laboratorio Universitario de Proteo´mica, Instituto de Biotecnologıá, UNAM Av. Universidad, 2001, Col. Chamilpa, CP 92210, Cuernavaca, Mor., Me´xico www.ibt.unam.mx The lack of efficacy of conventional antibiotics against an increasing number of pathogenic microorganisms constitutes a serious public health problem. One of the strategies applied in order to overcome microorganism resistance resides in the search for novel antimicrobial agents. Antimicrobial peptides (AMPs) are widespread in living organisms and constitute an important component of innate immunity against microbial infections. Amphibians are known to produce a large number of amphipathic peptides which manifest a broad range of 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. Lithobates 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 its skin secretions. At present, liquid chromatography coupled with 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 liquid chromatography coupled with an Orbitrap Velos mass spectrometer to determine the molecular mass of all components and the primary structure of most of these. Primary structure comparison was searched against a public data bank showing that L. pustulosus skin secretion is mainly composed of ranatuerin, brevinin, esculetin and bradykinin related-peptides. Analysis of large proteins by gel electrophoresis was also performed and many tryptic peptides were de novo sequenced using CID and HCD dissociation methods.

Anti-inflammatory effect of poly-L-lysine in human intestinal epithelial cells mediated by calcium-sensing receptor activation Hua Zhang and Yoshinori Mine Department of Food Science, University of Guelph, Guelph, ON, N1G2W1, Canada The calcium-sensing receptor (CaSR) is involved in maintaining cellular homeostasis and promoting recovery of damaged intestinal epithelial cells (IECs). Poly-L-lysine is a basic polypeptide which has been identified to activate CaSR through allosteric binding. The primary goal of the current study was to identify anti-inflammatory effect of poly-L-lysine on the human intestinal epithelial cell system, and verified that these effects are mediated by CaSR activation.

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S8 Human intestinal epithelial cell lines, Caco-2 and HT-29, were used as in vitro model to study intestinal inflammation. The CaSR activation induced by poly-L-lysine was determined by measuring intracellular calcium releasing. The Caco-2 or HT-29 was pre-incubated with poly-L-lysine for 2 h and the following inflammation was induced by recombinant human tumor necrosis factor (TNF)-a (2 ng/ mL). The secretion level of interleukin (IL)-8 from IECs which is recognized as a biomarker indicating the inflammation was measured by sandwich enzyme linked immunosorbent assay (ELISA). The poly-L-lysine reduced the IL-8 secretion from TNF-a-induced IECs. The gene expressions of several pro-inflammatory cytokines, including TNF-a, IL-6 and IL-1b, were also inhibited by poly-L-lysine supplementation. Both anti-CaSR antibody and antagonist (NPS2143) were used to block poly-L-lysine-mediated CaSR pathway and the suppression of IL-8 secretion in both IECs was abolished. Our current results demonstrated the anti-inflammatory activity of poly-L-lysine in vitro in IECs, and indicated that the activity was primarily via CaSRmediated activation.

Arginine as a novel regulator of macroautophagy in H4-II-E Cells Motoni Kadowaki1,2, Kenji Kaneshiro1, Yasuhiro Daigaku1, Masatoshi Kubota2, and Shinobu Fujimura1,2 1

Graduate School of Science and Technology, and Center for Transdisciplinary Research, Niigata University, Japan

2

Backgrounds and objectives: Macroautophagy is a major system of intracellular bulk degradation and is regulated physiologically by a number of amino acids. However, the signaling mechanism of amino 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 contribution of NO pathway as the signaling mechanism of Arg on macroautophagy was investigated. Materials and methods: H4-II-E cells were incubated in HBSS with complete amino acid mixture, regulatory amino acids, and Arg. Longlived proteolysis was measured by 14C-Val release and calculated as %/h, and presented as % of HBSS (control) value. As an autophagy marker, LC3 in the cytosol fraction was separated by western blotting and detected by anti-LC3 antibody. The amount of LC3-I and LC3-IIs were quantified by densitometric scanning and calculated as cytosolic LC3 ratio, a quantitative autophagy index. The NOS inhibitors, aminoguanidine (AG, 1 mM), L-NIL (100 lM), L-NMMA (100 lM) were employed for testing the contribution of NO pathway for the signaling. Results: Since Arg significantly suppressed proteolysis and cytosolic LC3 ratio equally, the effect was autophagic. The suppressive effect of Arg was inhibited by these NOS inhibitors, so the signaling mechanism by Arg was through NO pathway, independent from that by other amino acids.

Atherogenic transformation of LDL and impaired function of HDL by glycation Naila Rabbani and Paul J Thornalley Warwick Medical School, Clinical Sciences Research Institute, University of Warwick, University Hospital, Coventry CV2 2DX, U.K Glycation by glucose of plasma proteins—including lipoproteins low density lipoprotein (LDL) and high density lipoprotein (HDL)—has

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12th International Congress on Amino Acids, Peptides and Proteins been studied for many years. Recent research has shown that glycation of plasma proteins by the physiological dicarbonyl compound, methylglyoxal, forms quantitatively important advanced glycation endproducts (AGEs). We investigated the impact of glycation by methylglyoxal on the structure and function of LDL and HDL—particularly in relation to the atherogenic transformation of LDL and the decreased stability in HDL associated with increased risk of cardiovascular disease. We studied the effect of glycation of LDL, HDL2 and HDL3 by methylglyoxal to physiological extent on particle size, aortal trapping and functional activities. LDL and HDL subfractions were purified from venous plasma of healthy human volunteers by density gradient centrifugation and purity confirmed by SDS-PAGE. LDL and HDL modified minimally by methylglyoxal were prepared. Cholesterol and triglyceride contents and electron microscopy particle size were analysed. Plasma clearance and aortal trapping of control and modified LDL and HDL was studied in male rats. The particle sizes of both LDL and HDL were decreased by 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. Glycation of HDL2 by methylglyoxal decreased particle size. Glycation by methylglyoxal leads to atherogenic transformation and aortal trapping of LDL, facilitating atherosclerosis in renal failure. Quantitation of methylglyoxal-modified LDL may improve epidemiological cardiovascular disease risk models. Glycation of HDL2 by methylglyoxal in may contribute to dyslipidemia by increasing HDL degradation. Small HDL2 is more readily hydrolysed by hepatic and endothelial lipases with subsequent increased renal degradation of apolipoprotein A1, decreasing HDL levels. Therapeutic agents to decrease methylglyoxal may improve current treatment to decrease the risk of cardiovascular disease in high risk groups where protein glycation by methylglyoxal is increased—particularly the elderly, patients with diabetes and patients with renal failure.

Biological characterization and structure based prediction of IGFBP-5 Minkyung Sung, Eun Young Hwang, Mi Suk Jeong, and Se Bok Jang* Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Korea. *Corresponding author: E-mail: [email protected] The insulin-like growth factor binding protein (IGFBP) family has been shown to play a role in various functions such as cell growth, cell death, cell motility, and tissue remodeling. Among the 7 IGFBP family members, IGFBP-5 was recently shown to play an important role in breast cancer biology, especially in breast cancer metastasis. 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 with expression and crystallization of full-length IGFBP-5, fragments have more frequently been studied. In this study, IGFBP-5 structures containing N, L, and C domains were separately modeled from solved structures in protein data bank (PDB). In addition, the L domain of IGFBP-5 was expressed in Escherichia coli and purified for studying its structural characterization. Despite very low sequence homology, the novel L domain structure of IGFBP-5 was unexpectedly similar to that of the corepressor of repressor element-1 silencing transcription factor (CoREST) linker in the lysine-specific demethylase 1 (LSD1)CoREST complex. The purified L domain existed as a homogenous dimer in glutaraldehyde cross-linking and exhibited a typical a-helix

12th International Congress on Amino Acids, Peptides and Proteins structure in the circular dichroism (CD) assay. This study has potential applications in medicine and other fields such as drug design, mutational study, and disease prediction.

Citicoline influences kynurenic acid formation: an in vitro study Halina Baran1 and Berthold Kepplinger1,2,3 1

Neurochemical Laboratory, Karl Landsteiner Research Institute for Pain Treatment and Neurorehabilitation, LKM Mauer-Amstetten, 2 Department of Neurology, Neuropsychiatric Hospital Mauer, Amstetten-Mauer, 3 Department of Neurology, General Hospital Amstetten, Amstetten, Austria Corresponding author: [email protected] Citicoline (cytidine-50 diphosphocholine) is a neuroprotectant and neurorestorative drug and is used in the treatment of acute stroke. Citicoline is involved in the formation of phosphatidylcholine, a 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 125 mg citicholine/ml is called startonyl, Firma Torrex Chiesi Pharma GmbH Austria) with respect to kynurenic acid formation in an in vitro study. Kynurenic acid is an endogenous metabolite of tryptophan degradation along the kynurenine pathway and acts as an antagonist at the glutamate ionotropic excitatory amino acid and at the nicotine cholinergic receptors and exerts neuroprotective activity. The activities of the KYNA synthesising enzymes kynurenine aminotransferase I, II and III (KAT I, KAT II and KAT III) in rat liver homogenates were analysed in the presence 0, 15, 35 and 75 ll of startonyl, respectively. KAT I, II and III activities were measured using a enzymatic method in the presence of 1 mM pyruvate and 100 lM L-kynurenine. Citicoline, dose-dependently and significantly increased KAT I activity of rat liver homogenate, whereas KAT II activity was dose-dependent and moderate reduced, while KAT III was not affected significantly. The present study for the first time demonstrates the ability of citicoline to influence kynurenic acid formation in rat liver homogenates. The effect of citicoline varied notably between KAT I, II and III activities. We suggest that the neuroprotective effect of citicoline could be in part due to influence of kynurenic acid formation involving KAT I.

Cloning and functional analysis of afsR, a global regulatory gene for secondary metabolism in Streptomyces acidiscabies Min-Jeong Kim, Hye-Yun Park, Hyo-Ji Kim, Sun-Uk Choi, and Yong-Il Hwang Department of Food Science and Biotechnology, Kyungnam University, Changwon, 631-701, Republic of Korea

S9 Potato scab disease is caused by several Streptomyces strains producing thaxtomin such as S. scabies, S. acidiscabies, and 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 the regulation of secondary metabolism and morphological differentiation 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 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. acidiscabies was cloned by genomic Southern hybridization with PCR product as a probe and then studied. To identify the function of the cloned afsR gene, it was inserted into pSET152ET to construct a high expression vector, and then was introduced into Streptomyces strains. The exconjugant including the high expression vector of afsR was constructed and its phenotype was compared with the control strain. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20100008443).

Compartmentalization of proteins in lipid droplet biogenesis Horst Robenek, Insa Buers, Oliver Hofnagel, Mirko J. Robenek, David Troyer and Nicholas J. Severs Leibniz-Institute for Arteriosclerosis Research, Department of Cell Biology and Ultrastructure Research, Domagkstr. 3, 48149 Mu¨nster, Germany Our existing understanding of the protein organization and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve protein composition. The electron microscopic technique of freeze-fracture replica immunolabeling (FRIL) overcomes these problems. Because of the property of frozen lipids to deflect the fracture plane, en face views of the lipid droplet and its component layers are revealed for high resolution visualization. By means of immunogold labeling, proteins involved in the accretion and mobilization of lipids, notably the PAT family proteins, can be localized at and in the droplet. This approach demonstrates that, contrary to prevailing wisdom, the PAT family proteins 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 phospholipid monolayer, is not substantiated. Instead, lipid droplets appear to develop externally to both ER membranes at specialized sites in which the ER enwraps the droplet, and the facing leaflets of the ER membrane and droplet surface are enriched in adipophilin. PAT family proteins are not, as often stated, specific to the lipid droplet, but are widely present in the plasma membrane where, after lipid loading, they adopt a similar configuration to that of specialized sites in the ER. These examples highlight the contribution of the FRIL technique to critical appraisal and development of concepts in the lipid droplet field.

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Conformational stability of exchangeable apolipoproteins in solution and associated with phospholipid A. D. Dergunov

12th International Congress on Amino Acids, Peptides and Proteins The results of this study show that the photoproperties of pheomelanin can be modulated by various experimental conditions, ranging from the photoprotection to the triggering of potentially damaging photochemical reactions. Noteworthy, the observed photosensitizing effect of pheomelanin on nitrative modification of 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 in human plasma apoA-I, apoA-IV, apoE3 and three recombinant apoE isoforms in solution and discoidal complexes of apoA-I, apoA-II, apoA-IV and apoE3 with phosphatidylcholine was studied. The protein conformational stability ðDGH2 O Þ 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 threestate model, thus evidencing two-domain structure both in solution and in complexes. The predicted folding rate of the four apoE in solution correlated with their conformational stability, but the dependence disappeared at the inclusion of apoA-I and apoA-IV into analysis. In addition, the m-values, adjusted for residue number in helices (mrh), differed between those for apoE and apoA-I/apoA-IV. However, if analyzed together, the mrh-values for six proteins correlated positively with the fractional change in accessible surface area at unfolding for Phe, Lys and Asn, while negatively for Arg, Ala and Gly residues. The difference between the adjusted DGH2 O values for apolipoproteins in complexes and in solution decreased at the increase of reduced temperature Tred, calculated as Tred = (Tobs - Tt)/Tt. Two-domain structure of apolipoproteins in complexes was much more unified. The induction of intrinsic disorder by arginine residues may be of primary importance in metabolism and function of exchangeable apolipoproteins, while their stability in nascent discoidal HDL is controlled by the physical state of phosphatidylcholine (RFBR grant 10-04-00270).

Effect of pheomelanin on UVB radiation-induced oxidation/nitration of tyrosine Mario Fontana, Alessia Baseggio Conrado, Simonetta Maina, Luciana Mosca and Laura Pecci Department of Biochemical Sciences, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy Pheomelanin is a natural yellow-reddish pigment derived from tyrosinase-catalyzed oxidation of 5-S-cysteinyldopa. Generally, the formation of melanin pigments is a protective response against the damaging effects of UV radiation in skin. However, pheomelanin increases UV-induced lipid peroxidation in liposomes, suggesting that this pigment could also act as pro-oxidant. The photoproperties of this natural pigment have been studied by analyzing the effect of pheomelanin on photooxidation of tyrosine induced by UVB radiation (kmax 313 nm, total dose 4,800 J m-2). After exposure to UVB radiation, tyrosine is converted to 3,30 -dityrosine and, in the presence of nitrite, also to 3-nitrotyrosine. It has been observed that pheomelanin plays a protective role on the oxidation/nitration of tyrosine at pH 7.4. The formation of 3,30 -dityrosine and 3-nitrotyrosine is completely inhibited at 4.2 lg/mL pheomelanin concentration. Conversely at pH 5.5, pheomelanin acts as photosensitizer and a 60% increase of the production of 3-nitrotyrosine is observed with respect to control experiments without pheomelanin. The photosensitizing action of pheomelanin on the production of 3-nitrotyrosine, is further increased in D2O, suggesting a predominant role of singlet oxygen (1O2) in the reaction mechanism.

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Effects of T-2 toxin on low selenium state rats’ chondrocyte expression of collagen II, matrix metalloproteinase (MMP-1, MMP-3, and MMP-13) and TIMP1 XiaoRong Zhou, Jinghong Chen, and Haojie Yang The Second Hospital of Medical School of Xi’an Jiaotong University, Xi’an 710004, Shaanxi, People’s Republic of China Corresponding Author: Zhilun Wang Institute of Endemic Diseases, Medical School of Xi’an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi’an 710061, Shaanxi, People’s Republic of China Kashin–Beck disease (KBD) is a chronic, endemic, deforming osteoarticular disease with unclear etiology and severer damage to sufferers’ working ability and quality of life. Low selenium and T-2 toxin are regarded as the most important etiological factors related to the take place of the disease. But laboratory and epidemiologic experiments of either selenium or T-2 toxin separately cannot well explained the endemically and wavy characteristic of KBD. So we established a new hypothesis that the KBD is due to an complicated 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 of KBD by animal experiment to validate this hypothesis and tried to depict the molecular mechanism as well.

Function comparisons between harpinXoo and hpaXm in the elicited ROS in tobacco against TMV Lin Li, Wenbo Liu, Tingya Yang, Xiaoxi Lan, Xiang Li, Liang Sun, XiaoYan Wu, Weiguo Miao*, and Fucong Zheng* College of Environment and Plant Protection, Hainan University, Haikou 570228 China *Corresponding author: Weiguo Miao E-mail: [email protected] Fucong Zheng E-mail: [email protected], [email protected] Harpin is a hypersensitive response elicitor. HarpinXoo and hpaXm encoded by hpa1/G from Xanthomonas oryzae pv. oryzae and X. citri subsp. malvacearum, respectively. Although harpins (HarpinXoo and hpaXm) are able to trigger the resistances of tobacco to Tobacco mosaic virus (TMV), it is unknown whether harpins induce the sequential generation of reactive oxygen intermediates (ROI) in tobacco. In this study, respective methods in detection of H2O2 and relative enzyme activity, DAB (Diaminobenzidine) staining and quantitative real time PCR were used for confirming the ROI generation in tobacco compared with untreated tobacco. The results showed that after the leaves were inoculated by TMV, compared with untreated tobacco leaves, H2O2 content in harpins-treated leave tissue exhibited two peak values during 0–8 h; PAL and POD activities were significantly enhanced,

12th International Congress on Amino Acids, Peptides and Proteins DAB staining revealed visual reddish-brown coloration spots, and expressions of ghAOX1 and hsr203J genes were significantly induced. However, the first peak of ROI induced by harpinXoo was detected ahead compared with that induced by hpaXm. There are no significant deference between harpinXoo and hpaXm in the expression level of ROI related genes. These evidences indicated that harpins possessed the molecular basis for expression of some defense genes, and the expression of harpinXoo and hpaXm was responsible to the generation of ROI and the expression of the related defense genes in tobacco plant, and conferred the resistance of tobacco to TMV. Acknowledgments: This work was supported by grants from the NKBRPC (2003CB114204 and 2006CB101902), NKPC (2004BA901 A36), RFDP (20104601110004), CARS-34-GW8 and KYQD1006 (China).

Functional genomics of amino acid transporters in the human pathogen Leishmania: the story of proline and alanine Dan Zilberstein1, Ehud Inbar1, Doreen Schlisselberg 1, Marianne Suter Grotemeyer 2 and Doris Rentsch2 1

Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Haifa, Israel and 2 Institute of Plant Sciences, University of Bern, 3013 Bern, Switzerland Protozoan parasites of the genus Leishmania are the causative agents of a wide range of visceral and cutaneous diseases in humans. Leishmania species cause morbidity and mortality in 88 counties worldwide. Increasing cases of Leishmania-HIV co-infection is the major reason for the recent emergence of leishmaniasis. Unlike all other organisms, Leishmania (as well as all members of the Trypanosomatid family) maintain a large cellular pool of proline that, together with alanine, serves as an alternative carbon source and as a reservoir of organic osmolytes. We cloned and characterized a new neutral amino acid transporter, LdAAP24 that translocates proline and alanine across the L. donovani plasma membrane. By knocking out the gene that encodes for LdAAP24 we showed that this transporter fulfills multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates transport and homeostasis of glutamate, arginine and glycine, none of which are its substrates. Interestingly, loss of cellular proline signal for parasites in the insect to develop virulence, a process called metacyclogenesis; Dldaap24 mutants transiently express gene markers of metacyclogenesis, 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 susceptible to oxidative stress than wild type. We conclude that by controlling the cellular pool of proline LdAAP24 plays a key role in virulence development of Leishmania parasites inside the vector.

Genetically encoded nonnatural amino acids: novel methods for bioengineering structure–function into biomaterials

S11 amino acids (NAAs) are genetically encoded in vivo using a suitable host organism. The amino acids are encoded site-selectively in response to a recoded amber stop codon. The overall goals of our research are to develop novel amino acids, but also to advance the understanding of the mechanism and function of bioorthogonal translation elements which is described in a recent publication from my group. Using the fundamentals of chemistry, NAAs may be chemically tailored to possess specific properties for highly specialised applications, for example, in the charting of biological pathways or in unique post-translational modifications. In these scenarios, there is a synergy between the chemistry of the NAA and the biochemistry of the host organism used to heterologously express the labeled protein. By increasing the sophistication of the amber suppression method, a host of multidisciplinary applications related to drug delivery and biomaterials becomes possible, and preliminary work in these directions is presented.

Glycation by methylglyoxal in physiological systems— the damage caused and strategies to prevent it Paul J Thornalley and Naila Rabbani Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, University Hospital, Coventry CV2 2DX, U.K Increased glycation of cellular and extracellular proteins by methylglyoxal occurs physiological systems. Steady state levels of glycated proteins are generally low yet often has significant physiological effect—particularly linked to ageing, diabetes development and progression of vascular disease, neurological disorders and arthritis. Identification of sites activated toward damaging modifications or ‘‘hotspots’’ in functional domains within proteins appears key to assessing targets of functional impairment. Disease progression is likely linked to instances where change in low level of hotspot damage influences metabolic control or physiological function. Examples discussed are: type IV collagen modification leading to endothelial cell detachment and anoikis, mitochondrial protein modification leading to oxidative stress, and apolipoprotein B100 modification in low density lipoprotein leading to vascular retention and atherosclerosis. The role of mathematical systems biology, bioinformatics and proteome dynamics in future investigations is discussed. Therapeutic interventions to decrease glycation by methylglyoxal may be achieved by agents that decrease triosephosphate precursors—such as high dose thiamine therapy in diabetes, scavenging agents and inducers of enzymes that metabolize methylglyoxal such as glyoxalase 1.

High pH solubilization and chromatography based renaturation and purification of recombinant human granulocyte colony-stimulating factor from inclusion bodies** Ming Li, Jiahua Liu, Lili Wang, and Chaozhan Wang*

Cardiff University School of Chemistry, Cardiff United Kingdom CF10 3AT

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 *Corresponding author: Dr. Chaozhan Wang, College of Chemistry and Materials Science, Northwest University, 229 Tai Bai North Road, Xi’an 710069, People’s Republic of China. Tel: +86-29-88302604, Email: [email protected]

New approaches for the optimization of translational and post-translational modification of proteins are presented. We show that nonnatural

Recombinant human granulocyte colony-stimulating factor (rhGCSF) is a very efficient therapeutic protein drug which has been

Eric M. Tippmann

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S12 widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was 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 employed buffer, alkaline pH drastically favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured by using weak anion exchange, strong anion exchange and hydrophobic interaction chromatography separately, with simultaneous purification. The results indicated that the rhG-CSF solubilized by high pH combined with low concentration of urea had much higher mass recoveries than the one solubilized by 8 M urea in all three employed refolding methods. In the case of weak anion exchange, the high pH solubilized rhG-CSF could get a mass recovery of 73%. A strategy combining solubilization of inclusion bodies at high pH and refolding of protein using liquid chromatography may become a routinely method for protein production from inclusion bodies. ** This work was supported by the National Natural Science Foundation in China (No. 20705028).

Hydroquinone regulates tumorigenic responses through Src activation by binding to cysteine residues 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 1

Department of Genetic Engineering, Sungkyunkwan University, Suwon 446-746, Korea; 2 College of Natural Science, Kangwon National University, Chuncheon 200-701, Korea; 3 Research Institute and Hospital, National Cancer Center, Goyang 410-769, Korea; 4 College of Pharmacy, Chonnam National University, Kwangju 500-757, Korea The hydroxylated benzene metabolite hydroquinone (HQ) is mainly generated from benzene, an important industrial chemical, and is also a common dietary component. Although few reports have addressed the immunosuppressive effects of hydroquinone, numerous papers have explored its roles in tumorigenic responses. In this study, we characterized Src-targeted tumorigenic responses by hydroxylated benzene metabolites, hydroquinone and its derivatives, in activating various cancerous responses using cellular, molecular, biochemical and immunopharmacological approaches. HQ up-regulated transcriptional activation of NF-B and ARE responses so that hemeoxygenase-1 and pro-inflammatory gene expressions were increased. Several of evidence have raised a possibility that HQ directly activates Src kinase activity. Thus, Src knockdown or deficiency by siRNA treatment and infection with retrovirus expressing shRNA to Src as well as exposure of PP2, a Src kinase inhibitor, strongly abrogated HO-1 expression. Interestingly, HQ directly targeted and bound to the sulfhydryl group of Cys-483 and Cys-400 of Src, potentially leading to breaking intracellular disulfide bonds. Indeed, Src kinase activity was dramatically enhanced by the mutation of these Cys sites, implying that these sites may play an important role in Src activity regulation. Therefore, our data suggest that Src and its target site Cys-483 can be considered as a prime molecular target of HQ-mediated tumorigenic responses and for strong Src kinase regulatory loop.

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Illuminating the interactions of the C-terminal region of TRPV1 channel with membrane phospholipids Lenka Grycova, Blanka Holakovska, and Jan Teisinger Institute of Physiology, Academy of Sciences of the Czech Republic TRPV1 channel is non-selective Ca2+-permeable cation channels activated by a wide range of stimuli. As a result of its activation, the cellular level of Ca2+ increases, which initiates a wide variety of cellular responses. It is assumed that TRPV1 channel has six transmembrane domains with a pore domain between the fifth and the sixth segments. Both C- and N-termini are located intracellularly and have been shown to be involved in the regulation of the channel activity. Both tails recruits agonist and regulatory molecules such as ATP, calmodulin (CaM), phosphatidyl inositol 4, 5 bisphosphate (PIP2). In this study using the combination of biochemical and biophysical approaches the highly purified C-terminal fusion protein TRPV1 (TRPV1-CT) was shown to interact directly with PIP2 and this interaction has been characterized in detail. As this sequence contains several basic 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. This work was supported by Grant GAAV IAA600110701, GACR P205/10/P308, GACR P301/10/1159, Centre of Neurosciences No. LC554 MSMT CR3

Induction of actinorhodin production from Streptomyces lividans TK24 by high expression of the two-component system derived from Streptomyces acidiscabies Hye-Yun Park, Min-Jeong Kim, Hyo-Ji Kim, Sun-Uk Choi, and Yong-Il Hwang Department of Food Science and Biotechnology, Kyungnam University, Changwon, 631-701, Republic of Korea Streptomyces acidiscabies produces thaxtomins that cause a scab disease of potatoes and have structure of cyclic dipeptide consisting of L-4-nitrotryptophyl-L-phenylalanyl. In this study, the two-component system, which performs an important role in secondary metabolite production of actinomycetes, cloned from Streptomyces acidiscabies, and its antibiotic production-increasing activity was confirmed. Genes (tcsK and tcsR) encoding the sensor kinase (TCSK) and the response regulator (TCSR) were acquired via PCR. PCR was carried out using the primers designed from the two highly conserved regions of previously studied two-component system of Streptomyces strains, and gave a 300-bp and 498-bp products for tcsK and tcsR. The obtained PCR products showed a high homology to the two-component systems of Streptomyces strains. By Southern hybridization using PCR products as a probe, intact 1.6-kb tcsK gene and 0.7-kb tcsR gene were obtained from genomic DNA of S. acidiscabies. In order to identify the effects of the cloned genes, the high expression vectors were constructed by using pSET152ET and introduced into S. lividans TK24, which was employed as a host for the confirmation of inducer gene activity for secondary metabolite production. And then the actinorhodin production of the exconjugants was assessed. As a result, it was determined that the expression of tcsK and tcsR induced 5.4-fold and 5.2-fold increases in actinorhodin production from S. lividans TK24, respectively, compared to the control strains.

12th International Congress on Amino Acids, Peptides and Proteins This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0008443).

Interaction of microcystin-LR with human serum albumin Chao Song and Hong-Wen Gao1 State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, China 1 To whom correspondence should be addressed, Fax: (86) 21-6598-8598. E-mail: [email protected] The binding of microcystin-LR (MC-LR) onto human serum albumin (HSA) was investigated by fluorescence and UV–visible spectrometry. The results showed that MC-LR can quench strongly the intrinsic fluorescence of HSA. The binding number of MC-LR was determined to be 1 per HSA and the thermodynamic constants (DH, DS and DG) calculated. The electrostatic attraction and hydrophobic stack are the main binding forces in the MC–LR/HSA interaction. From the synchronous and three-dimensional fluorescence spectra, the different concentrations of MC-LR exposed in HSA solution can cause the different quenching peak. For example, the low concentration of MCLR mainly caused the quenching of peak 3 (kex 230.0 nm, kem 330.0 nm), indicating a change of the peptide strands structure. The high concentration of MC-LR caused the quenching of peak 4 (kex 280.0 nm, kem 330.0 nm) and MC-LR interacted with the Trp and Tyr residues by hydrophobic stack. Therefore, the interaction of MC-LR with HSA exhibited a two-step reaction: first to change the polypeptide backbone structure, and then to combine with the hydrophobic amino acids.

Leucine modulated the effects of Walker factor in C2C12 myotubes E. M. Gonçalves and M. C. C. Gomes-Marcondes Depart Physiology and Biophysics, Biology Institute, State University of Campinas, Saõ Paulo, Brazil. Financial support: Fapesp, CNPq. 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 muscle during cancer cachexia is also mediated by a proteolysisinducing factor (PIF). The three main proteolytic systems responsible for proteolysis in skeletal muscle, especially during cachexia, are the lysosomal, the calcium-dependent, and the ubiquitin–proteasome pathways. In skeletal muscle, the effect of PIF on protein degradation is mediated through an increased expression and activity of the ubiquitin–proteasome proteolytic pathway. The branchedchain amino acid leucine has been investigated as an important cellular signalling and studies have shown that this amino acid can inhibit the activity of the ubiquitin–proteasome pathway in muscle of cachectic rats and can improve protein synthesis of muscle in tumour-bearing rats. In this study were investigated the effects of leucine in C2C12 skeletal muscle cells exposed to the proteolysisinducing factor (PIF)-like protein, purified from the ascitic fluid of the Walker 256 tumour-bearing rat, called Walker Factor (WF),

S13 which is immunologically identical to PIF. The WF has no cytotoxic effect in C2C12 cells after 24 h incubation. Higher WF concentrations 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 phosphorylated Erk in parallel to increased protein synthesis. Taken together, these results strongly suggest an important modulatory effect of leucine when acting previously under the WF effects on C2C12 muscle cells.

Mimicking a biological cellular structure with incorporation of interior organelles and protein cytoskeletal network in the cytoplasm Dooho Lee1, Sujin Park1, and Kwanwoo Shin1,2,3* Department of 1Chemistry and 2 Interdisciplinary program of integrated Biotechnology, Sogang University, Shinsu dong 1, Mapo gu, Seoul 121-742, South Korea 3 Sogang-HANARO Joint Center for Biological Interfaces, KAERI, Daijeon, Korea *Email: [email protected] A giant unilamellar vesicle (GUV) have received much attention recently because they, as a biological cell mimic in size and shape, can be utilized as a biological mimics. Up to now, many researchers have reported the dynamics and physical properties of GUV, for examples, dynamics of GUV with shear elasticity, or lipid phase behaviors of GUV, etc. However, most biological cells contain organelles, and cytoskeletal network in their cytoplasm, and it is not hard to expect that the physical properties of GUV will be greatly affected when a cellular vesicles contains complex sub-cellular structures (organelles and protein networks) in the cytoplasm. Initially, we mimicked various non-spherical structures of cellular organelle mimics (tadpole, snowman, dumbbell, etc.) by varying major membrane components. We could successfully injected the organelle mimics into a GUV. Furthermore, we introduced the collagen-mediated fibers into the cytoplasm, resulting in the fiber network in the inner volume of GUVs. We, then could study how the mechanical/physical properties of GUV membranes were affected with the incorporation of interior-organelles and protein-cytoskeletal network.

Molecular imprinting of thymopentin in aqueous media Liang Zhao, Yan-Ping Huang*, and Zhao-Sheng Liu* College of Pharmacy, Tianjin Medical University, Tianjin 300070, China Correspondence: Dr. Yan-Ping Huang, College of Pharmacy, Tianjin Medical University, Tianjin 300070, China, E-mail: [email protected] Molecular imprinting of peptides in aqueous media is always a challenge in the synthesis of artificial receptors. In this work, a new molecularly imprinted polymer (MIP) for thymopentin (TP-5) was prepared by the combined use of N-isopropylacrylamide (NIPAm) and N-t-butylacrylamide (TBAm) as functional monomers in the presence of N, N0 -methylenebisacrylamide (BIS) as cross-linker, and the imprinted nanoparticles can be synthesized using a simple

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S14 precipitation polymerization method. The preparation conditions were optimized by investigating the effect of the concentrations of the monomer and cross-linker on the adsorption properties. Dynamic light scattering (DLS) was used to investigate the dynamic process of particle growth. The resulting MIPs nanopartile were average diameter of 300 nm with the polydispersity (PDI) of 0.217. The adsorption properties of resultant imprinted polymers were evaluated by equilibrium rebinding experiments in an aqueous media. The highest binding capacity of TP-5 achieved from the optimized imprinted polymer was 194.72 lmol/g with an imprinting factor of 2.04. The results showed that the method established owns potential promises. This work was supported by the National Natural Science Foundation of China (Grant No. 21075090).

Monitoring of NTH1 phosphorylation sites important for BMH binding D. Veisova1, E. Macakova1, L. Rezabkova1,2, and V. Obsilova1 1

Institute of Physiology Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic 2 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic [email protected] Trehalase (EC 3.2.1.28) is an intrinsic glycoprotein of the small intestine and renal brush-border membranes that hydrolyzes a,a- trehalose (1-a-D-glucopyranosyl a-D-glucopyranoside) to two glucose molecules in animals. Three trehalases have been identified in Saccharomyces cerevisiae so far: neutral trehalase 1 (NTH1), neutral trehalase 2 (NTH2) and acidic trehalase 1 (ATH1). NTH1 is responsible for trehalose degradation, which is accumulated after stress. The activity of the NTH1 enzyme was just recently found to be mediated by BMH1 and BMH2 binding in yeast. Yeast BMH1 and BMH2 proteins (yeast 14-3-3 isoforms) form a complex with neutral trehalase after its phosphorylation by PKA. Either one of the two 14-3-3 yeast isoforms are required for complete activation of neutral trehalase (NTH1). However, details concerning the mechanism of BMH-dependent activation of NTH1 remain still unknown. We showed that PKA phosphorylates NTH1 in vitro on four Ser residues: 20, 21, 60 and 83. To find out which site or sites are essential for the 14-3-3 binding we produced NTH1 WT (both phosphorylated and non-phosphorylated), four NTH1 mutants containing single phosphorylation site, one double phosphorylated NTH1 mutant (at Ser20 and 21) and a mutant containing none of these studied phosphorylation sites as well. The interaction between BMH1 and BMH2 protein with enzyme NTH1 was monitored using native electrophoresis and sedimentation velocity measurements. The sedimentation equilibrium analysis was used to define the stoichiometry of NTH1/BMH complexes. Finally, we used enzyme kinetic measurements to monitor the BMH-dependent activation of NTH1. Acknowledgments: This work was funded by Grant P207/11/0455 of the Grant Agency of the Czech Republic and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic and by Grant 350111 of the Grant Agency of the Charles University.

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Mutation of amino acid within the n terminus of human O-GlcNAcase results in impaired function Lin Lin1, Zhonghua Li1, Jing Li1*, and Peng George Wang1,2,* 1 College of Pharmacy and State Key Laboratory of Element-Organic Chemistry, Nankai University, Tianjin 300071, China 2 Departments of Biochemistry and Chemistry, the Ohio State University, Columbus, OH 43210 *Corresponding author. Tel./Fax: +86 22 23507760 E-mail Addresses: [email protected] (J. Li), [email protected] (P. G. Wang)

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 events, including cell-cycle progression, cellular development, cellular stress and extracellular stimuli, but its faulty regulation is involved in the etiology of type II diabetes, cancer, and neurological disorders. O-G1cNAcase (OGA), belongs to GH84 family (http//: www.cazy.org), is the only enzyme responsible for removal of O-GlcNAc from protein and thus plays a key role in O-GlcNAc metabolism. Due to unstable expression and poor purification, until recently the structure and reaction/substrate recognition mechanisms of human OGA have not yet been understood. Using deletion mutation, our previous study revealed that the N-terminal region (a.a. 1-350) of OGA (sOGA, the smallest OGA) contains the catalytic site for glycoside hydrolase. Here using Geno 3D and the crystal structure of human OGA orthologue in GH84 from C. perfringens (CpOGA), we modeled sOGA and identified key amino acids involved in glycosidase hydrolysis. Key amino acids were subjected to site-directed mutagenesis, and the mutated enzymes were expressed in E. coli BL21 cells and assayed with 4-MU-GlcNAc (4-methylumbelliferyl-2-acetamido-2-deoxy-b-Dglucopyranoside) as substrate. D174N showed a severely impaired catalytic activity (about 2,000-fold reduction in Kcat), consistent with its role in polarized and oriented the 2-acetamido group for a nucleophilic attack. D175N showed only modest 26-fold decrease in Kcat, in good agreement with its role in general acid/base catalyst. D285 interacts with the O4/O6 hydroxyls through tight hydrogen bonds, which aid in formation of the 4E envelope conformation of the pyranose ring in the transition state, providing a possible explanation for the large mutational effect on Km (about twofold increase) and Kcat (about 120-fold reduction). N280 is involved in stabilizing the conformation of the acetamido group and the oxazolinium intermediate, compatible with its 300-fold reduction in Kcat. C215 and Y219 are hydrogen bonded to the oxime nitrogen (approximately equivalent to the position of the substrate glydosidic oxygen), in agreement with this, mutations of C215A and Y219F show modest increase in Km. In contrast, the Y69F mutant enzyme displayed comparable level of O-GlcNAcase activity as that of the wild type enzyme-sOGA, indicating Y69 may not be an essential residue in substrate binding and catalysis. This study provides, to our knowledge, the fist data on the threedimensional homology modeling to analyze the essential role of six essential residues in human OGA catalysis. Keywords: O-GlcNAcylation, OGA, Site-directed mutation This research was supported by the National Basic Research Program of China (973 Program, grant No.2007CB 914403) the National Natural Science Foundation of China (31000371) and the Fundamental Research Funds for the Central University (65011091).


Preparation and characterization of the yeast enzyme Neutral trehalase 1 and its complex with BMH proteins E. Macakova1, D. Veisova1, L. Rezabkova1,2, and V. Obsilova1 1 Institute of Physiology Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic, 2 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic

Neutral trehalase (EC 3.2.1.28) is a trehalose hydrolyzing enzyme, found in the yeast Saccharomyces Cerevisiae. It hydrolyzes a,a-trehalose (1-a-D-glucopyranosyl a-D-glucopyranoside,) which plays an important role as a reserve and stress metabolite in yeast cells, to two glucose molecules. According to former results, the activity of NTH1 is mediated by BMH1 and BMH2-yeast isoforms of 14-3-3 proteins. When the NTH1 is phosphorylated by PKA, it forms a complex with one of the 14-3-3 isoforms, which is required for activation of NTH1. However, the exact mechanism of BMH controlling the NTH1 activation remains still elusive. BMH proteins were expressed as described previously. All mutants of NTH1 were expressed as 6xHis tag fusion proteins and were purified from Escherichia coli Rosetta cells using ChelatingSepharose Fast Flow, cation-exchange chromatography on HiTrap SP column and using gel filtration on Superdex 200 column afterwards. Purified NTH1 mutants were phosphorylated by cyclic AMP-dependent protein kinase (PKA). The completeness of the phosphorylation reaction was checked using MALDI-TOF mass spectrometry. The interaction between BMH1 and BMH2 protein with enzyme NTH1 was monitored using native electrophoresis and sedimentation velocity measurements. Limited proteolysis was used to show how is NTH1 in complex with BMH protected from cleavage. Finally, we used enzyme kinetic measurements to monitor the BMH-dependent activation of NTH1.

Regulatory effect of nicotine on Poly (I:C)-induced signaling using PCR and antibody arrays: from expression to activation Wen-Yan Cui State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University Nicotine, the primary psychological stimulant in tobacco, exerts broad physiological and pharmacological effects on both neuronal and peripheral systems. In recent years, the anti-inflammatory function of nicotine has drawn particular attention. However, most of the previous studies on the immunomodulatory effect of nicotine are limited to single gene level. In this study, we used multiple high throughput molecular techniques to characterize nicotine’s antiinflammatory actions during viral infections at both gene expression and pathway activation level. We examined RNA expression of 51 key genes in TLR (Toll-Like Receptor) pathways using quantitative RT-PCR array, and determined the protein levels for TNF-a and IL-6 as indicators of the pathway using ELISA assay, in Poly (I:C)-induced macrophages. We found that Poly (I:C)-induced innate immune signaling was significantly modulated by nicotine. We further demonstrated that such modulation was mediated by the a7 nicotine acetylcholine receptor (nAChR). We also employed an antibody array containing 1,318 antibodies to determine nicotine’s effects on Poly (I:C)-triggered kinases at the phosphorylation level, and found a

S15 cluster of calcium-elicited kinases was suppressed by nicotine. These findings were further supported by Western blotting and calcium imaging experiments. Taken together, our results indicate that nicotine has significant down-regulatory effects on Poly (I:C)-induced pathways, and that calcium signaling is actively involved in nicotine’s immunomodulatory effects during viral-induced inflammation.

Replication study of prior GWAS candidate genes/loci for coronary artery diseases in the genetic isolated Newfoundland and Labrador population Y.-G. Xie1, J. Cui1, E. Randell1, J. Renouf5, S. Li1, A. Pope4, G. Sun2, W. Gulliver2, B. Sussex2 and F.-Y. Han1 Disciplines of 1Laboratory Medicine, 2 Medicine, Memorial University, St. John’s, NL, Canada 3 Laboratory Medicine Program, Eastern Health, St. John’s, NL, Canada 4 Dept. of Molecular Genetics, Newfound Genomics, St. John’s, NL, Canada Genome-wide association studies (GWAS), have provided some promising achievements for a number of multifactor diseases including coronary artery disease (CAD). However, many of these GWAS candidate genes failed to been replicated in studies using different ethnic populations which indicates the variety of genetic modifiers among different ethnic populations. Additionally, the genetic heterogeneity in the studied population can reduce the sensitivity in detection of weak genetic effects and leads to false negative results in replication studies. The population of Newfoundland and Labrador (NL) is a well known genetic isolated population, and this population has a high prevalence of CAD in Canada. As a part of our ongoing study, 15 genetic variants from 12 selected prior GWAS candidate genes/loci 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. Genotyping was carried out by using the Sequenom’s MassARRAYTM system. Among the 12 studied genes/loci, only the 9p21 locus (rs 133049, rs 10757274, rs238306 and rs238307) was successfully associated to the patients (P \ 0.000 in all SNPs). This association was further confirmed in another study with enlarged sample size (1,000 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 susceptibility for CAD in the NL population. The failure of replicating other 11 GWAS candidate genes for CAD in NL patients strongly suggests the diversity of genetic modifiers for CAD in NL population.

Structural insight into interactions of calmodulin with TRPV2 and TRPV5 C-termini Blanka Holakovska, Lenka Grycova, and Jan Teisinger Institute of Physiology, Academy of Sciences of the Czech Republic TRPV2 and TRPV5 ion channels belong to vanilloid subfamily of transient receptor potential channels (TRPs). These channels are ubiquitously expressed in all eukaryotic cells and are involved in many cellular processes like transduction of sensory signals and regulation of Ca2+ and Mg2+ homeostasis. TRPV2 falls within the so called thermoTRPs and has been proposed to mediate high-threshold noxious heat sensation. TRPV5 is a rather distinct member of TRPV subfamily and features quite different properties then the rest of this subfamily. It is strictly Ca2+ selective and involved in renal Ca2+ absorption/reabsorption.

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S16 It is known that calmodulin (CaM) serves as an important TRP channels regulator via binding to their intracellular termini in a calcium dependent manner. We identified CaM binding sites on the C-termini of TRPV2 (654683) and TRPV5 (587-616) corresponding to the consensus CaM binding motif 1-5-10 using steady-state anisotropy measurement, CD spectroscopy and computer homology modeling. Moreover, it is the first time that such sequence has been recognized on the intracellular parts of TRPV2. We also investigated the role of basic residues present in the region. The exchange of positively charged residues to neutral alanine resulted in some cases in total loss of binding ability to CaM. Also the data from the CD spectroscopy experiment showed changes in the distribution of the secondary structure elements in the mutants compared to the wild type. Based on these results we concluded that basic residues play crucial role in TRP channels binding to CaM. This project was supported by Grant GAAV IAA600110701, GACR 301/10/1159, GACR P205/10/P308, project No. 305/08/H037, Centre of Neurosciences No. LC554 MSMT CR.

The binding of hepatitis B virus X protein to GLI1 and its biological characterization in vitro So Young Park, Young-hoon Park, Se Bok Jang and Mi Suk Jeong Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Korea Hepatitis B virus (HBV) X protein (HBx) is a 17-kDa transcriptional coactivator that plays a significant role in the regulation of genes involved in inflammation and cell survival. It has been known to be involved in the development of liver cancer and alteration of the cellular HBx level may influence the pathogenesis of HBV-induced liver diseases. The transcription factor GLI1, a member of the gliomaassociated oncogene homologue (GLI) subfamily of Kru¨ppel-like zinc finger proteins is involved in signal transduction within the hedgehog (Hh) signaling pathway, which is involved in the development of many human malignancies. GLI activation is important for cell proliferation and anti-apoptosis in various cancers. To investigate whether the transcriptional coactivator HBx binds to the zinc finger transcription factor GLI1, recombinant HBx and GLI1 were isolated. Expression and purification of the HBx and GLI1 proteins were successfully performed in Escherichia coli. The binding of HBx to GLI1 was detected by surface plasmon resonance spectroscopy (BIAcore), fluorescence measurement, and a His-tagged pull-down experiment. After measuring the fluorescence emission spectra of purified HBx and GLI1, it was found that the interaction of these proteins is accompanied by significant conformational changes in one or both. This study provides important clues for the structural identification of signal transduction pathways involving the HBx and GLI1 proteins.

Toward a protein profile of the zooplankton Daphnia pulex exposed to the pesticide cypermethrin, using ATR–FTIR spectroscopy as a novel method S. B. Akkasa, M. Severcanb, M. Beklioglua, and F. Severcana a

Department of Biological Sciences, Department of Electrical and Electronic Engineering Middle East Technical University, 06800 Ankara, Turkey

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12th International Congress on Amino Acids, Peptides and Proteins Pesticides have become an integral part of ecosystems, although many of them are extremely toxic, even to non-target species. Hence, it is crucial to detect pesticide-induced molecular alterations in exposed species. In this study, Attenuated Total Reflectance Fourier Transform Infrared (ATR–FTIR) spectroscopy was utilized as a novel method to better understand the impact of cypermethrin toxicity (0.04–3.60 lg/L) on the protein profile of Daphnia pulex— key-stone species in lake ecosystems. Spectral analysis revealed a decrease in protein content at all cypermethrin concentrations from the peak area values of both the amide I and II bands. In order to determine protein secondary structures, Neural Network (NN) method using the 1,700–1,600 cm-1 spectral region of the FTIR spectra put forward a cypermethrin-induced protein denaturation at high cypermethrin doses (0.90–3.60 lg/L) through a decrease in ahelix and turn content and an increase in total b-sheet and random coil content. Increased random coil content in high-dose groups was observed also with vector normalization in the same spectral region. Vector normalization method further suggested that the increase in 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. Supporting the findings on the different attitude of high cypermethrin doses, hierarchical cluster analysis successfully differentiated the highdose groups from the control and low-dose groups. Therefore, the ATR–FTIR spectroscopic results put forward that the protein profile of Daphnia pulex are adversely affected by cypermethrin.

Two-dimensional glycoproteome maps: a new insight into protein glycosylation patterns during larval development and metamorphosis in marine invertebrates K. H. Chandramouli, Y. Zhang, Y. H. Wong, S.Y. Mok, and P.-Y. Qian Section of Marine Ecology and Biotechnology, Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR. *Presenting author Email: [email protected] The life cycle of most marine invertebrates has two distinct stages: the pelagic larvae and the adults attached to marine surface. The transition between these two stages (called ‘‘larval settlement and metamorphosis’’) is often abrupt and may involve differential protein modifications. Glycosylation, a very important post-translational modification of proteins plays fundamental roles in controlling various biological processes. However, to date, no study has addressed glycosylation patterns of proteins during larval settlement and metamorphosis in marine invertebrates. To understand the common molecular patterns of protein glycosylation associated with larval metamorphosis, we used comparative approach to investigate glycosylation patterns in barnacles, bryozoan, and polychaete species. We applied a fluorescence-based multiplexed proteomics technology which included two-dimensional gel electrophoresis (2-DE) followed by the fluorescence staining of glycoprotein and mass spectrometry identification of proteins. Twodimensional proteome maps showed that protein glycosylation pattern changed markedly from larval to juvenile stages of selected marine species. We identified twenty abundant and differentially expressed glycoproteins, of these cytoskeleton proteins showed distinct glycosylation in all three species. Oxidative stress proteins revealed distinct glycosylation in B. amphitrite and energy

12th International Congress on Amino Acids, Peptides and Proteins metabolism proteins were glycosylated in B. neritina. Expression patterns of selected proteins were confirmed at the protein level by Western blot analysis while some proteins were further studied at the mRNA level by real-time PCR. The identified glycoprotein proteins were shown to be involved in development, cell differentiation and integrity, apoptosis, oxidative stress and energy metabolism. The protein glycosylation alteration provide essential basis to understand molecular processes and pathways involved in larval development and metamorphosis of multiple marine species.

Bioinformatics Application of fluorogenic substrate libraries containing natural and unnatural amino acids for profiling of proteolytic enzymes Marcin Drag1,2, Anna Gajda1, Paulina Kasperkiewicz1, Marcin Poreba1, Rafal Latajka1, Malgorzata Pawelczak3, Aline Marschner4, Christian Klein4, Matthew Bogyo5, Jonathan A. Ellman6, and Guy S. Salvesen2 1

Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wroclaw University of Technology, Poland 2 Apoptosis and Cell Death Research Program, Sanford Burnham Medical Research Institute, La Jolla, CA, USA 3 Institute of Chemistry, University of Opole, Poland 4 Department of Medicinal Chemistry, Universita¨t Heidelberg, Germany 5 Department of Pathology, Stanford School of Medicine, Stanford, CA, USA 6 Department of Chemistry, Yale University, New Haven, USA Proteases are known to participate in many cellular processes due to their ability to process the peptide bond. They are key players in several cascades taking place in the cell with apoptosis, fibrinolysis, blood clotting, complement fixation, gastrulation or general cell cycle being only a few examples. However, proteases can be also bad guys and participate in the cellular events, which leads to severe diseases such as cancer, diabetes, pathogens infections or hypertension. Each protease recognise only substrates, which can fit into the recognition pockets. There are several methods of determination of substrate specificity. One of the most versatile is Positional Scanning Substrate Combinatorial Library approach, which allows fast and reliable determination of substrate preferences for most of the proteases. To date used libraries contained only natural amino acids and only very occasionally unnatural amino acids were incorporated into the structure of synthesized substrates. To get better insight into substrate specificity, we have applied several different tailored to certain type of protease approaches using broad range of unnatural amino acids. This allowed us to obtain much better substrates comparing to natural amino acids derivatives for several different aminopeptidases, dipeptidylpeptidases and endopeptidases. Here we will present the latest findings for each respective group of proteases. Acknowledgments: The Drag laboratory is supported by the Foundation for Polish Science and the State for Scientific Research Grant N N401 042838 in Poland.

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Computational studies of protein phosphorylation: from site-specific prediction to systematic network analysis Zexian Liu1, Xinjiao Gao1, Jun Cao1, Qian Ma1, Jian Ren2, and Yu Xue1,* 1

Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, Anhui 230027, China 2 Life Sciences School, Sun Yat-sen University (SYSU), Guangzhou, 510275, China 3 Hubei Bioinformatics and Molecular Imaging Key Laboratory, Department of Systems Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China *Correspondence: [email protected] Phosphorylation catalyzed by protein kinases precisely regulates cell signaling pathways. Comprehensive elucidation of kinase-specific phosphorylation sites will reveal a complex protein phosphorylation network (PPN), which provides a systematic insight for understanding cell dynamics and plasticity. Recent progresses on phosphoproteomics with high-throughput mass spectrometry (MS) produce hundreds of thousands of phosphorylation sites. However, the upstream kinases are difficult to be determined, while further in vitro and in vivo studies were greatly hampered by lack of accurate and efficient computational techniques. Here we show that kinases and substrates form highly complex PPNs in eukaryotes. Based on a general model, we accurately predict 188,288 site-specific kinase-substrate relationships between 9,247 targets and 1,079 kinases for 44,290 phosphorylation sites. An integrated and automated computational platform was developed for analyzing various organism- or tissue-specific PPNs. Particularly, we discovered a small potential core PPN specifically in liver conserved between human and mouse. Furthermore, we found that PPNs evolved rapidly even among near species, and consistent with other recent observations. Our results demonstrate that MS-based large-scale phosphoproteomic analysis with subsequently computational prediction could generate flood of useful information, which might be helpful for further experimental manipulation. We anticipate our study to be a starting point for more sophisticated analyses of PPNs. For example, human PPNs influenced by the individual polymorphisms could be tested. Furthermore, tissues under different developmental stages might also exhibit distinct PPNs, while some key pathways will be rewired in diseases and cancer.

New alignment and software package for sequence analysis *Toshihide Hara, Keiko Sato, and Masanori Ohya We develop a new alignment for bio-sequences called MTRAP by introducing a measure based on entangled correlation in two consecutive residues. We show that the alignment accuracy could be improved by our approach not only for pairwise sequences but also multiple sequences.

The code structure of the receptor binding site of influenza A virus hemagglutinin *Keiko Sato, and Masanori Ohya Information of life is stored as a sequence of nucleotides, and the sequence composed of four bases seems to be a sort of code. The

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hemagglutinin (HA) of influenza A viruses is responsible for binding the virus to host receptors. In the coding theory, information is properly coded for its transmission. The artificial codes in information transmission were applied to find the code structure of the left and right edge of the receptor binding site in HA. Various influenza A viruses are classified according to the method above.

Walking on the thin line between order and randomness in conformational transitions in polypeptides: chiral bifurcation Wojciech Dzwolak Department of Chemistry, University of Warsaw, Pasteur 1, 02-093 Warsaw, Poland Recently, we have described an unexpected phenomenon consisting in vortex-induced formation of two conformational variants of insulin amyloid fibrils with opposite chiral biases, as revealed 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 biophysics, although similar phenomena have been reported in condensed matter physics—e.g. upon crystallization of NaClO3. Our findings point to a new aspect of topological complexity of protein fibrils: a chiral feature of hierarchically assembled polypeptide chains, which is not determined by the innate lefthandedness of amino acids. Because altering chirality of a molecule changes dramatically its biological activity, these findings may have important ramifications in the context of structural basis of ‘amyloid strains’. Hydrational forces may play an important role in defining pathways of conformational transitions of misfolded proteins both in vitro and in vivo.

perspective, we proposed a model to explain the DAAO/pLG72/DSer association with the onset of the pathology: an anomalous hypoexpression of pLG72 yields to an increase of DAAO activity, then to a decrease of D-Ser released at the synapse, finally leading to hypoactivation of NMDA receptors. This intriguing mechanism 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 newly synthesized DAAO is located in the cytosol where it can interact with pLG72, placed on the external mitochondrial membrane. Subcellular immunolocalization and FRET analyses strongly support this proposal. Furthermore, in cotransfected cells pLG72 overexpression decreases the half life of DAAO. Since neosynthesized cytosolic DAAO was demonstrated to be catalytically active, we suggest that pLG72 binding to DAAO (and ensuing inactivation and degradation) might play a protective role against excessive D-Ser depletion. Our results uncover basic molecular aspects of the D-Ser degradation pathway and open novel perspective for therapeutic strategies of schizophrenia by targeting DAAO and the metabolism of D-Ser.

Levels of amino acids in the brain and peripheral organs of serine racemase knock-out mice Mao Horio1, Tamaki Ishima1, Mami Kohno1, Yuko Fujita1, Ran Inoue2, Hisashi Mori2, and Kenji Hashimoto1 1 Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan 2 Department of Molecular Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan

D-Serine,

D-Amino

acids

D-Serine

level in glial cells: spotlight on the role of D-amino acid oxidase S. Sacchi, P. Cappelletti and L. Pollegioni ‘‘The Protein Factory’’ Research Center, Universita` degli Studi dell’Insubria (Dip. Biotecnologie e Scienze Molecolari) and Politecnico di Milano, via Dunant 3, 21100 Varese, Italy; [email protected] D-Serine

is a brain enriched transmitter-like molecule that physiologically activates NMDA receptors, playing a main role in neuronglia communication and excitatory neurotransmission. D-Ser is synthesized by serine racemase and degraded by D-amino acid oxidase (DAAO). We demonstrated that D-Ser cellular concentration depends on the expression of active DAAO and of its negative regulator pLG72. Genetic evidences indicated that both pLG72 and DAAO are related to schizophrenia susceptibility. In this

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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 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, glycine, glutamine and glutamate in the frontal cortex, hippocampus, striatum and cerebellum as well as in peripheral tissues of blood, heart, pancreas, spleen, liver, kidney, testis, epididymis, heart, lung, muscle and eyeball, in wild-type (WT) and Srr-knockout (Srr-KO) mice. Levels of D-serine in the frontal cortex, hippocampus, and striatum of Srr-KO mice were significantly lower than in WT mice, while levels in the cerebellum stayed the same. In contrast, levels of L-serine, glycine, glutamine and glutamate remained the same in all tested brain regions. In vivo microdialysis using free-moving mice showed that extracellular levels of D-serine in the hippocampus of Srr-KO mice were significantly lower than in WT mice while the other amino acid levels remained the same between mice. In peripheral organs, levels of D-serine in the liver, kidney, testis, epididymis and eyeball of Srr-KO mice were significantly lower than in WT mice. Tissue levels of the other tested amino acids in peripheral organs were not altered. These results suggest that SRR is the major enzyme responsible for D-serine production in the mouse forebrain, and that other pathways of D-serine production may exist in the brain and peripheral organs.


Pain biology of spinal D-amino acid oxidase (DAAO) Yong-Xiang Wang, Nian Gong, Xiao-Lin Chen, Jin-Lu Huang, Yan-Chao Wang, Xin-Yan Li and Ai-Niu Ma King’s Lab, Shanghai Jiao Tong University School of Pharmacy, 800 Dongchuan Road, Shanghai 200240, China. Tel.: 86-21-3420-4763; Email: [email protected] 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 stereospecificity. DAAO is restrictedly distributed in the kidneys, liver 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. Systemic and intrathecal injections of a series of DAAO inhibitors, blocked formalin-induced tonic pain and bone cancer-induced mechanical allodynia, with maximum inhibition of 50–60%, in rats and mice, correlated to their inhibition of spinal DAAO activity. Intrathecal administration of siRNA/DAAO delivered by PEI complexation or adenovirus vector prevented tonic pain in rats. DAAO gene mutation also caused similar inhibition of tonic pain in ddy/DAAO-/- mice. DAAO inhibitors produced analgesia via the blockade of raised spinal hydrogen peroxide. The effects of DAAO inhibitors in chronic pain were specific as they were not effective in acute pain such as escape responses in the tail-flick and hot-plate tests, and thermal hyperalgesia in the carrageenan model. In addition, DAAO inhibitors interacted with morphine in an additive manner in their analgesia, and did not produce either self-tolerance to analgesia or cross-tolerance to morphine after 7 days exposure, in contrast to morphine. Thus, spinal DAAO is a potential new target molecule for discovery and development of novel medicines for chronic pain including cancer pain, with high efficacy, pain signal transduction and tissue expression specificity, and without tolerance to algesia. The study was supported by NSFC grants: No 81072623 and 30973581, China.

Glycation Mouse models of accelerated aging by carbonyl and oxidant stress

S19 the homozygous mouse. Methionine oxidation and protein disulfide formation in crystallins are highly increased at 6 months of age. Mice develop nuclear opacities as in human lens. In the NEGSKO mouse, molecular, cellular and behavioral phenotypes resembling amyotrophic lateral sclerosis (ALS) develop at 1 month of age, which include low pan-neuronal levels of GSH, increased apoptosis, diminished neuronal cell count, increased tau phosphorylation, and cleavage of the ALS 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.

Protein glycation, aggregation and cognitive impairments Yan Wei, Chanshuai Han and Rongqiao He* State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China D-Ribose,

one of the most important reducing monosaccharides, is present in all living cells and also in blood, cerebrospinal fluid as well as the human brain. Recently, D-ribose has been found to be very active in glycation of proteins such as bovine serum albumin, a-Synuclein and Tau protein, resulting in advanced glycation end products (AGEs). Ribosylation occurs much more rapidly than glucosylation in vitro. That is, in the presence of D-ribose, serum albumin, a-Synuclein and Tau protein are rapidly glycated, generating globule-like aggregations which can induce apoptosis and oxidative stress in several neuronal cell lines. However, we did not observe that the globule-like aggregations could advance into fibrils deposits after incubation with D-ribose over 2 weeks. We also find that D-ribose, but not D-glucose under our experimental conditions, can decrease cell viability and accelerate protein glycation and AGEs accumulation in SHSY5Y cells, HEK-293 cells and primary cultured hippocampal neurons. To investigate the effect of D-ribose and ribosylation in vivo, C57BL/6J mice were intraperitoneally 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 mice administrated with D-glucose or saline control. More interestingly, the ability of spatial learning and memory of D-ribose-treated mice declined in the Morris water maze test. These data demonstrated that Dribose, as an efficient glycation agent, could rapidly glycate proteins and 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 University, Cleveland, OH 44106, USA Aging is associated with progressive protein damage in lens, collagenrich tissues, neurodegenerative diseases and conditions with weakened antioxidant defenses. While the nature of the chemical protein changes is getting better understood, it has been difficult to directly implicate damage in the disease process itself. To close this gap, we developed novel mouse models of accelerated tissue aging, by selectively overexpressing or knocking out genes controlling carbonyl and oxidant stress. The first model was the hSVCT2 mouse in which we showed that expression of the human vitamin C transporter 2 in the lens accelerated the protein modifications observed during lenticular aging. Two new models of accelerated protein aging by oxidation have now have been generated by conditionally targeting glutathione synthesis in lens (LEGSKO mouse) (to mimic the low GSH levels in the core of the human lens) and nerve (NEGSKO mouse). c-Glutamyl cysteine ligase mRNA, activity and glutathione (GSH) levels are severely depressed in

Medicine Abundances of intestinal apical neutral amino acid transporter B0AT1 and exchanger ASCT2 proteins are reduced in pigs with dextran sodium sulfate-induced colitis Chengbo Yang,1,2 Dale Lackeyram,1 Tania Archbold,1 Yoshinori Mine,2 and Ming Z. Fan1 1

Center for Nutrition Modeling, Department of Animal and Poultry Science, Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1

2

We investigated changes in the expression of B0AT1 and ASCT2 genes as affected by inflammation in a porcine model of colitis

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S20 induced with dextran sodium sulfate (DSS). Real time RT-PCR analyses showed no difference (P [ 0.05) in the relative abundance of the B0AT1 mRNA between the DSS and the control groups in the colon. However, lower jejunal B0AT1 mRNA abundance was observed (P \ 0.05) in the DSS group. There were no differences (P [ 0.05) in the relative abundances of the ASCT2 mRNA in both jejunum and the colon between the DSS and the control groups. Both B0AT1 and ATB0 protein abundances were lower (P \ 0.05) in the jejunal homogenate and cytosol, and on the apical membrane in the DSS group compared with the control. Abundances of the B0AT1 and ATB0 proteins on the colonic apical membrane was lower (P \ 0.05) in the DSS group than in the control, whereas there were no differences (P [ 0.05) in abundances of the B0AT1 and ATB0 proteins in 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 decreased in the small intestine and colon in pigs with colitis induced by DSS. Considering the reduced B0AT1 and ATB0 protein 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 membrane may have the potential for the treatment of colitis.

12th International Congress on Amino Acids, Peptides and Proteins reduced the BSCB breakdown, edema formation and cell injury. Functional recovery was also markedly improved by treatment with this combination of neurotrophins. Expression of HO-2 in the spinal cord was significantly reduced. However, co-application of CNTF with either BDNF or GDNF was not that effective in inducing neuroprotection or downregulation of HO-2 expression in SCI. These observations suggest that (i) a combination of CNTF with BDNF and GDNF is necessary to induce effective neuroprotection even during the later phase of SCI (i.e., 120 min after primary insult), and (ii) the neuroprotective effects of neurotrophins in SCI are some how interrelated with increased CO production. Taken together our novel observations suggest an active interaction of BDNF, CNTF and GDNF with CO system for inducing neuroprotection in the spinal cord after trauma.

Affibody molecules. New targeting scaffold proteins for radionuclide molecular imaging and therapy of cancer Anna Orlova 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 recovery and reduces hemeoxygenase-2 (HO-2) expression, blood-spinal cord barrier permeability, edema formation and cellular damage in the rat 1

Hongyun Huang, 2Aruna Sharma, and 2Hari Shanker Sharma

1

Neuroscience Institute of Taishan Medical University, China; Email: [email protected], 2 Cerebrovascular Research Laboratory, Department of Surgical Sciences, Anesthesiology & Intensive Care Medicine, University Hospital, Uppsala University, SE-75185 Uppsala, Sweden, Email: [email protected] Previous reports from our laboratory show that neurotrophins when applied topically over the injured spinal cord in combination is able to reduce cord pathology and improves functional recovery. In this regard, a combination of BDNF and GDNF showed remarkable neuroprotection even when applied 90 min after spinal cord injury (SCI). However, a combination of BDNF or GDNF with NGF was not so effective. In present investigation, we wanted to know the role of CNTF in enhancing neuroprotection in SCI in combination with BDNF or GDNF treatment. Previously, we have shown that BDNF is able to attenuate nitric oxide (NO) production in the spinal cord after injury that correlates well with its neuroprotective ability. Since carbon monoxide (CO) is also a free radical gas and has many similarities with NO in inducing cell damage, in present investigation, we explored the role of neurotrophins in modulating CO production in the spinal cord in relation to neuroprotection. For this purpose, we used immunohistochemistry of the constitutive isoform of CO synthesizing enzyme, hemeoxygenase-2 (HO-2) to understand the functions of CO in SCI in relation to neurotrophins treatment. A focal SCI on the right dorsal horn on the T10–11 segment 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 (BSCB), edema formation and cell changes were seen in several spinal 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 from a solution of 1 lg/ml BDNF, GDNF or CNTF solution) 60, 90 and 120 min after injury over the exposed surface of the cord significantly

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Specific recognition of cancer-associated molecular abnormalities, i.e. targeting, is a promising way to improve diagnostics and treatment of malignant tumors. Affibody molecules represent a new class of affinity proteins based on a scaffold of B-domain of staphylococcal protein A. Affibody molecules combine small size (*6.5 kDa) with high affinity and specificity. Selection of high-affinity (low nanomolar or subnanomolar) Affibody molecules binding to cancer-associated molecular targets, such as HER2, EGFR, IGF-1R, HER3 and PDGFbR, has been reported. The small size and simple structure of Affibody molecules allow their production by chemical synthesis. This permits the use of unnatural amino-acids, site-specific incorporation of chelators for labeling with variety of nuclides, or toxins and drugs for therapy. As Affibody scaffold does not contain cysteine, an introduction of a single cysteine by gene-engineering enables site-specific conjugation of linkers and chelators to recombinantly produced Affibody molecules using thiol-directed chemistry. Fusion of Affibody molecules with streptococcal albumin-binding domain (ABD) permits modulation of their residence time in the blood circulation. Pre-clinical studies have demonstrated that Affibody molecules can be labeled with different nuclides for imaging (PET and SPECT) and for radionuclide therapy. In vivo, Affibody molecules provide rapid and specific binding to tumors and rapid renal clearance for nonbound tracer, permitting high contrast in vivo images already a few hours after injection. A pilot clinical study confirmed that [111In]- and [68Ga]-labeled Affibody molecules can visualize HER2-expressing metastases. In conclusion, Affibody molecules constitute a promising class of targeting proteins for tumor targeting.

Altitude hypoxia on the ultrastructure of rat brain and Hsp70 expression Li Wenhua, Yuan Dongya, Zhang Min, Li Yang, Sun Zhenqi, Zhao Fengcang Medical School, Tibet Institute of Nationalities, Xianyang, Shanxi 712082, China; Email: [email protected] Objective means of cell biology and molecular biology of high altitude adaptation in rat, the rat heat shock response, heat shock protein in the organism to understand high altitude adaptation in biological

12th International Congress on Amino Acids, Peptides and Proteins significance. Methods 90 male SD rats were randomly divided into 10 groups, three experimental animals brought by the Golmud, Qinghai, 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 24 h of slaughter; three different experimental groups 1d directly to 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 group (in Xi’an, elevation 5 m), with the Western blot and conventional RT-PCR and real-time fluorescence quantitative PCR (realtime PCR detection) measured at different altitudes SD Heat shock protein in rat brain 70 (Hsp70) expression and brain differences in the natural expression of Hsp70 gene, light microscopy and transmission electron microscopy observation of animals in each group structural changes in brain tissue. Results Mammals have a different elevation of heat shock response genes, stress, high altitude when the rapid increase in the expression of mammalian Hsp70; Hsp70 can be high altitude (high altitude) induced. Conclusion: Hsp70 heat shock response in the rapid synthesis of high altitude hypoxic stress is conducive to maintaining the normal physiological function when the cells, Hsp70 and cell formation is proportional to hypoxia tolerance. Keywords: High altitude, SD rats, Heat shock protein 70 (Hsp70), Western blot, RT-PCR real time quantitative PCR Project: Tibet, the Office of Science and Technology Research Fund (2010), the State Ethnic Affairs Commission research project (2011), initial results.

Antibodies to hemeoxygenase-2 tagged with nanowires enhances neuroprotection in traumatic brain injuries Hari S Sharma, and Aruna Sharma Department of Surgical Sciences, Anesthesiology & intensive Care Medicine, University Hospital, Uppsala University, SE-75185 Uppsala, Sweden. Email: [email protected] Previous reports from our laboratory showed that traumatic brain or spinal cord injuries upregulates hemeoxygenase-2 (HO-2) proteins in 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 neuroprotective capabilities, influence of nanowired HO-2 antibodies was also examined in present investigation. 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 separate group of rats HO-2 antibodies (1:20, monoclonal) were applied 10, 30 or 60 min after TBI. Another group of rats received identical HO-2 antibodies but tagged with TiO2 nanowires applied over the lesion at the same time intervals after trauma. Topical application of HO-2 antibodies given 10 or 30 min after TBI resulted in marked neuroprotection in terms of reduction in the blood–brain barrier (BBB) breakdown to Evans blue and radioiodine traces, edema formation and neuronal injuries. However, normal HO-2 antibodies did not give any effects when they are applied 60 min after TBI. On the other hand nanowired HO-2 was able to significantly reduce neuronal injuries and BBB disruption or brain edema even applied after 60 min TBI. The neuronal damages were tightly correlated with upregulation of HO-2 expression in both untreated and following HO2 antibodies treatments with or without nanowiring in TBI. Taken together our results for the first time show that HO-2 antibodies could

S21 induce marked neuroprotection in TBI and these effects are further potentiated by nanowiring of the HO-2 antibodies.

Arginine transport in the human pathogen Leishmania and its possible role in parasite-host interactions Adele Goldman-Pinkovich1, Ilona Darlyuk1, Doris Rentsch2 and Dan Zilberstein1 1

Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel, 2 Institute of Plant Sciences, University of Bern, 3013 Bern, Switzerland Arginine is an essential amino acid for the intracellular parasitic protozoan Leishmania but not for its host. Thus, maintaining cellular homeostasis of arginine is critical for parasite survival and virulence. Previously, we cloned and functionally characterized a high affinity arginine-specific transporter, LdAAP3, from Leishmania donovani. Here we characterized the relationship between arginine transport via LdAAP3 and amino acid availability. Exposing parasites to amino acids starvation decreased the cellular level of most amino acids including arginine, while the abundance of LdAAP3 mRNA and protein greatly 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 established that LdAAP3 expression and activity changed whenever the cellular level of arginine changed, and thus hypothesized that L. donovani promastigotes have a signaling pathway that senses cellular concentrations of arginine and subsequently activates a mechanism that regulates LdAAP3 expression and activity. Although starvation for amino acids is an artificial condition examined in an in vitro system, it is likely that Leishmnania also experience starvation in vivo. Inside macrophage phagolysosomes the parasites proliferate as non-motile amastigotes that need to compete for host arginine, possibly by employing their starvation response mechanism. Therefore, we hypothesize that LdAAP3 plays a vital role in the parasites survival within its host.

Association study of hypoxic gene single nucleotide polymorphism with the susceptibility of acute mountain sickness Li Wenhua, Yuan Dongya, Zhao Fengcang, Sun Fangyun Medical School, Tibet Institute of Nationalities, Xianyang, Shanxi 712082, China. Email: [email protected] Background and objective: Acute mountain sickness (AMS) is apotentially serious affliction to health in immigrants to Tibetan Plateau above 3,000 m. There are no effective treatment and prevention strategy of AMS now. Interindividual variation in acclimatization and adaptation to high altitude suggest that the probability of developing AMS depends on genetic and environmental factors. So we speculated that genetic factors may be associated with AMS susceptivity. The research aimed to explore the association between single nucleotide polymorphism (SNP) of hypoxic gene and AMS by comparing the difference of hypoxic gene SNP between AMS-susceptible and acclimatized individuals.

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S22 Methods: The study enrolled Han Chinese students into Tibet in our hospital for the first time flew to high altitude (3,658 m, Lhasa) from lowland (505 m). AMS was diagnosed on the basis of the AMS Questionnaire. Venous blood was collected and then analyzed by ELISA for VEGF and for extraction of genomic DNA and peripheral arterial oxygen saturation (SP02) was recorded at low altitude and after 24–48 h at high altitude. By case–control study method, we selected 200 cases of AMS as AMS group and 200 individuals which not developed AMS as control group randomly from these soldiers respectively. The selected subjects were genotyped for the fourteen polymorphisms of the hypoxic genes by primer extension of multiplex products with detection by matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectroscopy. These hypoxic genes include vascular endothelial growth factor (VEGF), hypoxia inducible factor I alpha subunit (HIF I A), Glutathione S-transferase mu 3(GSTM3), Glutathione S-transferase pi 1(GSTPI), Nitric oxide synthase 2(NOS 2), nitric oxide synthase 3 (NOS 3), Angiotensin I converting enzyme (ACE) and heat shock 70 kDa protein 4 (HSPA4). The association between SNP and AMS was analysed by genetic statistical software. Results: Morbidity of AMS in Han Chinese students into Tibet in our hospital for the first time flew to high altitude was 35.68%. SP02 fell from 98.0211.69% at 505 m to 85.16 ± 5.42% at 3,658 m in subjects 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 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. 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 the baseline level. The plasma VEGF concentration were significantly 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, 55.58 ± 20.19 vs. 16.98 ± 11.61 pg/ml, respectively, P \ 0.01). All 13 gene locuses exist polymorphisms and were in Hardy–Weinberg equilibrium in both AMS group and controls except for HSPA4 which only have a allele T. The T allele of rs3025039 of VEGF was significantly associated with AMS (P = 0.012, OR = 0.62, 95% CI 0.43–0.90). The rs3025039 of VEGF genotypic frequency distribution differed significantly between AMS and control group (P = 0.0297). CT genotype of rs3025039 was significantly associated with AMS (P = 0.010, OR = 0.56, 95% CI 0.36–0.87) assuming a additive effect of the T allele; CT + TT genotype of rs3025039 was significantly associated with AMS (P = 0.008, OR = 0.56, 95% CI 0.37–0.86) assuming a dominant effect of the T allele. The C allele of rs3025030 of 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 associated with AMS (P = 0.018, OR = 1.66, 95% CI 1.10–2.45) assuming a dominant effect of the T allele. In terms of NOS2, TT genotype of rsl137933 was significantly associated with AMS (P = 0.049, OR = 3.045, 95% CI 0.96–9.68) assuming a additive effect of the T allele. TT genotype of rs1137933 was significantly associated with AMS (P = 0.04, OR = 3.13, 95% CI 0.99–9.87) assuming a recessive effect of the T allele. For ACE, CC genotype of rs4309 was significantly associated with AMS (P = 0.049, OR = 1.83, 95% CI 1.00–3.34) assuming a recessive effect of the C allele. Genotypic and allelic frequencies of the rs2297518 of NOS2; rs8005745, rs2301108, rs 10873142 of HIF 1 A; rs 1695 of GSTP 1;rs7483 of GSTM3; rsl799983, rs3918188 and rs7830 of NOS3 and rs35853823 of HSPA4 were not significantly different between AMS and control group (P [ 0.05). At the haplotype level, a haplotype GC/ CT frequencies consisting of rs3025030 (G/C) and rs3025039 (C/T) of VEGF was significantly differences between the AMS group and control group (P = 0.029). There were no statistically significant differences in the haplotype AGT/TAC/AGC frequencies consisting of

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12th International Congress on Amino Acids, Peptides and Proteins rs8005745, rs2301108 and 10873142 of HIF 1 A between the AMS group and control group at high altitude (P [ 0.05). Conclusions: 1, Morbidity of AMS in Han Chinese students into Tibet in our hospital for the first time flew to high altitude was 35.68%; 2, SPOZ and VEGF is down-regulated significantly in AMS group and control at athigh altitude but VEGF level of AMS group still higher significantly than control not only at high altitude but also at sea level; 3, Polymorphism of rs3025039 and rs3025030 in VEGF gene were significantly associated with AMS. Individuals carrying the allele T of rs3025039 and C of rs3025030 significantly decrease the risk of AMS in a Chinese population. 4, Polymorphic loci of rs1137933 in NOS2 and rs4309 in ACE were significantly associated with AMS. Individuals carrying the genotypic TT of rs1137933and genotypic CC rs4309 significantly increase the risk of AMS. 5. This study did not provide evidence that SNPs of the rs2297518 of NOS2, rsl799983,rs3918188, rs7830 of NOS3, rs1695 of GSTP1, rs7483 of GSTM3, rs8005745, rs2301108, rs10873142 of HIFIA and rs35853823 of HSPA4 gene are associated with susceptibility to AMS in a Chinese population. Keywords: VEGF, Hypoxic gene, Single nucleotide polymorphism, AMS Project: Tibet, the Office of Science and Technology Research Fund (2010), the State Ethnic Affairs Commission research project (2011), initial results.

Autoantibodies against heat shock proteins are increased in patients with amyotrophic lateral sclerosis I-Lin Liao1,2#, Chi-Shin Hwang3,4#, Guan-Ting Liu1,5, Yi-Chen Wu1,6, Margaret Dah-Tsyr Chang3*, and Hao-Teng Chang1,5,7* 1

Graduate Institute of Molecular Systems Biomedicine, Department of Medical Laboratory Science and Biotechnology, 3 Institute of Molecular and Cellular Biology & Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China., 4 Department of Neurology, Taipei City Hospital–Zhong Xiao Branch, Taipei, Taiwan, Republic of China. E-mail: [email protected]; [email protected] 5 Graduate Institute of Basic Medical Science & Ph.D. Program for Aging, 6 Department of Biological Science and Technology, 7 Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan, Republic of China, 2

Amyotrophic lateral sclerosis (ALS) is one of severe neurodegenerative disorders. With the loss of motor neurons in the brain and spinal cord, it causes progressive muscular atrophy. Therefore, ALS patients die due to respiratory failure caused by bronchial muscular dystrophy within 2–5 years postdiagnosis. The exact pathogenesis of ALS remains unclear to date. Though there are ways to slow down the progression of the disease, no absolute treatment is effective. In addition, discovery of ALS biomarkers is needed both for early diagnosis and to monitor disease progression. Previous studies showed that the increasing of intracellular oxidative stresses would raise the rate of suffering ALS. Similar with those studies, Western blotting showed heat shock protein 60 (HSP60) and HSP70, which belong to the damage-associated molecular patterns (DAMPs), were elevated in sera of patients with ALS. However, in our cases it was fail to distinguish the difference between patients and control using enzyme-linked immunosorbent assay (ELISA) to detect HSP60. We proposed that HSP60 might be neutralized by the autoantibody against itself. Thus, the levels of autoantibodies against HSP60 and against HSP70 would be measured to distinguish the

12th International Congress on Amino Acids, Peptides and Proteins patients with ALS from the controls. Compared with 40 subjects with ALS and 40 age-matched controls, serum levels of the autoantibodies against HSP60 and HSP70 were 1.64 and 1.37 folds higher in patients with ALS than in controls. The ROC curve also revealed that the AUC of 0.7651 and 0.6179 for autoantibodies against HSP60 and 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.

Clotting activity changes of fibrinogen induced by peroxynitrite Yunjing Luo, Zhiguo Han, Qi Zhu, Shuang Cui, and Rugang Zhong College of Life Science and Bioengineering, Beijing University of Technology, 100124 People’s Republic of China. E-mail address: [email protected] Fibrinogen plays an important role in blood clot formation. The injury of fibrinogen induced by perxoynitrite is related to thrombotic and cardiovascular disease. In this paper, we investigated the changes on the secondary structure and clotting activity of fibrinogen treated by peroxynitrite with FTIR spectra and Von Clauss method. With the increase of peroxynitrite, the content of a-helix in fibrinogen reduced from the original 38.03 to 29.69% whereas b-sheet increased from 29.89 to 43.66%. The increase of b-sheet structure makes folded fibrinogen looser, consequently, the ability of fibrinogen to release fibrin clot is changed. Although the clotting activity of fibrinogen decreased with the increase of peroxynitrite in overall trend, trace of peroxynitrite could enhance the clotting activity. We speculated slight nitration could make fibrinogen release fibrin monomers easier in the presence of thrombin. When fibrinogen treated with hydrogen peroxide, the clotting activity of fibrinogen decreased sharply, which suggested fibrinogen is more susceptible to oxidation than nitration.

Development of O-GlcNAcase inhibitors for Alzheimer’s disease therapy Zhonghua Li1, Tiehai Li1, Lin Lin1, Jing Li1*, Wei Zhao1* and Peng George Wang1,2,3* 1 College of Pharmacy and State Key Laboratory of Element-Organic Chemistry, Nankai University, Tianjin 300071, China2Departments of Biochemistry and Chemistry, the Ohio State University, Columbus, OH 43210

O-GlcNAcylation is an abundant, essential and dynamic protein posttranslational modification of cytosoic proteins in metazoans and can compete with phosphorylation at similar Ser/Thr sites of target proteins. The enzyme of O-GlcNAc metabolism (OGT/OGA) are enriched in the brain and many proteins important for neuronal function are modified by O-GlcNAc including microtubule-associated protein tau, b-amyloid precursor protein (AP), clathrin-assembly proteins and neurofilaments. Pathological hyperphosphorylation of tau is characteristic of Alzheimer’s disease (AD) and the associated tauopathies. So a potent and specific O-GlcNAcase inhibitor can be used to enhance O-GlcNAc level and downregulate phosphorylation of tau and further provide a new drug for AD therapy. Here, using Cu(I)-catalyze ‘‘click’’ cycloaddition reactions between glycosyl azides and alkynes, 16 candidate compounds were synthesized

S23 and screened for inhibitory activity against human O-GlcNAcase and human lysosomal b-hexosaminidase (Hex A & B). As a result, 4-pyridyl1-(20 -deoxy-20 -acetamido-b-D-glucopyranosyl)-1,2,3-triazole, displayed specific inhibitory activity against human O-GlcNAcase, with Ki = 48 lM and 200-fold selectivity against Hex A & B.

Diabetes-induced structural alterations in rat liver proteins and the recovery effect of selenium: an FTIR microspectroscopy and neural network study Ozlem Bozkurt1, Sevgi Haman Bayari2*, Mete Severcan3, Christoph Krafft4, Ju¨rgen Popp4,5, and Feride Severcan1 1

Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey 2 Department of Physics, Hacettepe University, 06800 Ankara, Turkey 3 Department of Electrical Engineering, Middle East Technical University, 06531 Ankara, Turkey 4 Institute of Photonic Technology, 07745 Jena, Germany 5 Institute of Physical Chemistry, University Jena, 07743 Jena, Germany Diabetes mellitus is a major endocrine disorder and a growing health problem in the world, characterised by hyperglycemia resulting from defects in insulin secretion, insulin action or both of them. In the current study, the changes in Streptozotocin (STZ)-induced diabetes in content and structure of proteins in rat livers were studied by Fourier transform infrared microspectroscopy (FTIRM). The potential role of selenium in recovery of diabetes induced alterations was also investigated. FTIRM results demonstrated an increase in protein content of diabetic group. In order to monitor the changes in other macromolecules with respect to protein, different ratios, such as lipid 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 NN results, the content of a-helical structures was decreased, while the b-sheet content was increased in diabetic group with respect to the control. Restoring effect of selenium was observed in all of the spectral parameters investigated. Furthermore, the hierarchical cluster analysis performed in fingerprint region (1800–900 cm-1) resulted in the successful differentiation between control, diabetic and selenium treated diabetic groups. The sensitivity and specificity values calculated from the cluster revealed the power of FTIRM in characterizing diabetic liver tissues. The results of this study established that FTIRM together with chemometric methods can be used as a powerful tool in determination of diabetes-induced alterations at molecular level.

Dialyl trisulfide affects sulfurtransferases and Bcl-2 expression in human cancer cells SH-SY5Y and U-87 MG Maria Wro´bel and Halina Jurkowska Chair of Medical Biochemistry, Jagiellonian University Medical College, Krako´w, Poland Dialyl trisulfide (DATS), a component of garlic extract, contains three sulfur atoms with two allyl groups. It acts as the direct precursor of

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S24 sulfane sulfur atoms (oxidation state 0 or -1) and exerts a potent antiproliferative effect on several human cancer cell lines. The cellular sulfane sulfur levels depend on cysteine availability and the activity of two sulfane sulfur-generating enzymes: c-cystathionase and 3-mercaptopyruvate sulfurtransferase. The response of the two cell lines: SH-SY5Y, a human derived neuroblastoma, and U-87MG, 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 expression of 3-mercaptopyruvate sulfurtransferase in the U-87 MG cells and c-cystathionase in the SH-SY5Y cells, and the inhibition of proliferation in case of both cell lines. In the SH-SY5Y cells an increased expression of gene for Bcl-2 protein suggested induction of apoptosis, while in U-87 MG cells, the formation of apoptotic bodies was observed by fluorescence microscopy. In both cell lines, DATS potency to enhance endogenous levels of GSH was shown; this allowed for maintaining intracellular redox status measured as the concentration ratio of reduced to oxidized glutathione. Increased levels of cystathionine and cysteine after 24 h of culturing the U-87 MG cells in the presence of 2 mM DATS suggested that the pathway towards the glutathione formation through the cystathionine b-synthase and c-cystathionase reactions was stimulated in these cells.

Designing radiolabelling strategies for proteins and peptides. How peptide based chelators can improve radionuclide imaging and therapy Vladimir Tolmachev Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Sweden;Email: [email protected] An increasing number of scaffold proteins are being evaluated for in vivo tumour targeting. One important application area of scaffold proteins is radionuclide tumour targeting for imaging and therapy. Selection of an optimal labelling chemistry for scaffold proteins is essential, since it can influence their affinity, cellular processing of the tracer by cancer cells and cellular retention of a radionuclide, biodistribution of radiolabeled tracers and predominant excretion pathway. A promising approach for labelling chemistry is the use of peptide-based chelators for radionuclides 99mTc for radionuclide imaging and rhenium isotopes 188Re and 186Re for radionuclide therapy. The use of peptide-based chelators permits site-specific labelling of recombinant proteins without additional chemical processing. An experience with Affibody molecules suggests that optimising composition and order of amino acids in peptide-based chelators might appreciably improve radionuclide tumour targeting. For example, the use of hexahistidine tags for labelling of proteins using 99mTc(CO)3 is associated with elevated liver uptake of radiotracer. However, a substitution of every second histidine with glutamate provides HEHEHE-tag, which permits IMAC purification and stable labelling with 99mTc(CO)3, but does not cause high hepatic uptake. Increasing hydrophilicity of amino acids in meracptoacetyl-containing chelators at N-terminus of Affibody molecules suppress undesirable hepatobiliary excretion of tracers. Moreover, a careful optimisation of amino

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12th International Congress on Amino Acids, Peptides and Proteins acid composition in chelators permits substantial decrease of renal retention of radioactivity. In conclusion, the use of peptide based chelators for technetium and rhenium radioisotopes permits not only stable coupling of nuclides to scaffold proteins, but also improvement of their targeting properties.

Effects of arsenic exposure from drinking water on the expression of MMP-1 and TIMP-1 in hepatic tissue in rats XU Zhao1,2, WANG Zhou3, LI Jian-jun2, ZHANG Ping-chuan1, DONG Lu1, ZHANG Xiao-tian1, WANG Shu-mei1, and WANG Zhi-lun1 1

Xi An Jiao Tong Univ, Sch Med, Minist Educ, Key Lab Environm & Genes Related Dis, Xian 710061, People’s Republic of China 2 Xian Jiaotong Univ, Sch Sci, Dept Chem, Xian 710049, People’s Republic of China 3 Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, People’s Republic of China Objective: Chronic toxicity of arsenic from drinking water has become a global health problem that affect millions of people. Epidemiological studies indicate that chronic exposure to arsenic can cause varying degrees of liver damage, liver fibrosis, cirrhosis and 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 exposure from drinking water on the liver function and the expression of MMP-1 and TIMP-1 in hepatic tissue in rats. Methods: Sprague–Dawley rats were divided into three groups at 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 drinking water. The rats were sacrificed at 3, 5 and 7 months for pathologic, total arsenic levels, liver function, RNA and proteins examination. The levels of arsenic in blood and hepatic tissue were determined by atomic fluorescence spectrometry. The serum were collected and the glutamic–pyruvic transaminase (ALT), aspartate transaminase (AST), lactic dehydrogenase (LDH), and gammaglutamyl transpeptidase (GGT) were measured by automatic biochemistry analyzer. The RNA and proteins were extracted by Trizol reagent. Then the RNA were reversed transcription into cDNA, the mRNA expression of MMP-1, TIMP-1 and b-actin were detected by real-time fluorescence quantitative PCR. The protein expression of MMP-1, TIMP-1 and b-actin were detected by Western Blot Assay. Results: After 5 months of arsenic feeding, the serum ALT, AST, LDH of group B and group C were higher than that of group A, and pathologic examination showed that there were different degree of inflammatory penetration embellish and water-like lesions in hepatic tissue of group B and group C; After 3 months of arsenic feeding, the expression of MMP-1 mRNA and protein of group B and group C were lower than that of group A, and the expression of TIMP-1 mRNA and protein of group B and group C were higher than that of group A. Conclusion: Exposed to arsenic solution could result in hepatic injury in rats. The TIMP-1 and MMP-1 gene play significant effect in process of hepatic injury.


Effect of Panax notogino side on the p38MAPK pathway in lung tissue of hypoxic rats* LIN Li-na1, ZHAO Shan2#, LIANY Ying-qi2, LIU Ya-kun2, TANG Lan-lan2, WANG Shu-Jun2, LI Guan-Long2, WANG Yuan-yuan2, WU Cheng-yun3, and WANG Wan-tie2* 1

Department of anesthesiology, The Frist Affiliated Hospital, Department of Pathophysiology, 3 Department of Respiratory Disease, The Second Affiliated Hospital, Wenzhou Medical College, Wenzhou 325035, China Correspondence to: Wang Wan-tie, Department of Pathophysiology, Wenzhou Medical College, wenzhou325035, China (E-mail:[email protected]; Tel: 0577-86689817) 2

Objective: To observe the effect of PNS (Panax notogino side) on the pulmonary artery pressure and the p38MAPK (p38 Mitogen activated protein kinase) in lung tissue of rats treated with hypoxia and therefore to explore the mechanisms of PNS on HPH (hypoxic pulmonary hypertension). Methods: Thirty adult male SD rats were randomly divided into 3 groups. One group was exposed to normal conditions (N group), the second group was exposed to isobaric hypoxia (H group), and the third group was treated with PNS under the hypoxia (HP group), after 4 weeks, cardiac catheterization was used to measure the mPAP (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) in rat pulmonary arterioles was determined by immunohistochemistry and the content of p38MAPK mRNA was tested by RT-PCR. Results: The mPAP, RV/(LV + S) in HP group were higher than N group, The expression of p-p38MAPK in rat pulmonary arterioles and p38MAPK mRNA in the lung were higher than that in N group (P \ 0.05). The mPAP, RV/(LV + S). The expression of p-p38MAPK in rat pulmonary arterioles and p38MAPK mRNA in the lung were significantly lower than those in the H group (P \ 0.05). Conclusion: PNS was shown to prevent hypoxic pulmonary hypertension, the underlying mechanisms may relate to the decrease on p-p38MAPK and down regulation of p38MAPK mRNA. Keywords: Panax notogino side, Hypoxia, Pulmonary arterial hypertension, p38MAPK

Engineered nanoparticles from metals influence glutamate, aspartate, GABA and glycine neurotransmission in the normal and injured spinal cord 1

Jose´ Vicente Lafuente*, 2Ranjana Patnaik, 3Aruna Sharma, and 3Hari S Sharma

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Lab Neurociencias Clıńicas y Experimentales (LaNCE), Dpto. de Neurociencias, Universidad del Paı´s Vasco—EuskalHerriko Unibertsitatea, Apdo. 699, 48080-Bilbao, Espanã, 2 Department of Biomaterials, School of Biomedical engineering, Institute of technology, Banaras Hindu University, Varanasi-221005, India 3 Department of Surgical Sciences, Anesthesiology & intensive Care Medicine, University Hospital, Uppsala University, SE-75185 Uppsala, Sweden. Email: [email protected] Previous reports from our laboratory show that engineered nanoparticles from metals exacerbate spinal cord pathology following injury. However, the possible mechanisms of nanoparticles induced exacerbation of

S25 spinal cord damage are not well understood. Since spinal cord injury (SCI) induced widespread alterations in amino acid neurotransmitters e.g., glutamate, GABA, aspartate and glycine, in present investigation influence of engineered nanoparticles form metals on amino acid content of the spinal cord in normal rats and after SCI was examined. Rats were administered engineered nanoparticles from Cu and Ag (50–60 nm) once daily (50 mg/kg, i.p.) for one week. On the 8th day, one group of rats were subjected to SCI by making a longitudinal incision into the right dorsal horn of the T10-11 segments under Equithesin anesthesia. Five h after SCI, the spinal cord from the T9 and T12 segments were taken out and glutamate, aspartate, GAB and glycine were analyzed using HPLC technique. Another group of nanoparticles treated rats no injury was made. Normal rats served as control. Nanoparticles treatment resulted in a marked increase in Glutamate and aspartate (+50 to 70%) content in the T9 and T12 segment of the cord where as GABA and glycine showed a significant decline (-20 to 40%). Control from the control value. This effect was most pronounced with Ag nanoparticles. Subjection of rats to SCI in nanoparticles treated animals resulted in further enhancement of Glutamate and aspartate content in the cord (+150 to 180%) whereas, GABA and glycine showed marked decline (-60 to 80%). This effect on amino acid contents was most marked in Ag treated rats after SCI. Neurological dysfunction and cord pathology were also exacerbated in nanoparticles treated arts after SCI. These observations are the first to show that nanoparticles induced exacerbation of cord pathology following SCI is mediated through amino acids neurotransmission.

From single gene to pathway analysis: high-throughput screening for drug dependence Ju Wang Department of Psychiatry and Neurobehavioral Sciences, University of Virginia Drug dependence is a prevalent neurological disorder characterized by the structural, biochemical or electrical abnormality in the nervous system, which affects millions of people worldwide and is one of the most serious threats to public health. Although our understanding on the nature of drug dependence has greatly advanced in the past decades, 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 may be involved in the development of drug dependence, with each factor only has a relatively small effect. In recent years, the rapidly growing field of functional genomics and proteomics has played an increasingly important role in drug addiction research because of their ability of analyzing a large number of molecular targets simultaneously. In this study, we summarize the application of a multitude of high-throughput genomic and proteomic technologies, such as gene expression microarray, microRNA array, 2-D gel electrophoresis, and genome-wide association studies (GWAS), toward indentifying and characterizing the molecular factors involved in drug dependence. We first provide an overview of genomics and proteomics technologies and bioinformatics tools available to analyze the data harvested via these approaches. Then we summarize the recent applications of these technologies to profile the gene/protein expression pattern in animal or human brain tissues in response to substances of abuse, as well as identify the genes genetically associated with drug dependence in human. Further, we detect the biological pathways enriched in these genes/proteins. Our study demonstrates the promise and potential of high-throughput screening technology in drug dependence study that aims at deciphering the underlying molecular mechanisms and finding better targets for developing therapeutic intervention.

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Genetics analysis of complex traits in human: what we have learned from studying genetics on smoking dependence? Ming D. Li Department of Psychiatry and Neurobehavioral Sciences, University of Virginia 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 significantly to vulnerability to this complex trait, the susceptibility genes underlying it are largely unknown. To identify susceptibility genes for smoking dependence, almost all approaches commonly used in the genetic studies on complex traits such as genome-wide linkage analysis, candidate gene-based association analysis and genome-wide association analysis have been employed. These analyses have implicated several common genomic regions and genes in the etiology of smoking addiction. Although a significant number of reported genomic regions did not reach the level of ‘‘suggestive’’ or ‘‘significant’’ linkage and failed to be replicated in other independent studies, thirteen regions, located on chromosomes 3–7, 9–11, 17, 20, and 22, have been found to be suggestive or significant in at least two independent samples. Among them, the regions on chromosomes 9, 10, 11, and 17 have received the strongest support. In addition, several genome-wide association analyses including three meta analyses of smoking related behaviors have been conducted. A gene cluster on chromosome 15q24/q25.1 that encompasses the genes for nicotine acetylcholine receptor subunits a5, a3, and b4 was implicated in addiction to tobacco and other substances as well. Current efforts aim not only to replicate these findings in independent samples but also to determine the functional mechanisms of these associations.

High altitude hypoxia environment changes of the content of AVP and expression Li Wenhua, Yuan Dongya, Zhang Min, Sun Fangyun, Sun Zhenqi, Zhao Fengcang Medical School, Tibet Institute of Nationalities, Xianyang, Shanxi 712082, China; Email: [email protected] Objective: To study the environment of high altitude hypoxia in rats plasma arginine vasopressin (AVP) levels and the expression of gene transcription and its relationship with the relationship between altitude hypoxic acclimatization. Methods 50 male SD rats were 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 (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) and 1 y Nagqu (elevation 4,500 m), were sacrificed at different time points, application of radioimmunoassay of plasma AVP concentration, and compared with the control group. Using [35S] CTPRNA probe by in situ hybridization histochemistry was used to detect within the PVN of rats AVPhnRNA, AVPmRNA expression. Results Plasma AVP concentration increased after entering the plateau, then decreased to the lowest in the 2d, after rising gradually to 1w close to the level of control when, a month reached the highest level. Hypothalamic paraventricular nucleus (PVN) of small cells and AVPmRNA AVP hnRNA expression was positively correlated with the above results, large cells AVP hnRNA expression levels in

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12th International Congress on Amino Acids, Peptides and Proteins 1 month into the plateau increased significantly (P \ 0.01), AVP mRNA expression levels Quick access to the plateau in the early (2d) increased slightly (not statistically significant) after no significant change. Conclusion altitude chronic hypoxia, the plasma concentration of AVP with the time change hypoxia, coexist in the PVN large and small cells of the AVP gene transcription by different mechanisms, this may be related to degree of hypoxia and high altitude hypoxia Acclimatization processes. Project: Tibet, the Office of Science and Technology Research Fund (2010), the State Ethnic Affairs Commission research project (2011).

High dose simvastatin administration ınduces changes in the structure and concentration of liver microsomal membrane proteins 1

Kumsal OZGUN, 1Nihal SIMSEK OZEK, 2Mete SEVERCAN, and 1Feride SEVERCAN

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Department of Biological Sciences, Middle East Technical University, Ankara, Turkey 2 Department of Electrical and Electronics Engineering, Middle East Technical University, Ankara, Turkey Simvastatin is one of the most frequently prescribed cholesterol reducing drugs, due to its higher efficacy in reducing LDL cholesterol levels and its tolerability. The possible hepatoxic effect of this drug may be related to drug induced alterations in structure and function of liver microsomal membrane proteins. This is because it contains many enzymes that are involved in drug, cholesterol and fatty acid metabolism and any changes in the structure and amount of these enzymes may lead to protein malfunction. In this study, the simvastatin-induced 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 region). FTIR results revealed structural alterations in protein secondary structure and an increase in total protein amount in simvastatin treated group, deduced from changes in the band area, bandwidth, band frequency values of these bands and also their ratio values. Detailed protein secondary structural changes were obtained from intensity calculations from second derivative spectra and neural network (NN) analysis, using the amide I band of FTIR spectra. Chronic simvastatin treatment induced a reduction in b-sheet and an increase in the random coil and aggregated b content of liver microsomal membrane. Moreover, based on these alterations in proteins, successful discrimination of control and simvastatin treated groups were obtained by hierarchical cluster analysis. The results of this study approved the higher efficiency of FTIR technique in determination of alterations in protein structure and content induced by simvastatin administration.

Hypoxic preconditioning induced neuroprotection against cerebral iachemic injury of mice and its cPKCc-mediated molecular mechanism Nan Zhang, and Junfa Li* Department of Neurobiology and Beijing Institute for Neuroscience, Capital Medical University, #10 You An Men Wai Xi Tou Tiao, Beijing 100069, People’s Republic of China *E-mail: [email protected] As of yet, pharmacological treatments of stroke are only met with mediocre results, which are either ineffective or confounded by adverse

12th International Congress on Amino Acids, Peptides and Proteins effects, thus calling for a better understanding of endogenous neuroprotective mechanism. Previously, we have demonstrated that the translocated activation of conventional protein kinase Cc (cPKCc) is involved in the development of cerebral hypoxic preconditioning (HPC), one of the most profound neuroprotective strategies. This study was designed to substantiate the role of cPKCc and its signaling molecules in HPC-induced neuroprotection against subsequent middle cerebral artery occlusion (MCAO)-induced permanent cerebral ischemic injuries. The effects of HPC and cPKCc on cerebral ischemic injuries were studied by observing the changes in neurological deficits, infarct volume and neural cell apoptosis. cPKCc membrane translocation and its interacting protein synapsin in the ischemic brain were examined by Western blot analysis. Proteomic approaches were employed to identify the cPKCc-interacting proteins. We found that HPC could markedly attenuate MCAO-induced brain injuries and the decrease of cPKCc membrane translocation, but cPKCc inhibitor Go6983 could block HPC-induced neuroprotection. Among the 41 identified cPKCc-interacting proteins, 17 up- and 6 down- regulated proteins were observed in cytosol or particulate fraction during HPC. In addition, the up-regulated synapsin could reciprocally co-precipitated with cPKCc both in cytosol and particulate fractions, and Go6983 abolished HPC-induced inhibition on synapsin dephosphorylation in ischemic core and penumbra. This study is the first to report multiple cPKCc-interacting proteins in HPC mouse brain and suggested that cPKCc-synapsin pathway might be responsible for HPC-induced neuroprotection against cerebral ischemic injuries of mice.

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer Wei Liu, Mingxia Zhai, Zongyin Wu, Yuanming Qi, Yahong Wu, Chao Dai, Meng Sun, Lu Li, and Yanfeng Gao* Department of Bioengineering, Zhengzhou University, Zhengzhou 450001, China *Corresponding author: Dr. Yanfeng GaoDepartment of Bioengineering, Zhengzhou University, 100 Science Road, Zhengzhou 450001, ChinaTel.: +86-371-67739057; fax: +86-371-67783235. E-mail: [email protected] Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential to the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely-studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9 V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-c. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02+ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201+, PLAC1+) in vitro. When immunized in HLA-A2.1/Kb transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope to the development of peptide vaccines against PLAC1-positive breast cancer.

S27 Keywords: Breast cancer, PLAC1, Cytotoxic T lymphocyte, Epitope, Immunotherapy

Influence of cholesterol redistribution on APP processing and its relevance to Alzheimer’s disease pathology Satyabrata Kar Departments of Medicine and Psychiatry, University of Alberta, Edmonton, Canada Evidence suggests that cholesterol by regulating amyloid precursor protein (APP) processing can influence the generation of b-amyloid (A b) peptide—the key player in Alzheimer’s disease (AD) pathogenesis. However, most of these observations arise either from cell culture studies using drugs having multiple effects or animal models with dietary modulation of cholesterol, which does not cross the blood–brain barrier. At present, it remains unclear how cells/neurons genetically modified to accumulate cholesterol can influence APP processing and A b production. To address this issue, we developed novel in vitro and in vivo genetic models overexpressing APP in the absence of NiemannPick type C1 (Npc1) protein, required for intracellular cholesterol transport, and analyzed the effects of cholesterol accumulation on APP/ 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, exacerbate glial pathology and to some extent influence extracellular A b deposition. Additionally, cholesterol accumulation in brain neurons of ANPC mice and in cultured N2a cells can differentially regulate levels and distribution of APP C-terminal fragments (CTFs) and Ab1–40/1–42 with no evident alterations in APP mRNA levels. This is accompanied by changed levels of APP processing enzymes in ANPC mouse brains as well as in cultured N2a cells. Collectively, these results suggest that cholesterol sequestration in neuronal cells can reduce longevity, exacerbate degeneration of neurons possibly via the production/accumulation of Ab and APP CTFs which can influence ADrelated amyloid pathology. (Supported by funding from CIHR).

Interactions of acidic pharmaceuticals with human serum albumin: insights into the molecular toxicity of the emerging pollutants J. B. Chen1,2, Y. L. Zhang1, X. F. Zhou2, Y. J. Qian2, and H. W. Gao2,* 1 State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, China 2 Key Laboratory of Yangtze River Water Environment for Ministry of Education College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, China *Corresponding author: [email protected]

Acidic pharmaceuticals such as diclofenac, clofibric acid, ketoprofen have been frequently detected in the environment. In order to investigate the toxicity of such emerging pollutants, their interactions with human serum albumin (HSA) were investigated by capillary electrophoresis and molecular spectrometry. The binding constants and sites of these acidic pharmaceuticals with HSA were obtained. The thermodynamic parameters, e.g. enthalpy change (DH) and entropy change

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S28 (DS) of these interactions were calculated in order to characterize the acting force between acidic pharmaceuticals and HSA. From the fluorescence and UV–Vis spectra, the fluorescence quenching of HSA is due to the formation of ground-state complex with the pharmaceuticals. The three-dimensional fluorescence confirmed that the conformation of HSA changed after the interactions with the pharmaceuticals. Thus, the interactions between acidic pharmaceuticals and HSA may influence on the normal activity of HSA.

Investigation of the effects of different epileptic activities on cell membrane proteins reveals diagnostic information Sevgi Turker1, Mete Severcan2*, Gul Ilbay3, and Feride Severcan4 Department of Biology1, Physiology3 Medical School of Kocaeli University, Kocaeli, Turkey Department of Electrical and Electronics Engineering2, Biological Sciences4, Middle East Technical University, Ankara, Turkey In the current study, the effects of convulsive (pentylenetetrazol treated) 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 without isolation procedure, requiring less time and sample. The 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 amide II modes give information about total protein concentration which were dramatically decreased in the mixed group. The alteration in the amide I to amide II ratio implies structural variations in the proteins of the system. Since amide I band arises from overlapping individual peaks assigned to different protein conformations like ahelix and b-sheet, this band was used to determine secondary structural changes in protein using neural networks (NNs). This chemometric method has been previously shown to be a powerful tool for secondary structure prediction. The findings revealed that for both epileptic groups there was a significant increase (p \ 0.05*) in random coil and significant decrease (p \ 0.05*) in beta sheet structures. Moreover, cluster analysis based on amide I band provided successful differentiation of the epileptic groups in between and the control samples. Consequently, FTIR spectroscopy together with NNs method introduces promising approach for diagnosis of epilepsy by detecting the impacts of different epileptic models on brain cell membrane proteins.

LL-37 and hBD-3 elevate Xog1p activity resulting in adherence decrease of Candida albicans onto plastic surface Hsin-Hui Huang1,2, Pei-Wen Tsai3, Ting-Jia Chang1, Tzu-Shan Chien1, Chuan-Yi Lan3, and Hao-Teng Chang1,4,5* 1

Graduate Institute of Molecular Systems Biomedicine, Department of Medical Laboratory Science and Biotechnology, 3 Institute of Molecular and Cell Biology & Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic Of China.Correspondence: [email protected] 4 Graduate Institute of Clinical Medical Science, 5 Graduate Institute of Basic Medical Science & Ph.D. Program for Aging, China Medical University, Taichung, Taiwan, Republic Of China 2

Candidiasis is the common cause of opportunistic fungal infection in immunocompromised patients. For instance, Candida albicans

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12th International Congress on Amino Acids, Peptides and Proteins (C. albicans) causes oral thrush and vaginal candidiasis. On the cell walls of C. albicans, Xog1p serves as the major b-1,3-exoglucanase, which participates in the metabolism of b-glucan. The previous studies showed antimicrobial peptides (AMPs) such as LL-37 and hBD-3 could inhibit the cell adhesion and growth. We proposed that Xog1p is a target for AMPs binding, and involves in the C. albicans adherence. Sixty percent of the adherence of C. albicans on the plastic surface would be inhibited by LL-37 and hBD-3 in a dose dependent manner. Pull down assay and ELISA demonstrated that recombinant Xog1p-6H and deletion fragments could interact with both AMPs in vitro. Moreover, the b-1,3-exoglucanase activity of Xog1p-6H would be enhanced around twofolds by LL-37 and hBD-3. Thus, the elevated b-1,3-exoglucanase activity of Xog1p might destroy the cell wall integrity and decrease the C. albicans adhesion. To further investigate the hypothesis, the C. albicans was treated with 2 lM Xog1p-6H for 2 h at room temperature, and we found a decrease of the C. albicans adherence for 3.5-folds. Altogether, Xog1p is a cell wall protein serving as a target interacting with LL-37 and hBD-3, and the enhanced b-1,3exoglucanase activity leads to reduce the C. albicans adherence onto plastic surface.

Mixed linear approaches of mapping QTL, QTS and QTT with gene–gene interaction and gene-environment interaction for complex traits Jun Zhu, Zhihong Zhu, and Chenhao Zhang Institute of Bioinformatics, Zhejiang University, Hangzhou, 310058 It has been a challenge to develop efficient statistical methods for mapping genes underlying complex traits. Here, we report the development of mixed linear approaches that integrate the detection of gene-by-gene and gene-by-environment interaction for quantitative trait loci (QTLs) based on microsatellite markers, for quantitative trait nucleotides (QTNs) based on SNPs, and for quantitative trait transcripts (QTTs) based on variation in expression of transcripts. The genetic models include cofactors (i.e., sex, age), genetic main additive (A) and dominant (D) effects, epistasis effects including additive by additive (AA), additive by dominance (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 main effects, epistasis effects, and gene-environment interaction effects. The variation contributing to these effects can be estimated. Mapping software (QTLNetwork V3.0) has also been developed, which can be used under different operating systems. To illustrate our proposed approach, both phenotypic and transcriptome data on anxiety assay with alcohol and stress treatments from 71 strains of the BXD family were analyzed. Using 506 microsatellite markers, we detected eight QTLs including three main-effect QTLs (h2 = 0.27) and three pairs of epistasis QTLs (h2 = 0.23). By using 2,320 SNPs, 17 QTNs were detected for seven main-effect QTNs (h2 = 0.25) and six pairs of epistasis QTNs (h2 = 0.22). Further, we also used a small mapping population (188 individuals in 5 treatments), and detected one main QTT (h2 = 0.07) and two pairs of epistasis QTTs with relatively small effects. The mapping results by three methods were compared for chromosomes 1 and 11. Three QTSs were mapped onto the flanking marker intervals of three QTLs detected. The QTT was mapped into the QTLs on chromosome 1. In conclusion, the approaches we developed here are capable of identifying causal genes/loci associated with complex traits.


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Nanowired cerebrolysin restores imbalance of excitatory and inhibitory amino acid neurotransmitters in the CNS following hyperthermia induced brain pathology

cop_20e with diseases and it could be named as nanomedicine applied to difficult-to-treat diseases. As known, in this field of research, the most important goal to be reached is an increase in selectivity and specificity of drug action. Several results with stimulating findings in preclinical or clinical phases have been reached by using nanocarriers, delivering agents to targeted pathologies, and among them, it is known that neuropathologies represent a stimulating issue. In fact, the pharmaceutical treatment of Central Nervous System (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, non-invasive drug delivery systems for CNS are actively studied. In fact, the development of new delivery systems (nanoparticles and liposomes) started with the discovery that properly surface-engineered colloidal vectors, with a diameter around 200 nm, were shown to be able to cross the blood–brain Barrier without apparent damage, and to deliver drugs or genetic materials into the brain. During this talk, an overview will be presented considering the most recent literature results of nanomedicine applied to brain diseases, carried out with all the most popular kinds of nanoparticulate systems, focusing in particular on immunenanoparticles and peptide-decorated nanosystems able to target the CNS, with in vivo and in vitro evidences investigating the pathway for BBB crossing and CNS localization of engineered nanoparticles. The brain localization and the multi-modal pathways for BBB crossing highlighted the endocytosis as preferential pathway; moveover, in vitro test on hippocampal neurons showed the presence of cell-to-cell transport of nanoparticles.

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Dafin F Muresanu*, 2Ranjana Patnaik, 3Aruna Sharma, and 3Hari S Sharma 1

Department of Neurology, University of Medicine & Pharmacy, Cluj-Napoca, Romania 2 Department of Biomaterials, School of Biomedical engineering, Institute of technology, Banaras Hindu University, Varanasi-221005, India 3 Department of Surgical Sciences, Anesthesiology & intensive Care Medicine, University Hospital, Uppsala University, SE-75185 Uppsala, Sweden. Email: [email protected] Previous reports from our laboratory showed that hyperthermia induces marked increase in excitatory amino acid neurotransmitters, e.g., glutamate and aspartate in the cerebral cortex, hippocampus, thalamus, and hypothalamus and in spinal cord at the time of neuronal damages and blood–brain barrier (BBB) dysfunction. At this time, inhibitory amino acids such as GABA or glycine showed marked decrease in the above CNS regions. This suggests that an imbalance between excitatory and inhibitory amino acids in the CNS causes brain damage. Since cerebrolysin is a mixture of various neurotrophic factors and induces marked 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 hyperthermia in relation to brain damage. Rats were subjected to 4 h heat stress (HS) in a biological oxygen demand (BOD) incubator and amino acids were analyzed using HPLC in the above brain regions. Separate groups of rats were treated with cerebrolysin (2.5 ml/kg, i.v.) with or without nanowiring after 30, 60 and 90 min of HS. Rats received cerebrolysin after 30 min markedly thwarted the increase in glutamate and aspartate and reduction in GABA and glycine in the CNS resulting in neuroprotection. However, 60 or 90 min after cerebrolysin administration did not affect the amino acid levels and/or brain damage. On the other hand nanowired cerebrolysin if given at 60 or 90 min after HS, thwarted this amino acid imbalance and induced profound neuroprotection. These results show that nanowiring of cerebrolysin enhances its neurotherapeutic efficacy in hyperthermia induced neuroregeneration probably through modulating amino acid neurotransmission, not reported earlier.

Nanomedicine in neuro-diseases: engineered nanoparticulate systems for the drug brain delivery Giovanni Tosi*1, Lucia Bondioli1, Barbara Ruozi1, Andreas Grabrucker2, Antonietta Vilella3, Michele Zoli3, Francesco Rivasi4, Maria Angela Vandelli1, and Flavio Forni1 1

Department of Pharmaceutical Science, Univ. of Modena and Reggio Emilia, Modena, Italy 2 Department of Psychiatry and Behavioral Sciences, Stanford Univ., Stanford, CA, US 3 Department of Biomedical Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy 4 Department of Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy In the last years, the application of ‘‘nanotechnology ‘‘to the field of ‘‘medicine’’ surely represented the most innovative strategy to

Nanowired delivery of HOE-140 enhances neuroprotection in spinal cord injury and downregulates heat shock protein expression 1

Linyuan Feng*, 2Aruna Sharma, and 2Hari S Sharma

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Department of Neurology, Norman Bethune International Peace Hospital Army, 398 Zhong Shan Road West, Shi Jia Zhuang, Hebei Province, China 50082, Email: [email protected] 2 Department of Surgical Sciences, Anesthesiology & intensive Care Medicine, University Hospital, Uppsala University, SE-75185 Uppsala, Sweden. Email: [email protected] Previously, we reported that blockade of bradykinin B2 receptor with 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 after 30 min SCI did not affect nNOS expression or cell injury. Since SCI induces profound oxidative and cellular stress, in this investigation we examined effects of HOE-140 on heat shock protein expression in SCI. Furthermore, the role of nanodrug delivery of HOE-140 in attenuating cell damage and stress reaction was also examined in our rat model. SCI was produced by a longitudinal incision over the right dorsal horn of the T10-11 segments of the cord under Equithesin anesthesia. The animals were allowed to survive 5 h after injury. In separate group of rats HOE-140 was delivered tagged with TiO2 nanowires (0.1 mg/kg, i.v.) 30 min or 60 min after trauma and heat shook protein (HSP 72 kD), nNOS expression, cell injury and edema formation was examined using standard procedures. HOE140 without nano-labeling was used as control. Treatment with HOE-140 without tagging with nanowires did not influence massive upregulation of HSP 72 kD at 5 h after trauma or cellular damage. On the other hand, nano-drug delivery of HOE-140 markedly attenuated HSP expression, nNOS upregulation, edema formation and cell injury when given 30 or 60 min after injury. However,

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S30 TiO2 nanowires alone did not influence pathophysiology of SCI or HSP expression. These observations are the first to show that nanodrug delivery of HOE-140 even at a later stage of SCI is able to thwart stress protein response, nNOS expression and cell injuries. This indicates that nanowiring of drugs could enhance their therapeutic potentials in SCI.

Novel ionic polymer-platinum nano-composite for real-time control of drug metabolism Jun Lu1, Sang-Gyun Kim3, Sunwoo Lee4 and Il-Kwon Oh2 1

Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, China, corresponding author e-mail: [email protected] (J. Lu) 2 Division of Ocean System Engineering, School of Mechanical Aerospace and Systems Engineering, Korea Advanced Institute of Science and Technology, 335 Gwahangno (373-1 Guseong-dong), Yuseong-gu, Daejeon, 305-701, Republic of Korea, Corresponding author e-mail: [email protected] (I.K. Oh) 3 Membranes & Separation Research Center, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong, Daejon 305-600, Republic of Korea 4 Department of Chemistry, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwang-Ju, 500-757, Republic of Korea NADPH plays an important role in drug metabolism, which is an obligatory requirement for cytochrome P450-dependent drug oxidation. NADPH levels vary periodically in time in neutrophils, macrophages, and certain tumor cells, and it oscillates in an approximate sinusoidal pattern with a period between 3 and 4 min in all of these cells. In this study, a novel nano-composite with biocompatibility, composed of styrene-maleimide alternating polymer, vinylidene fluoride polymer and platinum, was fabricated, and its electromechanical properties were investigated at ultra-low frequency. Under the stimuli of sinusoidal wave signals with frequency from 0.1 to 0.005 Hz, the nano-composite with glycerol as a solvent displayed excellent harmonic response, and its deformation was observed to be improved continuously by a selfactivating process along with the time increased. The experiments also showed that the treatment conditions by glycerol had significant effect in determining its final response properties. The best harmonic response to the signals with ultra-low frequency was achieved when the nano-composite was immersed in anhydrous glycerol at 100°C for 4 days and then exposed to air for 8–10 days at ambient temperature. The current study on ionic polymer-platinum composite suggested it could be used in monitoring the metabolic oscillations so as to realize the real-time control of drug metabolism. Acknowledgments: Supported by the Korea Science and Engineering Foundation NRL Program grant funded by the Korea government (MEST) (No. R0A-2008-000-20012-0), National Natural Science Foundation of China (No. 50973089), and the Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

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Olfactory ensheathing cell neurorestorotherapy for amyotrophic lateral sclerosis patients: Benefits from multiple transplantations Hongyun Huang Beijing Hongtianji Neuroscience Academy, Beijing, 100144, People’s Republic of China Center for Neurorestoratology, Beijing Rehabilitation Center, Beijing, People’s Republic of ChinaDivision of Neurorestoratology, Yuquan Hospital, Tsinghua University, Beijing, People’s Republic of China E-mail address: [email protected] Our previous series studies have proved that OEC transplantation appears to be able to slow the rate of clinical progression after olfactory ensheathing cell (OEC) transplantation in the first four months and cell intracranial (key points for neural network restoration, KPNNR) and/or intraspinal (impaired segments) implant are benefit for the patients (both subtype of bulbar onset and limb onset) with amyotrophic lateral sclerosis (ALS). Here we report the results of cell therapy in patients with ALS on basis of the long-term observation following multiple transplants. From March of 2003 to January of 2010, 507 ALS patients received our cellular treatment. Among them, 42 patients underwent further OEC therapy by the route of KPNNR for 2 or more times (2 times in 35 patients, 3 times in 5 patients, 4 times in 1 patient, and 5 times in 1 patient). The time intervals are 13.1 (6–60) months between the first therapy and the second one, 15.2 (8–24) months between the second therapy and the third one, 16 (6-26) months between the third therapy and the fourth one, and 9 months between the fourth therapy and the fifth time. All of the patients got partial neurological functional recovery after each cell-based administration. Firstly, the scores of ALS Functional Rating Scale (ALS-FRS) and ALS Norris Scale increased by 2.6 ± 2.4 (0–8) and 4.9 ± 5.2 (0–20) after the first treatment, 1.1 ± 1.3 (0–5) and 2.3 ± 2.9 (0–13) after the second treatment, 1.1 ± 1.5 (0–4) and 3.4 ± 6.9 (0–19) after the third treatment, 0.0 ± 0.0 (0–0) and 2.5 ± 3.5 (0–5) after the fourth treatment, and 1 point after the fifth cellular therapy, which were evaluated by independent neurologists. Secondly, majority of patients have achieved improvement in EMG assessments after the first, second, third and fourth cell transplantation. After the first treatment, among the 42 patients, 36 (85.7%) patients’ electromyogram (EMG) test results improved, the rest 6 (14.3%) patients’ electromyogram results showed no remarkable change. After the second treatment, of the 42 patients, 30 (71.4%) patients’ EMG results improved, 11 (26.2%) patients no remarkable change, 1 (2.4%) patient worse. After the third treatment, out of the 7 patients, 4 (57.1%) patients improved, the remaining 3 (42.9%) patients no change. Thirdly, the patients have recovered breathing ability partially which proven by pulmonary functional tests. After the first treatment, 20 (47.6%) patients’ pulmonary function ameliorated. After the second treatment, 18 (42.9%) patients’ pulmonary function improved. After the third treatment, 2 (28.6%) patients’ some pulmonary function recovered. After the fourth treatment and the fifth treatment, patients’ pulmonary function did not reveal significant change. The results show that the multiple cellular therapy definitely serves as a positive role in the treatments of ALS, and the repeated and periodic cell-based therapy which patients benefit from is strongly recommended for better controlling this progressive deterioration disorder.


Pain Neurorestoratology: a new idea for intractable ache? Lin Chen1-3*, Hongyun Huang1-3, Huancong Zuo3, and Hari Shanker Sharma4 1

Center for Neurorestoratology, Beijing Rehabilitation Center, Beijing, China; 2 Beijing Hongtianji Neuroscience Academy, Beijing, China; 3 Division of Neurorestoratology, Yuquan Hospital, Tsinghua University, Beijing, China; 4 Laboratory of Cerebrovascular Research, Department of Surgical Sciences Anesthesiology & Intensive Care Medicine, University Hospital, Uppsala University Pain Neurorestoratology (PN) is an important branch of Neurorestoratology, which study how to intervene pain, especially refractory neuropathic pain. The fundamental principles of PN are to promote nerve repair, immune regulation, improve microcirculation, and lessen clinical symptoms. New treatment options for PN includes: cellular therapies, close injection of neurorestorative drugs, ozone, local muscle deep hyperthermia, and neurotransmitter modulating agents. Cell medicine and transplantation is the core of this comprehensive PN therapy. Cells for nerve repair include glial cells (olfactory ensheathing cells, Schwann cells, etc.), mesenchymal cells (umbilical cord/blood mesenchymal stromal cells, bone marrow stromal cells, etc.), and chromaffin cells. The ways of transplanting cells include local parenchymal implanting, intrathecal injection and or intravascular routes. Four main directions of PN: (1) to treat chronic intractable pain with no way available currently, (2) to improve the outcome of traditional pain therapy, (3) to relief pain together with nerve system repair and vascular and blood circulation reconstruction in the patients with central pain after spinal cord trauma, stroke, neurodegenerative impairments, etc., (4) to provide new ideas for current effective treatments. For instance, can we treat the trigeminal neuralgia by cell therapy instead of the surgical vascular decompression? Could we control spinal cord pain using cell therapy with electric stimulation? Can we treat the acute pain by cell therapy? Here, we review the most recent clinical results of the PN therapy in patients with pain due to spinal cord injury, stoke, diabetes, aging, muscle strain, and sports injuries. Taken together, the proposing and implementing of the concept should be helpful for greatly improving the level of pain therapeutics.

Pancreatic b cell dysfunction in diabetes through induction of RAGE expression Dong Han*, Yasuhiko Yamamoto, Seiichi Munesue, Soh Motoyoshi, Hidehito Saito, Takuo Watanabe, and Hiroshi Yamamoto Department of Biochemistry and Molecular Vascular Biology, Kanazawa University Graduate School of Medical Science Objective: Elevated free fatty acids (FFA) and uncontrolled hyperglycemia could contribute to pancreaticb cell failure in diabetes. Pattern recognition receptor (PPRs) including receptor for advanced glycation end-products (RAGE) and toll-like receptor (TLR) 2 and 4 could be the main sensors of the triggers and danger signals inb cell dysfunction. In this study, we examined the

S31 relationship between PPRs, especially RAGE, and b cell failure in type 2 diabetes. Methods and results: Pancreatic islets were isolated and dispersed into single cells, from diet-induced obesity (DIO), ob/ob (leptindeficient), db/db (leptin receptor-deficient), RAGE-null (RAGE-/-), and the control (WT) mice, and then analyzed by flow cytometry (BD FACS Aria II) with insulin, RAGE and TLR2 and 4 antibodies. RAGE expression was detected in insulin-positive b cells from ob/ob and db/db mice, but not from DIO and WT as well as RAGE-/- mice, suggesting a possible linkage between inadequate leptin receptor signalling and RAGE expression. TLR2 was only detected in non b cell clusters. The expressions of RAGE and TLR2 were induced by palmitate in MIN6 cells, a mousebcell line, in a dose-dependent manner. Summary: RAGE expression on pancreaticb cells was demonstrated in diabetic ob/ob and db/db mice. FFA elevation with concomitant AGE formation may causeb cell damage through induction of RAGE expression in type 2 diabetes.

Pressure-crystallized polymer micro-spheres as delivery systems for therapeutic proteins Jun Lu1, Jiaojiao Tian1, Daopeng Zhang1, Daikun Xi1, and Rui Huang2 1 Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, China, corresponding author e-mail: [email protected] (J. Lu) 2 College of Polymer Materials Science and Engineering, Sichuan University, Chengdu 610065, Sichuan, China

Micrometer-sized polymer spheres are promising in controlledrelease drug delivery systems, which protect protein and peptide structures from rapid degradation in the bloodstreams and capture by non-specific receptors. In this study, two polymers, bisphenol-A polycarbonate (BAPC) and poly (ether ether ketone) (PEEK), were crystallized at high pressure with a piston-cylinder apparatus, and were investigated using wide-angle X-ray diffraction (WAXD), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The results showed that crystalline microspheres of BAPC and PEEK with folded-chain substructures were easily exposed and isolated with selective etching techniques. The 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 amorphous region was totally etched away. As for PEEK microspheres, they were endowed with an elliptical outline, and were consisted of flake-like lamellae with rugged surfaces. Further morphological observations suggested that such PEEK macro-spheres might be evolved from a novel dendritic crystal, which was isothermally crystallized and then accumulated into the final agglomerations. The as-prepared polymer micro-spheres, with high surface area, large pore volume, hydrophilic character, chemical and thermal stability, unique morphologies and toxicological safety, may diversify niche applications in achieving a controlled and sustained release system of proteins, as well as their adsorption and separation. Acknowledgments: This work was supported by the National Natural Science Foundation of China (Grant Number 50973089),, as well as the Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

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Proteins from Erwinia asparaginase ErwinaseÒ and E.coli asparaginase 2 MEDACÒ for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear

12th International Congress on Amino Acids, Peptides and Proteins purified and the structure of the protein will be determined as a potential biocontrol candidate. Acknowledgments: This work was supported by grants from the NKBRPC (2003CB114204 and 2006CB101902), NKPC (2004BA901A36), RFDP (20104601110004), CARS-34-GW8 and KYQD1006 (China).

Narkhyun Bae, and Gert Lubec L-Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; UniProtKB accession number P06608 [ErwinaseÒ]) and L-asparaginase 2 from E.coli (ASPG2_ECOLI; UniProtKB accession number P00805 [MedacÒ]), both L-asparagine amidohydrolases, are widely used for the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the protein chemistry of the medical products sold. Mass spectrometry data on ASPG_ERWCH and ASPG2_ECOLI have not been published so far and herein a gel-based proteomics study was performed to provide information on sequence and modifications of the commercially available medical products. ASPG_ERWCH and ASPG2_ECOLI were applied onto two-dimensional gel electrophoresis, spots were in-gel digested with several proteases and resulting peptides and protein modifications were analysed by nano-ESI-LC– MS/MS. Four spots were observed for ASPG_ERWCH, six spots were observed for ASPG2_ECOLI and the identified proteins showed high sequence coverage without sequence conflicts. Several protein modifications including technical and posttranslational modifications were demonstrated. Protein modifications are known to change physicochemical, immunochemical, biological and pharmacological properties and results from this work may challenge re-designing of the product including possible removal of the modifications by the manufacturer because it is not known whether they are contributing to the serious adverse effects of the protein drug.

Purification and preliminary function of diseaseresistance protein from Brevibacillus brevs A57 Tingya Yang, Wenbo Liu, Xiaoxi Lan, Lin Li, Xiang Li, Liang Sun, Xiaoyan Wu, Weiguo Miao*, and Fucong Zheng* College of Environment and Plant Protection, Hainan University, Haikou 570228 China *Corresponding author Weiguo Miao E-mail: [email protected]; Fucong Zheng E-mail: [email protected] Brevibacillus brevs A57 was isolated from cotton rhizosphere soil. It has been confirmed for A57 to have stronger antagonism against several important plant pathogens, such as Verticillium dahliae, Fusarium oxysporum f.sp. infectum, and Colletotrichum gloeosporioides Penz. The examination of antagonistic mechanism under light microscope showed that the antagonistic substance of A57 appeared to inhibit pathogens by leading to mycelium rupture, distortion, abnormality, and inhibition bourgeon of conidiophore, and leading to abnormality of conidiophore. Antifungal factor from A57 was further identified as a kind disease-resistance protein by ammonium sulfate precipitation method. This protein enable to accumulation in PDA (potato dextrose agar) medium, or in PDA medium against a pathogen. The accumulation content of A57 protein indicated higher peak in about 3 days, producing clear blank band between paper disk containing the protein and a pathogen. The protein could be isolated and purified from the medium with the blank band by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). It was able to inhibit the growth of pathogen, and promote plant development. In the future, the novel antifungal protein will be further

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Real-time studies of ultrafast electron transfer reactions of carbon tetrachloride with amino acids (tryptophan and tyrosine) and dGMP: free radical formation and implications for medicinal chemistry Ting Luo and Qing-Bin Lu* Departments of Physics and Astronomy, and Departments of Biology and Chemistry, University of Waterloo, Ontario, Canada Femtomedicine is an exciting emerging frontier, which involves a fusion of ultrafast (femtosecond, 1 fs = 10-15 s) laser techniques with biomedical methods to address important biological processes with close relevance to diseases, diagnosis and treatment. We have recently applied this innovative approach to obtain real-time observations of molecular reactions forming free radicals, which are highly relevant to the molecular pathways for DNA damage and cell death, the molecular mechanisms of cancer therapies and the development of new anticancer agents. Some halogenated compounds with a high affinity to DNA have been explored as radiosensitizers for cancer therapy, while carbon tetrachloride (CCl4) has served as a model substance for studying the mechanisms of hepatoxicity. It is generally believed that the mechanism of CCl4 hepatoxicity involves the cleavage of a carbonchlorine bond by the enzyme cytochrome P450 (CYP), generating free radicals such as CCl3 and CCl3OO. However, the mechanism for the 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 fsTRLS studies of the reaction dynamics of CCl4 with amino acids (tryptophan and tyrosine) and nucleotides. Our results show direct evidence of electron-transfer reactions of CCl4 with amino acids, leading to the formation of free radicals. This observation unravels a molecular pathway for CCl4 activation, i.e., through direct targeting the proteins at their Trp/Tyr sites, and provides a mechanistic understanding of the negative genotoxic and mutagenic effects of CCl4.

Redistribution of calcitonin-gene related peptide (CGRP) in the brain and heart tissues after cardiac arrest. Neuroprotection by methylene blue treatment 1

Lars Wiklund*, 2Ranjana Patnaik, 1Aruna Sharma, and 1Hari S Sharma 1 Department of Surgical Sciences, Anesthesiology & intensive Care Medicine, University Hospital, Uppsala University, SE-75185 Uppsala, Sweden. Email: [email protected] 2 Department of Biomaterials, School of Biomedical Engineering, Institute of technology, Banaras Hindu University, Varanasi-221005, India

Calcitonin-gene related peptide (CGRP) is altered in the brain, aorta, heart muscles and liver in spontaneously hypertensive rats. Furthermore, low dose of CGRP treatment in cardiac arrest results in

12th International Congress on Amino Acids, Peptides and Proteins improvement of heart function. However, the role of CGRP in cardiac arrest induced brain pathology is not understood. In present investigation, we measured CGRP in the cerebral cortex 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. The normal CGRP content in the cortex was 55 ± 2 pg/mg tissue, 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 decline in cortical CGRP content (14 ± 3 pg/mg) whereas, cardiac tissues showed a slight but significant increase (left 27 ± 4 pg/mg; right 23 ± 2 pg/mg). These changes in CGRP were further altered following 60 min after restoration of spontaneous circulation (ROSC) (brain CGRP 8 ± 2 pg/mg, ventricles left 34 ± 4; right 30 ± 2 pg/ mg). At 180 min after ROSC the heart tissues showed significant decline (left ventricle 8 ± 2; right 12 ± 4 pg/mg) whereas, cortical CGRP showed a mild elevation (64 ± 6 pg/mg) from the control values. Pretreatment with methylene blue significantly attenuated these changes in CGRP at 180 min as compared to the control cardiac arrest group (brain 40 ± 6 pg/mg; left ventricle 23 ± 4; right 33 ± 6 pg/ mg). Since neuronal changes are significant following 30–180 min after cardiac arrest and methylene blue markedly reduced these neuronal damages at 180 min, our results are the first to demonstrate that alterations in CGRP in brain and heart are crucial for neurological impairments. Further studies are needed to understand the molecular mechanisms of CGRP induced brain pathology in cardiac arrest.

S100 proteins and RAGE in cancer Jens Pietzsch Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmacy, Dresden, Germany E-mail: [email protected] RAGE, the receptor for advanced glycation endproducts, is a pattern recognition receptor that belongs to the immunoglobulin superfamily. In homeostasis, RAGE is expressed ubiquitously high only in the lung, and moderate to low in a wide range of cells such as endothelial cells, mononuclear phagocytes, smooth muscle cells, mesangial cells, and certain neurons. It is found either as a membrane-bound or soluble protein that is markedly and quickly upregulated by stress in both epithelial and inflammatory cells. RAGE binds multiple ligands, including advanced glycation end products (AGEs), amyloid fibrils, amphoterin, and various members of the S100 family of EF-hand calcium-binding proteins such as S100A4, S100A8/A9, S100A11, S100A12, and S100B. Activation and upregulation through a positive feedback loop of RAGE and, subsequently, perpetual RAGE S100 engagement effects the activation of diverse signaling cascades that initiate and stimulate chronic stress and survival pathways, depending on the distinct interacting S100 protein, environment, and developmental stage. This can result in chronic inflammation and is supposed a setting in which predominantly epithelial malignancies can arise. Therefore, exploring the function of RAGE and its panoply of S100 ligands in the setting of inflammation is critically important in understanding the role of this receptor in carcinogenesis and metastasis, but also in other pathological conditions such as radiation therapy-related vascular dysfunction. In this review, we summarize novel findings on RAGE S100 protein interaction and subsequently triggered signaling cascades from published reports and own ongoing studies. In particular, a comprehensive evaluation of S100 protein metabolism in rodent models using fluorine-18 radiolabeled recombinant S100 proteins and small animal positron emission tomography, further underlining the role of RAGE S100 protein interaction in

S33 normal and disease states in vivo, is demonstrated. These recent experiments also support the potential of RAGE and its S100 ligands as attractive theragnostic markers and targets, respectively.

Serum amino acid profiles and clinical purpose in patients undergoing CAPD Zheng li and Huang fengxian The First Affiliated Hospital of Sun-Yatsen University Fasting serum amino acid levels were measured in seven diabetic male, fourteen nondiabetic male, six diabetic female, and nondiabetic female patients on CAPD (RRF C 2 ml/min). Comparison of their serum amino acid values with fourteen controls showed that CAPD did not restore the serum amino acid levels of these patients to normal. High levels of phenylalanine and low levels of tryptophane showed up in essential amino acids of four group patients; among non-essential amino acids, taurine, glycine, aspartic acid, citrulline, glutamic acid, proline, ornithine were higher significantly, but tyrosine was lower significantly. Other 21 species of amino acids: asparamide, hydroxyproline, ethanolamine, 1-methyl-histidine, ASA arginine succinic acid, 3-methyl-histidine, Carnosine, c-aminobutyric acid, hydroxylation citrulline, a-amid adipic acid, b-aminoisobutyric 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 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.

Stochastic resonance therapy (SRT) and tryptophan metabolism Berthold Kepplinger1,2, Halina Baran2, Brenda Sedlnitzky-Semler2, Nagy-Roland Badawi1 and Helene Erhart1 1 Department of Neurology, Neuropsychiatric Hospital Mauer, MauerAmstetten 2 Neurochemical Laboratory, Karl Landsteiner Research Institute for Pain Treatment and Neurorehabilitation, Landesklinikum Mauer-Amstetten, Austria Corresponding author: [email protected]

Stochastic resonance therapy (SRT) is applied for treatment and rehabilitation of patients with various neurological and mental disorders e.g. Parkinson disease, Alzheimer’s, Multiple Sclerosis, stroke, depression and schizophrenia, also for the treatment of low back pain and also for prophylaxis of osteoporosis. The effectiveness of SRT in treatment of these diseases has been reported however the mechanism(s) of action is complex and needs further explanations. An involvement of tryptophan metabolism along kynurenine pathway has been demonstrated in various experimental and/or human neuroinflammatory and neurodegenerative diseases and during the aging process. Kynurenine metabolites show an ability to modulate the neuro-glia biochemical processes and they are involved significantly in neurophysiological and neuropathological events. The aim of this study was to search the influence of SRT as an exercise activity, on tryptophan metabolites in the serum of healthy subjects. The concentration of L-tryptophan, L-kynurenine, kynurenic acid and anthranilic acid was searched in the serum of volunteers 1 min before 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. L-tryptophan, L-kynurenine and kynurenic acid were time dependent significantly lowered up to 60 min of SRT. Changes of anthranilic acid were characterized by transient decrease accompanied by up a mild enhancement at 60 min. Revealed data demonstrate that exercise using SRT method affects tryptophan metabolism in the periphery significantly and it is likely that these influences take places in the central nervous system, as well. The lowering of tryptophan would suggest generally an activation of serotonin, protein and bone biosynthesis and might affect positively the immune system. The lowering of kynurenic acid due to exercise might increase the neurotransmission of dopamine- and cholinergic systems and therefore activates the anti-depressive, anti-dementive and anti-aging processes 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.

mitomycin C, known antitumor agents are identified as novel inhibitors and subversive substrates. Possible mechanism of subversive substrate is investigated. Both of these compounds show significant effect on redox homeostasis of the parasite and high leishmanicidal activity. Moreover, reactive oxygen species (ROS) determination using CM-H2DCFDA probe employing fluorescence spectroscopy and flow cytometric analysis show increase ROS generation in presence of these compounds. Additionally, our flow cytometric analysis also indicates change in cell morphology due to ROS. Our toxicity studies as well as available toxicity data in literature indicate these compounds to have acceptable toxicity in limited dose. The current report points out toward potential application of doxorubicin and mitomycin C as therapeutics against leishmaniasis.

Study on effects of Baicalin Bai on the repaired of injured podocytes in diabetic nephropathy rats

Daren Wang1, Alex Kuan2, David Hui3 and Dao Pan1,4

Targeted delivery of IDUA across the blood–brain barrier for MPS I disease

1

Division of Experimental Hematology and Cancer Biology Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA 3 Department of Pathology 4 Department of Pediatrics, University of Cincinnati, Cincinnati, OH 45219 2

SU Ning, ZHAO Ping, ZHOU Le-quan, DU Biao-yan, LUO Hui, and LANG Jian-ying School of Basical Science, Guangzhou University of TCM, Guangzhou 510006, China Objective: To Study Effects of Baicalin Bai on the repaired of injured podocytes and FN and IV-C expression in diabetic nephropathy (DN) Rats, which to explore its preventive and therapeutic effect on treating DN. Methods: DN rat model was established substances in kidney of rat of streptozotocin. DN model rats were randomly divided into model group and Baicalin treatment group, Normal control group was set up additionally. Rats in Baicalin treatment group were injected Baicalin Bai solution 40 mg/kg/day abdominally, rats in the model group were injected water 1 ml/rat/day abdominally, 6 weeks later, the weight of their bodys and kidneys was monitored. The right kidney tissues was fixed in 3% glutaraldehyde solution, and electronic scopy was performed. The left kidney tissues were fixed in 10% formalin solution, and pathological scopy was performed. Observe the morphology changes in podocytes and FN and IV-C expression in kidneys. Results: The kidney index decreased in the Baicalin treatment group, and there was significant difference compared to the model group, (p \ 0.05); under electric scopy, incrassation injured podocytes; expression of FN and IV-C ware decreased, and there was significant difference compared to the model group, (p \ 0.05). Conclusion: Baicalin reduce the expression of FN and IV-C in glomerular or tubule, to repaired podocytes, witch prefect the kidneys in DN Rats.

Subversive substrates of trypanothione reductase of Leishmania parasite: an alternative approach for chemotherapy Anil Kumar Shukla and Vikash Kumar Dubey* Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India- 781039, E-mail: [email protected] Trypanothione reductase is a key enzyme of unique redox metabolism of Leishmania parasite and validated drug target. Doxorubicin and

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Mucopolysaccharidosis (MPS) type I, resulting from the deficiency of the enzyme a-L-iduronidase (IDUA), is one of the most common lysosomal storage disorders affecting the central nervous system (CNS) that cannot be cured. We investigate in this study if the fusion of receptor binding domain (Rb) of Apolipoproteins (apo) to IDUA can enable the modified protein to bind members of low-density lipoprotein receptor family (LDLRf) on BBB and transcytose to the CNS. To determine the accessibility of IDUA protein for genetic modification, we first evaluated myc-tag IDUA using Western blot analysis, immunoprecipitation and IDUA enzyme assay, and identified IDUA3’Myc not only retained normal biological function, lysosomal enzyme trafficking, and endogenous receptor-mediated uptake pathway, but also acquired additional myc-antibody binding ability. We then constructed seven fusion IDUA-Rb candidates containing various amino acid residues within the receptor-binding domains of apolipoproteins, and evaluate receptor-specific up-take pathway in cells that overexpressed LDLR-associated protein 1 (LRP1) (highly expressed on BBB). Two out of seven fusion protein candidates introduced LRP1-specific protein uptake. The specific binding of fusion IDUA-Rb to LRP1 was further confirmed by a pause-chase assay. Using an in vitro BBB model, these two candidates could introduce IDUA levels significantly higher than 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 liver-specific expression of fusion proteins. Elevated IDUA activities (22–120-fold of normal plasma IDUA levels) were found in plasma of all MPS I mice 2 days after injection. Moreover, capillary-depleted brain tissues from well-perfused animals injected with IDUA-Rb exhibited significantly higher IDUA activities than those from IDUA3Myc controls. Immunofluorescent study has identified IDUARb positive cells mostly in non-endothelium perivascular cells, neurons, and less so in astrocytes of cerebra of injected mice. In summary, we have identified two Rb candidates by in vitro and in vivo evaluation that could mediate efficient IDUA delivery across the BBB via LRP1 receptor mediated transcytosis, thus open a door for novel and non-invasive approaches in treating brain diseases.


Targeting COX-2 for anticancer drug development, tumor radiosensitization, and molecular imaging of cancer Frank Wuest Department of Oncology, University of Alberta, Edmonton, Canada. E-mail: [email protected] Two isoforms of cyclooxygenase (COX-1 and COX-2) control the complex conversion of arachidonic acid to prostaglandins and thromboxanes, which trigger as autocrine and paracrine chemical messengers many physiological and pathophysiological responses. As a housekeeping enzyme, COX-1 is constitutively expressed in resting cells of most tissues, whereas COX-2 is an inducible isoform which is significantly upregulated as part of acute and chronic inflammatory conditions. Moreover, various immunohistochemical studies have confirmed COX-2 overexpression in several malignant or pre-malignant human tumors including colon, breast, lung, gastric 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 evident role of COX-2 in carcinogenesis has stimulated research activities towards molecular targeting of COX-2 and the COX-2 pathway as a promising strategy for the prevention and treatment of solid tumors. This included various preclinical and clinical studies using combined treatments of either chemotherapy or radiotherapy with COX-2 inhibitors. Another recent development is aimed at the development of non-invasive imaging probes for assaying COX-2 expression in vivo as a potential valuable diagnostic tool to identify and select cancer patients for combined tumor therapy with selective COX-2 inhibitors. In addition, molecular imaging of COX-2 could also be applied to monitor disease progression and to evaluate the efficacy of therapeutic interventions. The present review will survey recent research activities towards molecular targeting of COX-2 for molecular imaging and therapy of cancer. This will include the discussion of combined chemo- and radiotherapy with COX-2 inhibitors, and the development of molecular probes for imaging COX-2 expression in vivo.

The critical role of glutamic acid in chronical beryllium disease Shaodong Dai, Guinevere A. Murphy, Frances Crawford, Douglas G. Mack, Michael T. Falta, Philippa Marrack, John W. Kappler, and Andrew P. Fontenot Integrated Department of Immunology. National Jewish Health and School of Medicine of University of Colorado, Denver, CO 80206 Chronic beryllium disease (CBD) is a fibrotic lung disorder caused by beryllium (Be) exposure, characterized by granulomatous inflammation and the accumulation of Be-responsive CD4+ T cells in the human lung. Genetic susceptibility to CBD has been associated with certain alleles of the MHCII molecule, HLA-DP,

S35 especially HLA-DPB1*0201 (DP2) and other alleles that contain a glutamic acid residue at position 69 of the b-chain (b69Glu). Importantly, the HLA-DP alleles that can present Be to T cells match those implicated in the genetic susceptibility, confirming that the HLA contribution to disease is based on the ability of those molecules to bind and present Be containing compounds to T cells. ˚ crystal structure of DP2 whose antigenHere we show a 3.25 A binding groove is occupied by a self peptide derived from the HLADR a-chain. The most striking feature of the structure is a unique large solvent exposed pocket formed between the peptide backbone and the DP2 b chain a helix. This pocket is acidic due to the presence of three glutamic acids from the b chain including b69Glu. In the crystal packing, this pocket has been filled with the guanidinium group of an arginine from a neighboring molecule. This positively charged moiety has formed an extensive H-bond network that offers a plausible model for how Be containing complexes might occupy this site. This idea is strengthened by the demonstration that mutation of any of the three glutamic acids in this pocket results in loss of the ability of DP2 to present Be to T cells.

The effects of high altitude hypoxia environment on ultrastructure and the expression of HIF-1a in the rat lung Li Wenhua, Yuan Dongya, Zhang Min, Sun Fangyun, Zhao Fengcang Medical School, Tibet Institute of Nationalities, Xianyang, Shanxi 712082,China; Email: [email protected] Objective: To study the effects of high altitude hypoxia environment on rat lung ultrastructure and the expression of HIF-1a in the rat lung. Methods: 50 SD rats were randomly divided into 5 groups, namely group 1 d Golmud (altitude 2,700 m), 2 d Tanggula group (altitude 5,000 m), 3 d Nagqu group (altitude 4,500 m) and 30 d Nagqu group (altitude 4,500 m), the control group (in Xi’an, elevation 5 m). 4 time-consuming experimental animals 1 d from Xi’an to the Golmud, Qinghai (altitude 2,700 m), 2 d to the Tibetan Tanggula (altitude 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 expression, RT-PCR method for detection of high altitude hypoxic group, lung tissue expression of hypoxia inducible factor-1amRNA change. Results: 2d Tanggula acute hypoxia in lung tissue microstructure and ultrastructure of apparent high altitude pulmonary edema, and after hypoxic group after 30d Nagqu significantly reduce high altitude pulmonary edema, lung tissue was no Canon of Western blotting, HIF-1a Protein expression, RT-PCR detection of HIF-1amRNA 30d Nagqu group in the expression of a certain amount, the remaining three experimental groups the expression of HIF-1amRNA not significant (P [ 0.05), 30d expression of HIF-lamRNA Nagqu group were significantly higher (P \ 0.01). Conclusion: The hypoxia acclimatization for improved HIF-la mRNA expression is beneficial to the hypoxic altitude pulmonary edema. Keywords: SD Rat, High altitude hypoxia, Transmission electron microscopic, HIF-1a

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The key residues of active sites located on beta-tubulin forming an active pocket for paclitaxel binding Sichuan Xu1,2,*, Shaoming Chi1, Yi Jin1, Qiang Shi2, Maofa Ge2, Shu Wang2 and Xingkang Zhang2 1

Key Laboratory of Education Ministry for Medicinal Chemistry of Natural Resource, College of Chemical Science and Technology, Yunnan University, Kunming 650091, People’s Republic of China 2 State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Beijing National Laboratory for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China Paclitaxel (PTX) is used to treat various cancers but causes heavy side effect and resistance as well. To better design similar compounds with less toxicity and more activity against drug-resistant tumors, a significant issue is to clear understand the PTX-binding pocket organized by key residues as active sites on b-tubulin. 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 sites responsible for interaction with PTX. Other two residues Leu371 and Gly279 also likely serve as the active sites. Most of them contact with the ‘‘southern hemisphere’’ of PTX. Only one key residue interacts with the ‘‘northern hemisphere’’ of PTX. These key residues are composed of four groups and serve as active compositions to form an active pocket for PTX binding to the surface of b-tubulin. This active binding pocket provides the very potent interaction, whose strength is predicted to be in the range of -327.8 to -365.7 kJ/mol between b-tubulin and PTX using different orientated conformations. The potent interaction makes PTX have high activity against cancer cells, which is in good agreement with the clinical mechanism of PTX. The disclosed PTX pocket associated with the key residues can be applied to probe the mechanism of tumor cell resistant to PTX and design novel analogues with superior properties. Acknowledgments: The work was supported by One-Hundred-Talents Project of Chinese Academy of Sciences, and Academic Talent Foundation of Yunnan Province, China (2006PY01-29).

The role of bilirubin as a nature antioxidant in clinical outcomes in patients with cardiovascular and cardiometabolic diseases Shao-Sung Huang, Shing-Jong-Lin, and Jaw-Wen Chen National Yang-Ming University School of Medicine and Taipei Veterans General Hospital, Taipei, Taiwan Accumulating in vitro or in vivo evidence suggest that oxidative stress and inflammation could contribute to both the development and progression of atherosclerosis. Increased oxidative stress and vascular inflammation have been demonstrated in patients with cardiac syndrome X (CSX). Bilirubin, once considered simply the metabolic end product of heme degradation, has emerged as a potential endogenous inhibitor of atherosclerosis. We conducted this study to evaluate the prognostic role of serum bilirubin in disease progression and clinical outcome in patients with CSX. A total of 108 consecutive CSX patients were enrolled. Serum bilirubin levels were examined from blood samples collected before coronary

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12th International Congress on Amino Acids, Peptides and Proteins angiography. All patients were prospectively followed up. The primary end point was the combined occurrence of major adverse cardiovascular events (MACE) including cardiovascular and noncardiac death, nonfatal myocardial infarction (MI), nonfatal ischemic stroke, rehospitalization for unstable angina, and revascularization with percutaneous coronary intervention or coronary artery bypass grafting. There were 34 MACEs including 1 nonfatal MI, 7 ischemic stroke, 11 noncardiac death, and 15 rehospitalization for unstable angina in a duration of 88 ± 30 months. Patients with MACE had lower baseline serum bilirubin levels (P = 0.002). All patients were stratified into the high-, normal-, and low-bilirubin group. The patients in high-bilirubin group had the lowest incidence of MACE (P = 0.004). In a multivariate Cox regression analysis, serum bilirubin was the only independent predictor of MACE (HR, 0.066; 95% CI 0.008–0.562; P = 0.013). Accordingly, in patients with CSX, baseline serum bilirubin level was associated with longterm outcomes, suggesting that serum bilirubin may be a predictive and protective biomarker for disease progression and the development of cardiovascular events in patients with atherosclerosis as well as cardiometabolic diseases.

Thymosin beta4 treatment improves neurological functional recovery in experimental autoimmune encephalomyelitis mice Jing Zhang1, Zheng Gang Zhang1, Yi Li1, Mei Lu2, Dan Morris3, Stanton B. Elias1, and Michael Chopp1, 4 1

Department of Neurology Biostatistics and Research Epidemiology 3 Department of Emergency Medicine, Henry Ford Health System, Detroit, MI, 48202; 4 Department of Physics, Oakland University, Rochester, MI, 48309 2

Multiple sclerosis (MS) is the demyelinating disease of the central nervous system (CNS), which results from damage to oligodendrocytes that make myelin to encase axons. There is no cure for MS. Thymosin beta4 (Tbeta4) is a highly conserved 43-amino acid acidic 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 (OPCs) and thereby to enhance remyelination in MS. SJL/J mice were subjected to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated with saline 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 number of OPCs and mature oligodendrocytes, proliferated and differentiated OPCs were measured using antibodies against bromodeoxyuridine (BrdU), NG2 and 20 ,30 cyclic nucleotide 30 phosphodiesterase (CNPase), respectively. Triple immunofluorescent staining with antibodies against BrdU, CNPase and neurofilament heavy chain was employed to examine whether newly generated mature oligodendrocytes myelinate axons. Tbeta4 treatment (30 mg/ kgbw) significantly improved functional recovery after EAE. NG2+ OPCs, CNPase+ mature oligodendrocytes, BrdU+ with NG2+ OPCs, BrdU+ with CNPase+ mature oligodendrocytes were significantly increased in the EAE CNS with Tbeta4 treatment compared to those of saline controls. The alignment of newly differentiated OPCs along axons indicated the induction of remyelination. These data indicate that Tbeta4 treatment improved functional recovery and stimulated oligodendrogenesis after EAE. Tbeta4 is a potential therapy for MS.


TNF-a in carcinogenesis and cancer therapy Xia Wang Laboratory of Molecular and Translational Medicine, West China Second University Hospital, Sichuan University, Chengdu, People’s Republic of China; E-mail: [email protected] Tumor necrosis factor-a (TNF-a) was identified in the late 1970 s as a cytokine produced by immune cells capable of suppressing tumor cell proliferation and inducing tumor regression. TNF-a is a multifunctional cytokine that plays important roles in diverse cellular events 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 kinase (JNK), and apoptosis. NF-jB is a major cell survival signal that exerts its anti-apoptotic effect by activating a number of antiapoptotic target genes, whereas transient JNK activation contributes to cell survival and sustained JNK activation contributes to cell death. 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, TNFa is a double-edged sword. As it is an important pro-inflammatory cytokine, TNF-a may be involved in inflammation-associated carcinogenesis by serving as an endogenous tumor promoter. It could stimulate the growth, proliferation, invasion and metastasis, and tumor angiogenesis of cancer cells, almost all aspects of carcinogenesis, which is closely related to activation of NF-jB and JNK pathways. On the other hand, TNF-a is a potential cancer therapeutic agent as it has the property of inducing cancer cell death. However, using TNF as a chemotherapeutic drug has been hampered by its deleterious side effects, including systemic shock and widespread inflammatory responses. In addition, many cancer cells are resistant to TNF-induced cytotoxicity. Therefore, much work is needed to reduce its side effects and maximize the tumor-selective cytotoxicity. In this lecture, a comprehensive introduction about TNF-a and it’s roles in cancer development will be given, with focus on the role of TNF-a in cancer therapy including data from our lab.

Tryptophane supplements promote pregnancy success in mice challenged with pseudorabies virus (PRV) by regulating the expression of systemic cytokines, immunoglobulins, PRV-specific protein profiles and toll-like-receptors De Wu *, Zhengfeng Fang, Shixiu Qiu, Yan Lin, and Lianqiang Che Key Laboratory for Animal Disease Resistance Nutrition of the Ministry of Education of China, Animal Nutrition Institute, Sichuan Agricultural University, Ya’an 625014, People’s Republic of China *To whom correspondence should be addressed Email: [email protected] Tryptophane (Trp) plays an important role in regulating the maternal immune response, a key determinant of the success or failure of pregnancy, but whether Trp supplements can prevent a pseudorabies virus (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 the immunity and reproduction of PRV-challenged pregnant mice. PRV challenge resulted in decreased live embryo numbers, live litter sizes, serum progesterone and interleukin-10 (IL-10) concentrations, but increased the levels of serum immunoglobulins (PRV-specific antibody, IgG, IgA, IgM) and IL-1b. Live embryo numbers, live litter sizes, serum

S37 progesterone concentration, and IgG and PRV-specific antibody levels on day 9 of pregnancy were all increased dose-dependently by Trp inclusion in the diet of PRV-challenged mice. Increased Trp levels in PRV challenged mice promoted the upregulation of uterine and embryonic indoleamine 2, 3-dioxygenase expression, but attenuated the upregulation of uterine and embryonic Toll-like receptor 3 (TLR3) and TLR9 expression and increased serum interferon-gamma concentration. Collectively, Trp supplements might improve reproductive performance of PRV-challenged pregnant mice by downregulating TLR expression and proinflammatory cytokine synthesis, by upregulating PRV-specific antibody and immunoglobulin synthesis and by elevating the concentrations of anti-inflammatory cytokines and progesterone.

Metabolism A characteristic urinary metabolic profile in citrin deficiency Chunhua Zhang Dept. of Research and Development, MILS International, 3-1-1, Heiwa Machi, Kanazawa, 921-8105 Japan Citrin deficiency, which is due to the mutation in SLC25A13 gene, is characterized clinically by intrahepatic cholestasis in early infancy (neonatal intrahepatic cholestasis by Citrin deficiency; NICCD) and CTLN2 in adulthood. In NICCD, the clinical feature present jaundice, hypoglycemia, galactosemia, and multiple aminoacidemias. Gas chromatograph/mass spectrometry (GC/MS) analyzing, enables the simultaneous measurement of multiple categories of compounds and offer reliable and quantitative evidence for the screening or chemical diagnosis of more than 130 inborn errors metabolism (IEMs). Using GC/MS analyze patient urine, we can gait difference urinary metabolic 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, threonine, methionine, phenyalanine, tyrosine) like Funconi syndrome profile, (2) high level of galactose, galactitol and galactonate like galactosemie profile, combine (3) high level of 4-hydroxyphenyllactate and 4-hydroxyphenylpyruvate like tyrosinemia profile, and (4) decreased ratio of serine/threonine in the metabolic profile pattern. 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 within normal pattern. Therefore, the metabolic profile of Citrin deficiency depend sample collective condition. The final diagnosis of Citrin deficiency need refer genetic mutation analysis. However, urinary metabolic profile analysis is the effective procedure for screen of Citrin deficiency. The detail should be presentation.

Biochemical management in hepatorenal tyrosinemia: a report of 18 cases in Algerian children L. Yargui, S. Brahimi, L. Douaibia, S. Meherhera, and M. Berhoune Laboratoire Central de Biochimie, CHU Mustapha Alger. Place 1er mai. 16000 Alger, Algerie Hepatorenal tyrosinemia (TH1) is an autosomal recessive inborn disease caused by deficiency of fumarylacetoacetate hydrolase, which is the last enzyme in the tyrosine degradation.

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S38 This disorder is clinically severe and affects liver, kidney, and peripheral nerve. Hepatocarcinoma is usually a long term complication. TH1 was diagnosed in 18 cases (ages 4–36 months) on screening 1,268 children for aminoacidopathies, between December 2009 and January 2011. The commonest presenting features were hepatomegaly (37%) and cholestasis (35%). Other features included, hepatic failure (20%), rickets (3%), acute porphyria (3%) and renal manifestations (2%). Diagnosis was confirmed by an elevated serum tyrosine and urine succinylace´tone levels. Other investigations revealed mildly deranged liver functions and a markedly raised alpha feto protein. 8 tyrosinemic infants were treated with nitisinone plus diet restricted in tyrosine. We report the biochemical findings and the results of 1 year follow-up in tyrosinemic patients, in our laboratory.

Comparative mutagenicities of PAHs metabolites induced by peroxynitrite/Fe(III)porphyrin system through Ames test Yunjing Luo1*, Jing Dai1, Yuanbin She2, and Rugang Zhong1 1 College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China, 2 College of Environmental and Energy Engineering, Beijing University of Technology, Beijing, China, [email protected]

Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants in the environment because of their high potential toxicity, mutagenicity and carcinogenicity. Most of PAHs have no or less mutagenicity, which require activation to exert their mutagenic or carcinogenic effects. In our previous work, we took peroxynitrite/ Fe(III)porphyrin system instead of traditional enzyme system to induce their metabolic activation, then we successfully identified three major metabolites through high performance liquid chromatography coupled with mass spectrometry which were the quinone group, OH group and nitro group. The present study was undertaken to isolate three main PAHs metabolites and compare their mutagenicities through Ames test (Salmonella typhimurium TA98) without using the activation of S9 mix. At dose levels ranging from 5 to 50 lg per plate, quinone group, nitro group, PAHs metabolic mixtures all showed dose correspondence with their mutagenicities, while PAHs parent and OH-group were nonmutagenic. Moreover, it is clear that nitro group has the highest mutagenic level, PAHs metabolic mixtures rank the second, quinone group is the lowest one. It is suggested, therefore, nitro-PAHs plays the key role in mutagenicity of PAHs metabolic activation. This work was supported by National Natural Science Foundation of China (No. 20875006), and Beijing Natural Science Foundation (No. 2102005).

Is hydroxysteroid (17beta) dehydrogenase X (HSD10) deficiency an inborn error of isoleucine metabolism? Song-Yu Yang NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA

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12th International Congress on Amino Acids, Peptides and Proteins Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mitochondrial multifunctional enzyme encoded by the HSD17B10 gene. Missense mutations in this gene result in HSD10 deficiency (formerly MHBD deficiency) attributed to an inborn error of isoleucine metabolism. In fact, the detection of elevated levels of organic acids in patients’ blood and urine is a clinical marker for the screening of HSD10 deficiency, but not a critical pathogenic factor. Missense mutations of the HSD17B10 gene especially the most common one, p.R130C, abolished the HSD10 activity, interfering with not only isoleucine but also neuroactive steroid metabolism. Since affinities of HSD10 mutants to other peptides are probably altered, the pathogenesis of HSD10 deficiency may be also related to the formation of protein complexes by HSD10, which serves as a core of the mitochondrial RNase P for instance. These recent findings provided an explanation why the benefit of the proteinrestriction 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 degradation was not found in another X-linked mental retardation with choreoathetosis and abnormal behavior (MRXS10) resulting from a 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 affects male and some female patients, but female has usually milder phenotype. The establishment of our theory would lead to a new therapeutic strategy of this disease for ameliorating intellectual disability.

Lysine/ornithine decarboxylase: an enzyme catalyzing the first step of quinolizidine alkaloid biosynthesis in legume plants Somnuk Bunsupa1, Kae Katayama1, Akira Oikawa2, Kazuki Saito1,2, and Mami Yamazaki1,3 1

Graduate School of Pharmaceutical Sciences, Chiba University, RIKEN Plant Science Center, 3CREST, JST

2

Lysine decarboxylase (LDC) is the key enzyme involved in the first step of quinolizidine alkaloids (QAs) biosynthesis. We have cloned the lysine/ornithine decarboxylase (L/ODC) from alkaloid-containing cultivar of L. angustifolius by using PCR-select-subtraction and 50 /30 RACE techniques. The purified recombinant protein expressed in E. coli exhibited decarboxylase activities towards both L-ornithine and L-lysine with similar Km value. The decarboxylase activities toward both substrates were competitively inhibited by DL-a-difluoromethylornithine, which is a specific inhibitor of ornithine decarboxylase. We also characterized L/ODC genes from legume plants Sophora flavescens and Echinosophora koreensis which produce 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 preference for L-ornithine over L-lysine is only 0.5–1.5 times. The heterologous expression of L/ODC in Arabidopsis and tobacco resulted in the enhanced accumulation of cadaverine and tobacco alkaloids derived from lysine, indicating the actual function of this enzyme for the formation of cadaverine and subsequent production of alkaloids. This is the first report on an L/ODC involved in QAs biosynthesis from plants.


Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD): clinical analysis of 50 cases

S39 Supported by Grants: SAF 2010-17573 (Spanish Ministry of 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 1

Department of Pediatrics, The 1st Affiliated Hospital, Jinan University, Guangzhou 510630, China, 2 Department of Laboratory Science, The 1st Affiliated Hospital, Jinan University, Guangzhou 510630, China Aims: Citrin deficiency is resulted from dysfunction of citrin, a mitochondrial aspartate/glutamate carrier encoded by SLC25A13 gene. This study aims to investigate the clinical and laboratory features of Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD). Methods: Fifty NICCD cases confirmed by SLC25A13 analysis were enrolled as research subjects. Major clinical manifestations and the features of blood biochemistry, hepatopathology, medical imaging and metabolome were analyzed by means of a cross-sectional study. Results: Clinical presentations included jaundice, abnormal coagulation tests, chubby face, failure to thrive, steatorrhea, motor retardation, hepato/hepatosplenomegaly, anemia, echinocytosis, and light stool. Besides the elevated biochemical indices of cholestasis and dyslipidemia, reduced fibronectin, retinol binding protein and ceruloplasmin along with zinc deficiency were also revealed. Diffused fat deposition, cholestasis in hepatocytes and canaliculi, and varying degrees of inflammation and fibrosis were observed, suggestive of hepatopathological features of non-alcoholic fatty liver disease (NAFLD). Ultrasound, CT and MRI revealed fatty livers in some cases, respectively. Metabolome features included coexistence of indices of tyrosinemia type I and markers for galactosemia at urine samples, and abnormal amino acid spectrum and acylcarnitine profile in blood specimens, with dramatically elevated free, myristyl and palmityl carnitine. Conclusions: This study revealed motor retardation, reduced serum fibronectin, retinol binding protein and ceruloplasmin and zinc deficiency as novel clinical and biochemical features for NICCD, and proposed, for the first time, that NICCD might be a specific etiology for non-alcoholic fatty liver disease (NAFLD). Moreover, the changes of acylcarnitine profile in this study further expanded the metabolome feature of NICCD.

New insights into the roles of brain glutaminases

On the role of the mitochondrial 2-oxoglutarate dehydrogenase complex in the amino acid metabolism W. L. Araujo1, L. Trofimova2, Karavaeva Yu.3, D. Steinhauser1, L. Krall1, A. R. Fernie1, and V. I. Bunik3,4 1

Max-Planck-Institut fu¨r Molekulare Pflanzenphysiologie, 14476 Potsdam-Golm, Germany, 2 Department of Biophysics, 3 Department of Bioengineering and Bioinformatics, and 4 A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia Providing not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids, mitochondria are tightly linked to cellular nutrient sensing. Mitochondrial metabolic enzymes as generators and/or targets of signals could therefore be important players in the distribution of intermediates between the catabolic and anabolic pathways. The aim of this work was to characterize the specific impact of regulating the 2-oxoglutarate dehydrogenase complex (OGDHC), an essential system in both plant and animal mitochondria. Participating in the glucose oxidation via the tricarboxylic acid (TCA) cycle, this highly regulated complex occupies an amphibolic branch point of the cycle, where the energyproducing reaction of the irreversible decarboxylation of 2-oxoglutarate competes with glutamate synthesis via nitrogen incorporation into 2-oxoglutarate. A synthetic analog of 2-oxoglutarate, succinyl phosphonate (SP), forming a tight inhibitory complex with the coenzyme of the starting OGDHC component, was applied to living systems from different Kingdoms in situ and in vivo. Using a highthroughput mass-spectrometry-based approach, we show that the organisms possessing complete TCA cycle including OGDHC, respond to SP by significant changes in their amino acid pools. Increased glutamate and 4-aminobutyrate represent the most universal change accompanying the 2-oxoglutarate accumulation upon the OGDHC inhibition. Other amino acids were affected in a speciesspecific 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 evidence of a considerable role of OGDHC in amino acid metabolism.

Javier Ma´rquez Glutamine/glutamate homeostasis must be exquisitely regulated in mammalian brain and glutaminase is one of the main enzymes involved. Glutaminase is considered as the main glutamate-producer enzyme in brain and, consequently, it is essential for both glutamatergic and gabaergic transmissions. The classical pattern of glutaminase expression in mammals has been recently challenged by the discovery of novel isoforms and subcellular locations with particular relevance in brain. Furthermore, the interactome of brain glutaminases is also starting to be uncovered adding a new level of regulatory complexity. All these experimental evidences suggest new functions for brain glutaminases. In this talk, we summarize recent findings that point consistently towards glutaminase as a multifaceted protein able to perform different tasks. Some controversial issues about brain glutaminases will be also discussed.

The central nitrogen metabolism in Escherichia coli: coordinated regulations everywhere Dalai Yan Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA The central nitrogen metabolic circuit in Escherichia coli is a compact system that consists of only three enzymes. While either glutamate synthase and glutamate dehydrogenase may synthesize glutamate, glutamine synthetase (GS) catalyzes the sole pathway for glutamine biosynthesis, plays a central role in the system, and is under elaborate regulations. Ammonium is preferred sole nitrogen source,

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S40 supporting the fastest growth. Ammonium assimilation by the circuit yields the two central nitrogen intermediates that in turn supply 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 bifunctional adenylyltransferase/adenylyl-removing enzyme. We demonstrated that the opposing activities are dynamically balanced during cell growth. The study suggests that counterbalance by reversible covalent modification may be a general strategy for controlling the activity of enzymes like GS, whose physiological output allows adaptation to environmental fluctuations. The second is a differential but partially overlapping regulatory scheme governing GS expression and modification, achieved by two key metabolic effectors through regulatory cascades. We discovered that both GS expression and modification respond to a unique scaling combination of the internal concentrations of the metabolites, (glutamine)/(2-oxoglutarate)1/3. The third is a ‘‘kinetic’’ coordination for cell growth during ammonium depletion. We designed a new culturing apparatus which can continuously monitors cellular behavior during the nutrient excess to depletion transition with high temporal precision. With its application, we show that cells simultaneously elevate enzyme expression (GS) and concentrate the substrate (ammonium) internally, by the function of a unique active ammonium channel AmtB, to compensate the depleting external ammonium thus sustain the fastest possible growth before ammonium exhaustion. All the cases here demonstrate the power of quantitative studies for understanding the fine print of metabolic regulation.

12th International Congress on Amino Acids, Peptides and Proteins identification of clinically important bacteria on the genus and species level.

The fingerprint of microbial lipopeptides and their identification Shi-Zhong Yang, Jin-Feng Liu and Bo-Zhong Mu* State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China. School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China. *Corresponding to: Tel: +86-21-64252063; Fax: +86-21-6425 2485; E-mail: [email protected] The molecular weights of about 90 reported lipopeptides were calculated and the fingerprint of the lipopeptides, which can be used to distinguish microbial lipopeptides, was established. Based on the fingerprint, a lipopeptide could be identified through simple and easy methods, the ESI MS and the amino acid analysis. As an example, a kind lipopeptide from Bacillus subtilis was determined through ESI MS and amino acid analysis, the molecular weight and the amino acid composition of the lipopeptide revealed that the lipopeptide was surfactin. The ESI–MS and amino acid analysis are very useful for identifying the lipopeptides in virtue of the figerprint.

Microbiology Rapid genus- and species-specific identification of clinically important bacteria by MALDI-TOF in a routine laboratory

Vitreoscilla hemoglobin gene vgb improves the growth rate of Corynebacterium glutamicum ATCC13032 with atp gene inactivation 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] The human bowel contains a large and diverse bacterial community, termed as gut flora. These microorganisms perform a number of useful functions, such as making energy, training the host immune system, preventing growth of pathogenic bacteria, regulating the development of the gut, producing vitamins and hormones for the host. However, under certain conditions, some species are capable of causing diseases or increasing cancer risk for the host. Here we evaluated the application of MALDI-TOF MS for rapid genus and species identification of some clinically important members in gut flora. The strains provided mass spectra profiles covering a wide molecular mass range (2,000–30,000 Da) with high reproducibility and specificity. Genus- and species-specific biomarker protein mass patterns were determined, and validated by Spectral Archive and Microbial Identification System (SARAMIS) SuperSpectrum. For example, the mass spectrometry-based identification scheme yielded identical results of E. coli as with a PCR-based identification system, which was correctly identified. More importantly, the SuperSpectrum could be constructed manually, and will be imported into SARAMIS database for the optimal validations. This study demonstrated that MALDI-TOF MS was a reliable and powerful tool for the rapid

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Previously we reported that Corynebacterium glutamicum ATCC13032 with atp gene inactivation, designated as C.glutamicum Datp, showed an approximately 1.4 times higher L-glutamate production than the wild type strain, but manifested a low growth rate. In this study, vgb gene, which increases oxygen supply in Vitreoscilla sp., was introduced into C. glutamicum-Datp via constructed shuttle vector pJC1-vgb to enhance oxygen uptake. To determine the characteristics of recombinant C. glutamicum-Datp harboring vgb gene,namely C. glutamicum-Datp–pJC1-vgb, the fermentation with it as starter was performed in 25 L fermentor, and vgb activity were detected using CO-difference spectra. Under low dissolved oxygen (DO), vgb gene was expressed on vector pJC1 and regulated by the DO. vgb gene expression level increased rapidly as DO dropped. In 100 g/L glucose culture medium, the cell density of recombinant C. glutamicum-Datp-pJC1-vgb was more 22% than the original strain C. glutamicum-Datp. The results demonstrated that increasing oxygen supply via vgb gene expressed in recombinant C. glutamicum-Datp remarkably enhanced cell growth rate.It is suggested that using vgb in C. glutamicum-Datp could be an effective approach to improve the production of L-glutamate fermentation in high density culture involved in oxygen-limited bioprocess.


Neurobiology Carbon nanofiber (CNF) electrode and wireless instantaneous neurotransmitter concentration sensor (WINCS) system for fast scan cyclic voltammetry detection of neurochemicals Jessica E. Koehne1, Michael Marsh2, Adwoa Boakye1, Brandon Douglas1, Christopher J. Kimble3, Kevin E. Bennet3, M. Meyyappan1, and Kendall H. Lee2,4 1

Center for Nanotechnology, NASA Ames Research Center, Moffett Field, CA 2 Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 3 Division of Engineering, Mayo Clinic, Rochester, MN 4 Department of Neurologic Surgery, Mayo Clinic, Rochester, MN 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 (CNF) nanoelectrodes have shown great promise as stimulating electrodes due to their high surface area, low impedance, biocompatibility and capacity for highly localized stimulation. In addition, CNF nanoelectrodes have been used as sensing electrodes for the detection of many small biomolecules including DNA, proteins and neurotransmitters. This work describes recent developments in a novel approach based on CNF nanoelectrodes for localized neurochemical recording. Our approach combines a multiplexed CNF electrode chip, developed at NASA Ames Research Center with the wireless instantaneous neurotransmitter concentration sensor (WINCS) system, developed at the Mayo Clinic. The multiplexed 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 diameter CNFs that are spaced at 1 lm intervals. The CNFs are embedded in SiO2 so that only the CNF tips are exposed for sensing. The CNF nanoelectrode chip and a traditional carbon fiber microelectrode were utilized for neurochemical recording in a flowcell system and successfully recorded both dopamine and adenosine concentrations by fast scan cyclic voltammetry. The CNF nanoelectrode chip demonstrated similar performance to the CFM and has advantage in flexible multiplexed design architectures for highly localized neurochemical recording. In the future, combining CNF based stimulating and recording electrodes with WINCS may lay the foundation for an implantable ‘‘smart’’ DBS system that utilizes neurochemical feedback control while likely resulting in improved battery life and increased DBS application in various neuropsychiatric disorders.

Challenges in neurodegeneration drug discovery Bai Lu GlaxoSmithKline, R&D China, Building 3, 898 Halei Road, Zhangjiang Hi-tech Park, Pudong, Shanghai 201203 China Despite huge progress in neuroscience research, the number of approved drugs remains unchanged. Neurodegeneration (ND) is one of the most challenging areas in drug discovery. This is not only because the brain is the most complex organ in the body, but also

S41 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 task for drug development. Moreover, there is no good animal model that truly mimics human disease situations. In addition, lack of genuine biomarker and good clinical readout for AD or PD makes it extremely challenging for proof of concept studies in humans. To meet the challenges in ND drug discovery, many different approaches have been attempted in the biopharmaceutical industry. At GSK-R&D China, we have adopted three basic principles to guide our drug development efforts. First, our pipeline driver is strategic. Our ND programs are strategically positioned to address multiple disease mechanisms including, inflammation, neuronal cell death pathways and neuroprotection/repair. Second, our science is game-changing. We focus on cutting-edge science and emphasize on differentiation. Third, our approach is innovative. We do not constrain ourselves by the traditional ways of drug discovery, but try novel and creative approaches. These three principles are the bases of our overall strategy for the ND drug discovery program.

Efficacy of GluK1 receptor antagonists against somaninduced seizures and neuropathology: a potential new emergency treatment for nerve agent exposure T. H. Figueiredo, F. Qashu, J. P. Apland1, V. Aroniadou-Anderjaska, A. P. Souza, and M. F. Braga Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD 1 US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD The possibility of mass exposure to nerve agents by a terrorist attack necessitates the availability of antidotes that can be effective against nerve agent toxicity even when administered at a relatively long latency after exposure, because medical assistance may not be immediately available. Nerve agents induce status epilepticus (SE), which can cause brain damage or death. Antagonists of kainate receptors that contain the GluK1 (formerly known as GluR5) subunit (GluK1Rs) are emerging as a new potential treatment for SE and epilepsy from animal research, whereas clinical trials to treat pain have shown that the GluK1/a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist LY293558 [(3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid] is safe and well tolerated. Therefore, we tested whether LY293558 is effective against soman-induced seizures and 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 prevented neuronal loss in the basolateral amygdala (BLA) and the CA1 hippocampal area on both days 1 and 7 after soman exposure and reduced neuronal degeneration in the CA1, CA3, and hilar hippocampal regions, entorhinal cortex, amygdala, and neocortex on day 1 after exposure and in the CA1, CA3, amygdala, and neocortex on day 7 after exposure. It also prevented the delayed loss of glutamic acid decarboxylase-67 immuno-stained BLA interneurons on day 7 after exposure. LY293558 is a potential new emergency treatment for nerve agent exposure that can be expected to be effective against seizures and brain damage even with late administration.

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Linolenic acid, a dietary supplement, is an efficacious neuroprotective agent against soman-induced brain damage: Its possible use as a preventative strategy Hongna Pan1, Cynthia Chen1, David Jacobowitz1§, Kerry Van Shura2, Megan Lyman2, John McDonough2, and Ann M. Marini1 1 Department of Neurology and §Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD 2 US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD

Nerve agents represent a key threat to the United States military and civilian populations. The major mechanism of acute toxicity is the inhibition of acetylcholinesterase resulting in the accumulation of acetylcholine (ACh). Excessive ACh levels in synapses lead to the progression of toxic signs including hypersecretions, tremors, seizure activity, respiratory distress and ultimately death. Prolonged seizures induce severe brain damage and long-term cognitive impairment that is commensurate with the extent of damage caused by the nerve agent. The current treatment of nerve agent-induced seizures is only 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 improve outcome. Linolenic acid is an essential omega-3 fatty acid that can be obtained over the counter as a dietary supplement. The compound is safe, has no known side effects and exerts neuroprotective, antidepressant and anti-convulsant properties. Therefore, we tested whether linolenic acid may protect against soman-induced brain damage. Here, we report that linolenic acid is a highly efficacious neuroprotective agent against soman-induced neuronal cell death in four brain regions known to be damaged by soman: piriform cortex, hippocampus, amygdala and prefrontal cortex. Pretreatment or posttreatment administration of linolenic acid to rats reduces neurodegeneration 24 h after exposure to soman. Surprisingly, subcutaneous injection of linolenic acid afforded much higher levels of neuroprotection compared with the intravenous route. This is the first study to show significant protection against soman-induced neuropathology by linolenic acid. Its wide safety margin and neuroprotective efficacy against soman-induced neuropathology suggests its use as a pretreatment strategy.

Neuroscience [3H]CHIBA-3007: a new radioligand for glycine transporter 1 in rat brain Jichun Zhang, Jin Wu, Jun Toyohara, Yuko Fujita, Hongxian Chen, and Kenji Hashimoto Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan Glycine transporter-1 (GlyT-1) in glial cells regulates extracellular levels of glycine, which acts as an obligatory co-agonist at the N-methyl-D-aspartate (NMDA) receptors in the brain. In the present study, we developed a novel radioligand, [3H]3-chloro-N-((S)-((R)-1-

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12th International Congress on Amino Acids, Peptides and Proteins methylpiperidin-2-yl)(thiophen-3-yl)methyl)-4-(trifluoromethyl)picolinamide ([3H]CHIBA-3007), for studying GlyT-1 in the brain. The presence of a single saturable high-affinity binding component for [3H]CHIBA-3007 binding to the rat brain membranes was detected. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 1.61 ± 0.16 nM and a maximal number of binding 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 of GlyT-1 inhibitors, such as CHIBA-3007, desmethyl-CHIBA3007, CHIBA-3008, SSR504734, NFPS/ALX5407, LY2365109 and Org24598, consistent with the pharmacological profiles of GlyT-1 inhibitors. Interestingly, the potency of eight GlyT-1 inhibitors (CHIBA-3007, desmethyl-CHIBA-3007, NFPS/ALX5407, LY2365109, Org24598, SSR504734, sarcosine, and glycine) for blocking in vitro specific binding of [3H]CHIBA-3007 was significantly correlated with the potency of these inhibitors for inhibiting [14C]glycine uptake in the rat brain membranes. In contrast, the GlyT-2 inhibitor ALX1393 exhibited very weak at [3H]CHIBA-3007 binding. Furthermore, the regional distribution of [3H]CHIBA-3007 binding in the rat brain was similar to the previously reported distribution of GlyT-1. The present findings suggest that [3H]CHIBA3007 would be a useful new radioligand for studying GlyT-1 in the brain.

Activities of kynurenine aminotransferase I, II and III in the rat brain and heart tissues during the aging process Halina Baran1 and Berthold Kepplinger1,2 1

Neurochemical Laboratory, Karl Landsteiner Research Institute, Landesklinikum Mauer-Amstetten 2 Department of Neurology, General Hospital, Landesklinikum Amstetten, Amstetten, Austria Corresponding address: [email protected] The pattern of kynurenine aminotransferase (KAT) I, II and III, the biosynthetic enzymes of the excitatory glutamate amino acid receptor and nicotine cholinergic receptor antagonist kynurenic acid was examined in brain and heart tissues of 3, 12 and 26 months old male Wistar rats. KAT I activity at pH 9.6, KAT II at pH 7.2 and KAT III 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 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 heart homogenates between 3 and 26 months, whereas in the mitochondrial suspensions of brain and heart the KAT II and KAT III activities were moderately increased. Interestingly, no alteration of KAT I activities was found in the homogenates and mitochondrial suspensions prepared from brain and heart of 3, 12 and 26 months old animals. Obtained data indicate that under physiological condition the agedependent increase of kynurenic acid levels in the brain is rather due 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 of KAT I activities seen in Alzheimer’s brain is not related to aging process but probably predominantly to the pathological events. ¨ sterreichische Nationalbank Jubilaümsfonds ProSupported by O ject: Nr. 12316.


APPL1 mediates synaptic NMDA receptor-dependent neuroprotection Yubin Wang1, Shuang Qiu1, JieJie Wang, Shaohua Wang, and Jianhong Luo Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China Corresponding author E-mail: [email protected] 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 cause neuronal loss in acute trauma such as ischemia, as well as certain neurodegenerative diseases such as Huntington’s disease. However, physiological levels of synaptic NMDA receptor activity can promote neuronal survival through different signaling pathways under different circumstances. Among them, PI3K (phosphoinositide-3-kinase)/Akt kinase cascade is a key signaling pathway responsible for neuroprotective effects of NMDA receptor activity. It is still unknown how NMDA receptors are coupled with PI3K-Akt pathway. APPL1 (adaptor protein containing pH domain, PTB domain, and Leucine zipper motif), is an endosomal adaptor protein which are associated with various transmembrane receptors. Furthermore, APPL1 has been reported to interact with, and regulate the activity of, the kinase Akt. In the present study, using biochemical analysis and immunostaining, we found that APPL1 associates with NMDA receptor complex through its C-terminal PDZ binding motif. More importantly, APPL1 is involved in synaptic NMDA receptor mediated neuroprotective effect. Interruption of APPL1-NMDA receptor complex interaction by peptide against APPL1 C-terminal PDZ binding motif dissociates PI3K and Akt from NMDA receptor, blocks activation of Akt by synaptic NMDA receptor, and consequently, blocks NMDA receptor-dependent neuroprotection from starve-induced apoptosis. It suggests that in central nervous system APPL1, as an adaptor protein, connects synaptic NMDA receptor with intracellular PI3K/Akt cascade and down-stream prosurvival signaling pathway.

Correlation of VIP VPAC1 modulation of GluR1 phosphorylation and inhibition of protein kinases in theta-burst LTP experiments with data mining tools Cunha-Reis D1,2 and Carmo AJ2 1

Enzymology Group, CQB, FCUL and Autonomic Nervous System Unit, IMM, University of Lisbon, Lisbon, Portugal. [email protected]

2

VIP inhibits hippocampal CA1 long-term potentiation (LTP) through VPAC1 receptor activation in PKA and PKC independent process that may involve changes in AMPA GluR1 phosphorylation. We used data mining techniques to correlate VPAC1 modulation of LTP with GluR1 phosphorylation and activity of PKA, PKC and CamKII. Two independent data sets of experiments were used: G1-VPAC1 modulation of LTP, GluR1 levels and phosphorylation at Ser845 and Ser831; G2-impact of PKA, PKC and CamKII inhibitors on VPAC1 action on LTP. Grouping and distribution of data sets was studied by data clustering (k-means and hierarchical methods). Tree decision models targeting GluR1 phosphorylation versus of VPAC1 activation, LTP and kinase inhibition, were generated by supervised classification methods with Rattle 2.13.0. Clustering analysis targeting theta-burst stimulation evidenced weaker data clustering of LTP versus phosphorylation of GluR1 at

S43 Ser845 and Ser831 in the presence of a VPAC1 antagonist than in control conditions. Correlation between LTP and GluR1 phosphorylation at Ser 831 was also smaller. LTP was not correlated with GluR1 phosphorylation at Ser 845. This suggests that additional molecular mechanisms are recruited for LTP expression when VIP VPAC1 receptors are blocked.

D-Aspartate

and D-glutamate in the brain: from formation and localization to function

Jonathan V. Sweedler, Liping Wang, Ting Shi, Nobutoshi Ota, Lee Replogle, and Stanislav S. Rubakhin Departments of Chemistry and Neuroscience, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA chemistry.illinois.edu/faculty/Jonathan_Sweedler.html In the nervous system of animals ranging from mollusks to mammals, D-amino acids are present. As an important example, D-aspartate (D-Asp) and D-glutamate (D-Glu) are present at high levels in specific brain regions, although their functions are not well understood. Because neurochemistry is well conserved across metazoan life, we have selected the well-known physiological model Aplysia californica to probe the formation and functions of these D-amino acids in cell to cell signaling. Specifically, using capillary electrophoresis with laser induced fluorescence and radionuclide detection, we have determined which neurons synthesize D-Asp, and have measured the formation, transport and release of D-Asp in a stimulation dependent manner. We even make these measurements from individual indentified neurons. Once we determine which specific neurons synthesize D-Asp, we have located and characterized the enzyme responsible for D-Asp formation. The novel racemase has the ability to convert both L-Ser and L-Asp to their D-counterparts. We have also probed the electrophysiological effects of D-Asp and L-Asp on the activity of specific 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 studying the effect of D-Glu on neurotransmission, as well as facets of its formation and release.

Decreased number of oxytocinase/vasopressinasecontaining neurons in the hypothalamus of patients with schizophrenia Hans-Gert Bernstein1, Susan Mu¨ller1, Henrik Dobrowolny1, Uwe Lendeckel2, Johann Steiner1, and Bernhard Bogerts1 1 Department of Psychiatry, Medical School, University of Magdeburg. Leipziger Str. 44, 39120 Magdeburg 2 Institute of Medical Biochemistry and Molecular Biology, University of Greifswald, Germany

The insulin-regulated aminopeptidase (IRAP) is a membrane-spanning protein predominantly located in intracellular vesicles. We suggest that alterations of IRAP-levels in the brain could be involved in the pathophysiology of schizophrenia and depression, because: (1) its catalytic function as oxytocinase and vasopressinase. Altered levels of these neuropeptides are found in schizophrenia and depression. (2) The inhibition of IRAP by AngIV and LVV-H7 is associated with memory enhancement. Cognitive dysfunction is a

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S44 common symptom in both disorders. (3) IRAP is colocalized with Glut 4 and thus involved in the regulation of the glucose transport. (4) Polymorphisms in and close to the IRAP encoding gene are associated with schizophrenia and depression. We studied the densities of IRAP-immunoreactive neurons in the paraventricular and the supraoptic nuclei in post-mortem brains of 11 schizophrenic patients, 8 subjects with major depression, 7 patients suffering from bipolar disorder and 11 matched controls. Our study shows a significant decrease in cell densities of IRAPcontaining neurons in the paraventricular nucleus of schizophrenics in comparison with controls. In the supraoptic nucleus of individuals with schizophrenia a tendency towards reduced cell densities was seen. Depressive patients do not demonstrate significant alterations of IRAP-immunoreactive neurons densities in both nuclei. The decrease in IRAP cell density is most probably related to the disturbed oxytocin and/or vasopressin metabolism seen in schizophrenia.

Evidence of emergent patterns in GluR1 phosphorylation in theta-burst LTP experiments with data mining tools A. J. Carmo2 and D. Cunha-Reis1,2 1

Enzymology Group, CQB, FCUL and Autonomic Nervous System Unit, IMM, University of. Lisbon, Lisbon, Portugal. [email protected]

2

Hippocampal CA1 long-term potentiation (LTP) involves phosphorylation of AMPA GluR1 subunit that varies with stimulus strength and pattern. Kinases mediating this phosphorylation do not always affect LTP. We now used data mining techniques to correlate GluR1 phosphorylation patterns with proposed mechanisms for its occurrence. Two independent data sets of experiments were used: G1-LTP elicited by mild theta-burst stimulation (MTBS) versus GluR1 levels and phosphorylation at Ser845 and Ser831; G2-impact of PKA, PKC and CamKII inhibitors on LTP. Grouping and distribution of data was studied by data clustering (k-means and hierarchical methods). Tree decision models targeting GluR1 phosphorylation as a function of MTBS, LTP and kinase inhibitors, were generated by supervised classification methods with Rattle 2.13.0. Clustering analysis targeting MTBS evidenced stronger data clustering when comparing LTP and both phosphorylation of GluR1 at Ser845 and Ser831 (but not GluR1 levels). A stronger correlation was found between LTP and GluR1 phosphorylation at Ser831 than for Ser845. Phosphorylation at those two sites were not correlated neither LTP versus GluR1 levels. These observations suggest that MTBS causes LTP through phosphorylation of Ser831 and Ser845 of GluR1 subunits through independent pathways, and that the first is preponderant in this effect.

Food restriction in male and female rats: effects on the spatial memory of the hippocampus Amer Al-Ansari, Tarik Al- Shaibani, Hassan Al Aali, Alaa AbdulAmeer, Sahar Ashoor, Sara Al-Ghareeb, Reem Al Aradi, and Fatima Ahmed Arabian Gulf University, College of Medicine, Manama, Bahrain Purpose: Food restriction represents specific challenge for animal’s behavior. Strategies for food seeking, stress and availability of energy

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12th International Congress on Amino Acids, Peptides and Proteins sources can all affect the brains efficiency in this behavioral task. Studies showed clear effect of altered energy intake on cognitive function. In this study we tested learning and memory by Morris water-maze in males and females groups of rats after acute and chronic food restriction. Methods: Sprague–Dawley rats were used. The daily need of food was measured using a metabolic cage. Each animal was then fed with 40% of its daily need. Control animals were fed ad libitum. The rats were tested by Morris-water maze after 2 h (acute) and 2 weeks (chronic groups) of food restriction. During a series of trials the rats were trained to locate and reach a hidden platform to escape the necessity of swimming. The latency and distance swam by the animals to reach the platform, and the average speed of swimming, were all measured. Results: Our results showed that after acute stress of 2 h FR the male rats were learning to locate the platform better than the control animals. 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 platform. Interestingly, the female animals showed higher speed of swimming than the male rats. After 2 weeks of FR, both males and females were performing significantly inferior to the corresponding control rats. Conclusion: Acute stress by 2 h FR affected differently the cognitive behavior of male and female rats. The male animals showed enhanced learning and memory in the water maze tests. Both groups were performing inferior to control animals after chronic FR.

From potent AMPA/KA agonists to either selective NMDA antagonists or to inhibitors of the glutamate transporters Carlo De Micheli, Paola Conti, Andrea Pinto and Lucia Tamborini Dipartimento di Scienze Farmaceutiche, Universita` di Milano, via Mangiagalli 25, 20133 Milan, Italy Taking as a model kainic acid we designed a series of aspartic and glutamic acid analogues where the amino acid skeleton was embedded into the bicyclic isoxazolin-proline motif. Some compounds (i.e. 3-carboxy-isoxazolin-prolines, CIPs) proved to be very potent AMPA/KA agonists. Worth noting, the 3-hydroxy-isoxazolin-prolines (HIPs) turned out to be totally inactive at the glutamate receptors but efficient blockers of the excitatory amino acid transporters (EAATs). The EAATs under physiological conditions remove glutamate from its synapses but in pathological conditions (e.g. ischemia, neurotrauma), due to the reduction of energy levels and the collapse of the Na+ transmembrane gradient, release additional glutamate through the reversed mode of operation, thus contributing to neuronal cell death. Inhibitors of EAATs have been proposed as promising molecular targets for the development of novel neuroprotective agents. The design, synthesis and pharmacological characterization of two potent EAATs inhibitors HIP-A and HIP-B will be presented and their potential usefulness in the post-ischemic therapy will be pointed out. It is well known that overactivation of NMDA-type glutamate receptors generates an uncontrolled influx of calcium that triggers an excitotoxic cascade leading to neurodegeneration, a phenomenon associated to several acute and chronic neurological disorders (e.g. cerebral ischemia, epilepsy, Parkinson’s and Alzheimer’s diseases). Competitive NMDA antagonists are typically conformationally 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 xcarboxylate may be fruitfully replaced by the bioisosteric phosphonate group to increase the receptor affinity. According to these rules and

12th International Congress on Amino Acids, Peptides and Proteins taking advantage of the results obtained with the above mentioned CIP derivatives, we have designed a series of new amino acids acting as potent and selective NMDA receptor antagonists. In particular, two highly potent and selective NMDA receptor antagonists have been identified, characterized by a very good in vivo anticonvulsant activity and by a promising in vitro neuroprotective activity.

Functional coupling between D-serine synthesis and vesicular transport in astrocytes Magalie Martineau1, Ting Shi2, Jonathan Sweedler2, Reinhard Jahn3, and Jean-Pierre Mothet4 1

Institute for Medical Physics and Biophysics, University of Muenster, Muenster, Germany 2 Department of Chemistry, University of Illinois, Urbana, IL, USA 3 Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Go¨ttingen, Germany 4 Magendie Neurocenter, INSERM U862, Bordeaux, France Neuron-astrocyte reciprocal communication at synapses has emerged 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 Dserine. We focused on the molecular determinants of gliotransmission with a specific interest in D-serine, the endogenous co-agonist of Nmethyl-D-aspartate receptors. First, we showed that D-serine is confined to the regulated secretory pathway in cultured astrocytes and its release is dependent on calcium and on SNARE proteins, hence arguing in favour of an exocytotic release. Then, to explore the existence of a vesicular transport mechanism for D-serine, we developed a procedure to immunoisolate synaptobrevin 2-containing vesicles from astrocytes. The purified organelles are clear vesicles which possess the molecular machinery to undergo membrane fusion, and contain large amount of D-serine and glutamate. Furthermore, they can actively transport these two amino acids. Finally, we provided direct evidence for the presence of serine racemase, the D-serine biosynthetic enzyme, at the membrane of astroglial vesicles, and for the coupling between D-serine synthesis and its subsequent vesicular transport. Our results highlight for the first time the existence of a glia-specific vesicular transporter for D-serine yet to be discovered.

Glial D-serine gates NMDA receptors at excitatory synapses in prefrontal cortex Fabrice R. Turpin1,2, Pascal Fossat1,2, Silvia Sacchi3, Je´roˆme Dulong1,2, Ting Shi4 Jean-Michel Rivet5, Jonathan V. Sweedler4, Loredano Pollegioni3, Mark J. Millan5, Ste´phane H. R. Oliet1,2 and Jean-Pierre Mothet1,2 1

INSERM U862, Neurocentre Magendie 33077 Bordeaux, France Universite´ de Bordeaux, 33077 Bordeaux, France 3 Dipartimento di Biotecnologie e Scienze Molecolari, Universita` degli Studi dell’Insubria, and The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Universita` degli Studi dell’Insubria, Varese, Italy 4 Department of Chemistry and Beckman Institute, University of Illinois, Urbana, USA 5 Institut de Recherches Servier, 78290 Croissy-sur-Seine, France 2

S45 NMDA receptors (NMDARs) subserve numerous neurophysiological and neuropathological processes in the cerebral cortex. Their activation requires the binding of glutamate and also of a co-agonist. Whereas glycine and D-serine (D-ser) are candidates for such a role at central synapses, the nature of the co-agonist in the cerebral 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 degrade either D-ser or glycine, we demonstrate that under the 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) induction. Furthermore, blocking the activity of glia with the metabolic inhibitor, fluoroacetate, impairs NMDAR-mediated synaptic transmission and prevents LTP induction by reducing the extracellular levels of D-serine. Such deficits can be restored by exogenous D-ser, indicating that the D-amino acid mainly originates from glia in the mPFC, as further confirmed by double-immunostaining studies for D-ser and GFAP. Our findings suggest that D-ser modulates neuronal networks in the cerebral cortex by gating the activity of NMDARs, and that altering its levels is relevant to the induction and potentially treatment of psychiatric and neurological disorders.

Glutamate attenuated survival promoting effect and signaling of IGF-1 in neuronal cells in vitro and in vivo Chengming Sun, Dejun Wang, Yannan Ren, Lang Zhang, and Wenhua Zheng* Neuropharmacology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China *Corresponding author E-mail: [email protected] Impairing intracellular signallings induced by excess glutamate play important roles in the process of neurodegenerative diseases. However, the underlying mechanism(s) and its interrelationships 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 hippocampal neurons with glutamate concentration-dependently inhibited the tyrosine phosphorylation of IGF-1 receptors and the phosphorylation of Akt, GSK3b and FOXO3a. These inhibitory effects of glutamate were blocked by NMDA receptor antagonist MK801 but not by blockers of other glutamate receptor sub-types verifying the involvement of the NMDA receptor. These findings demonstrate that glutamate can block the effect of IGF-1 by decreasing IGF-1 receptor survival signalling in vitro. To verify this hypothesis further in vivo, similar experiments were performed in mouse brain and in ischemia mouse brain. Our results demonstrated that glutamate also inhibited the survival signaling of IGF-1 in ischemia brain via NMDA receptor. Put together these results suggest a novel mechanism by which glutamate can reduce cell viability and induce neurotoxicity via affecting the survival signaling of growth factors. Supported by National Natural Science Fund of China (No. 30711120565; No. 30970935).

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Modulation of nociceptin cleavage, b-endorphine liberation, glutamate release and ATP secretion by non-opioid analgesic drug metamizol M. Vlaskovska1, S. Surcheva1, A. Tsakova1, and L. Kasakov2 1

Department of Pharmacology, Medical University Sofia, 1143 Sofia, Bulgaria 2 Institute of Neurobiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Metamizol (Analgin) is a widely used non-opioid analgesic drug with a strong antinociceptive effect that involves possibly central and peripheral mechanisms. Aim: Study of central/peripheral neurotransmitter mechanisms of Metamizol. Methodology: Nociceptin cleavage was studied in primary brain cortical cell cultures from neonatal (\12 h) Sprague–Dawley rats by electrospray ionization mass spectrometry. b-Endorphine liberation was quantified by RIA in hypophyseal slices and plasma before and after i.m. injection of metamizol in mature male Wistar rats. K+-evoked release of glutamate and aspartate was quantified in primary brain cortical cell cultures from neonatal (\12 h) Sprague–Dawley rats by HPLC analysis. Urothelial release of ATP was quantified in whole urinary tract preparation with in situ urinary bladder in mature male Sprague–Dawley P2X3 ++ / and matching P2X3 -/- rats by chemiluminiscence. Results: (i) metamizol stimulated nociceptin biotransformation increasing the levels of fragments 1–6, 1–9, 1–12, 1–13, 1–17, (ii) metamizol increased b-endorphine liberation with fivefold maximum at 45 min, which faded to control levels at 240 min, (iii) metamizol diminished K+-evoked release of glutamate and aspartate, (iv) metamizol did not change distension-induced ATP release. However metamizol inhibited pelvic sensory nerve distension-induced firing in P2X3 +/+ but not P2X3 -/- rats. Conclusion: metamizol analgesic action involves central and peripheral mechanisms.

PKMf maintains drug reward, aversion and extinction memories Yan-qing Li, Yan-xue Xue, Yin-yin He, Fang-qiong Li, Li-fen Xue, Chun-mei Xu, and Lin Lu* National Institute on Drug Dependence, Peking University, Beijing 100083, China *Corresponding author E-mail: [email protected] During abstinence, memories of drug-associated cues persist for many months and exposure to these cues often provokes relapse to drug use. The mechanisms underlying the maintenance of these memories are 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 in the extinction memory of reward and aversion cues. We found injections of the PKMf inhibitor ZIP into accumbens core but not shell after CPP training blocked the expression 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 morphine withdrawal. On the contrary, intra-basolateral amygdala but not central nucleus of the amygdala injection of ZIP 1 day after CPP and CPA training impaired the expression of CPP and CPA for up to 14 days. The ZIP’s effect was mimicked by the PKC inhibitor chelerythrine that inhibits PKMf, but not by the conventional and novel

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12th International Congress on Amino Acids, Peptides and Proteins PKC inhibitor staurosporine that does not effectively inhibit PKMf. We also found that inhibiting PKMf activity in the infralimbic cortex, but not prelimbic cortex, disrupted the expression of the extinction memory of CPP and CPA. These results indicate that PKMf activity in accumbens core is a critical cellular substrate for the maintenance of drug reward memory, whereas PKMf in the basolateral amygdala is required for the maintenance of both drug reward and aversion memories, and PKMf in the infralimbic cortex is required for the maintenance of extinction memory of reward and aversion cues.

Role of low-conserved areas of extracellular vestibule in function of purinergic P2X4 receptor V. Tvrdonova1, M. Rokic1, P. Kuzyk1, S. S. Stojilkovic2, and H. Zemkova´1 1 Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology of the Academy of Sciences of the Czech Republic, Prague 4, Czech Republic 2 Section on Cellular Signaling, Program in Developmental Neuroscience, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD 20892, USA

Purinergic P2X receptor channels (P2XR) are formed by three subunits each containing two transmembrane domains (TM1 and TM2). Crystallographic data showed that the channel pore is formed by TM2 and surrounded by TM1 helices which are connected with a large ectodomain by the W50-V61 and F324-I337 linkers forming an extracellular vestibule. The aim of this study was to identify residues in this region that are of importance for the P2XR function. All residues of external vestibule segments of rat P2X4R were substituted one by one with alanine or cysteine, the wild-type and mutant receptors were expressed in HEK293 cells, and examined using whole-cell recording. This 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 showed absence of V49 mutants in the membrane. The receptor function was preserved in V49L, Y54F and G325P mutants, whereas Q55E, Q55T and Q55N mutants were non-functional. Ivermectin, a positive allosteric modulator of P2X4R, was unable to restore of the function of Q55 and G325 mutants. These experiments suggest that conserved residues Q55, adjanced to TM1, and G325, distal to TM2, are essential for P2X4R function. Glutamine in position 55 could interact with some partner in its vicinity to form the proper three-dimensional structure of the vestibule in the functional receptor. It is also possible that glycine in position 325 operates as a hinge during conformational changes of vestibule, transducing the signal from ectodomain to TM2. This study was supported by the grants No. IAA500110910, 305/07/0681, 305/08/H037, the Grant Agency of Charles University (Grant No. 3446/2011) and the Centrum for Neuroscience (LC554).

Roles of oxytocin in the control of social recognition Tatsushi Onaka, and Yuki Takayanagi Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Shimotsuke-shi, Tochigi-ken 329-0498, Japan Oxytocin and its receptors have been shown to be implicated in social recognition. Mice lacking the oxytocin or oxytocin receptor gene

12th International Congress on Amino Acids, Peptides and Proteins show socio-behavioral deficits, including impaired social recognition. Administration of oxytocin improves social recognition, while oxytocin receptor antagonists impair social recognition. On the other hand, secretin receptors have also been reported to be involved in social recognition. Secretin receptor-deficient mice show impaired social recognition. Secretin administration has been shown to increase oxytocin mRNA in the hypothalamus. However, relationship between oxytocin and secretin concerning social recognition remains to be determined. Here, we examined effects of secretin administration upon expression of Fos protein in oxytocin-secreting neurons. An intracerebroventricular injection of secretin induced expression of c-Fos protein in oxytocin-secreting neurons of the hypothalamus and facilitated oxytocin release into the blood. Secretin also facilitated oxytocin release from the isolated supraoptic nucleus. We then investigated whether secretin increases social recognition, and whether the effects of secretin are blocked by an oxytocin receptor antagonist. Secretion facilitated social recognition and the facilitative effects of secretin were blocked by an oxytocin receptor antagonist. These data suggest that secretin facilitates social recognition via activation of oxytocin-secreting neurons.

Spike timing-dependent long-term depression requires presynaptic NMDA receptors Antonio Rodrı´guez Moreno University Pablo de Olavide, 41013 Sevilla, Spain Spike timing-dependent plasticity (STDP) is a strong candidate synaptic mechanism involved in cortical development and map plasticity. In STDP, the temporal order and precise timing of preand postsynaptic action potentials (spikes) determine the direction and magnitude of synaptic change. Both timing-dependent longterm potentiation (t-LTP) and timing-dependent long-term depression (t-LTD) depend on NMDA receptors. How the same type of receptor can be involved in opposite changes in synaptic efficacy is not well understood. Whereas it is established that postsynaptic NMDA receptors are necessary for t-LTP, we investigated here whether presynaptic NMDA receptors are necessary for timingdependent LTD and LTP in layer (L) 4-to-L2/3 excitatory synapses in mouse barrel cortex. We used paired whole-cell recordings of synaptically connected L4 and L2/3 cells. In five pairs, a prebeforepost pairing protocol was applied with 1 mM MK-801 in the presynaptic pipette. Robust t-LTP was induced (154 ± 5%, n = 5; p \ 0.01, t test), of similar magnitude to that seen with extracellular stimulation (153 ± 9%, n = 5), suggesting that presynaptic NMDA receptors are not neccessary for induction of t-LTP. In contrast, t-LTD was completely blocked when MK-801 was included in the presynaptic pipette (104 ± 6%, n = 6), whilst in pairs of cells without MK-801 this protocol induced robust t-LTD (76 ± 6%, n = 6; p \ 0.01, t test), indicating that presynaptic NMDA receptors are necessary for t-LTD. The different sites of NMDA receptors necessary for induction of t-LTP and t-LTD may have important consequences for the computational operation of cortical microcircuits and map.

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The accessibility in the external part of the transmembrane domain 5 of the glutamate transporter EAAT1 is conformationally sensitive during the transport cycle Xiuping Zhang1 and Shaogang Qu2* 1

China-America Cancer Research Institute, Guangdong Medical College, Dongguan, Guangdong, China 2Department of Immunology, Southern Medical University, Guangzhou, Guangdong, China *Corresponding author E-mail: [email protected] Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. Moreover, it serves to keep the extracellular glutamate concentration below neurotoxic level. However the change of accessibility of transmembrane domain (TM) 5 during the transport cycle is not clear yet. We used cysteine mutagenesis with treatments with MTSET to investigation the change of accessibility of TM5. 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 of cysteine mutants by MTSET. Inhibition of transport by MTSET 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 sensitive reactivity pattern. The sensitivity of L296C, G299C and K300C to MTSET was potentiated by glutamate and TBOA. Our results indicate that the accessibility of some positions of the external part of the TM5 of EAAT1 is conformationally sensitive during the transport cycle.

The effect of glutathione and its analogue UPF17 on activity of frontal cortical Na,K-ATPase of Alzheimer’s disease in vitro Ceslava Kairane, R. Mahlapuu, U. Soomets, and M. Zilmer Department of Biochemistry, Medical Faculty, University of Tartu, The Centre of Excellence for Translational Medicine, 19 Ravila St, 50411 Tartu, Estonia Glutathione (GSH) carries an important role in the human body antioxidant defense system, as the most prominent low-molecular weight thiol that occurs in millimolar ranges in the cells. Various structural modifications in tripeptidic GSH molecule have been carried out to improve its stability and cellular uptake. In our lab small library of glutathione analogues (UPF peptides) was designed and synthesized. UPF17 is a tetrapeptide in which O-methyl-L-tyrosine is added to the N-terminus of GSH and characteristic c-glutamate bond is changed to the a-glutamate bond. In present study we have examined the effects of the GSH and its analogue UPF17 to frontal cortical Na,K-ATPase activity in human

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S48 brain without and with Alzheimer‘s diagnosis in vitro. Results showed that GSH at physiological concentrations inhibits the activity of healthy controls Na,K-ATPase in a time and concentration dependent manner, but UPF17 inhibits in a concentration dependent and time independent manner. In Alzheimer’s disease brain preparations GSH showed only a tendency of concentration and time dependent inhibition of Na,KATPase activity meanwhile 20% inhibition of Na,K-ATPase by UPF17 did not depend on peptide concentration nor incubation time. Coincubation of UPF17 with cardiac glycoside ouabain increased inhibition of Na,K-ATPase indicating to the different ouabain and UPF17 binding sites on protein.

The inhibitory effect of glutamate on the survival promoting effect and signaling of IGF-1 in vitro and in vivo Zheng Wenhua Neuropharmacology, School of Pharmaceutical Sciences, Sun Yat-Sen University, East Waihuan Road, Higher Education Mega Center, Guangzhou 510006, Guangdong, People’s Republic of China Impairing intracellular signalling induced by excess glutamate play important roles in the process of neurodegenerative diseases. However, the underlying mechanism(s) and its interrelationships 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 hippocampal neurons with glutamate concentration-dependently inhibited the tyrosine phosphorylation of 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 by antagonists of the NMDA receptor but not by blockers of other ionotropic or metabotropic glutamate receptor sub-types verifying the involvement of the NMDA receptor. These findings demonstrate that glutamate can block the effect of IGF-1 by decreasing IGF-1 receptor suvival signalling in vitro. To verify this hypothesis further in vivo, similar experiments were performed in mouse brain and in ischemia mouse brain. Our results demonstracted that glutamate also inhibited the survival signaling of IGF-1 in ischemia brain via NMDA receptor. Moreover, inhibition of NMDA receptor enhanced the phosphorylation of IGF-1 receptor in ischemia brain and the protective effect of IGF-1. Put together these results suggest a novel mechanism by which glutamate can reduce cell viability and induce neurotoxicity via affecting the survival signaling of growth factors. Supported by National Natural Science Fund of China (No. 30670652; No. 30711120565; No. 30970935).

The puzzling problem of glutamate transport for DA neurons: when too much glutamate uptake becomes also toxic Laurence Had-Aissouni IBDML, UMR 6216 CNRS-Aix Marseille University, Campus of Luminy, Marseille, France By transporting both glutamate and cysteine, the neuronal excitatory amino acid transporter EAAT3/EAAC1/SLC1A1 regulates

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12th International Congress on Amino Acids, Peptides and Proteins glutamatergic transmission and supplies neurons with precursors of the main brain antioxidant, glutathione. This last function may be crucial for dopamine (DA) neurons that degenerate in Parkinson’s disease. Indeed, we had previously found that EAAT dysfunction induced by L-trans-2,4-pyrrolidine dicarboxylate (PDC), a substrate inhibitor of EAATs, is preferentially toxic for these neurons, as the subsequent decline in antioxidant defenses increased their vulnerability to NMDA receptor-mediated excitotoxicity. Here, we further investigate the interactions between PDC and glutamate induced toxicity in immature (8-day-old) and mature (12-day-old) mesencephalic 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 neurons and for both immature and mature non DA neurons. In immature non DA neurons, glutamate-induced cell loss was independent 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 PDC or DL-threo-b-benzyloxyaspartic acid) or system xc- (by 4-carboxyphenylglycine), but not by the combination of both treatments. In mature mesencephalic cultures, glutamate excess was primarily excitotoxic. The protective effect of glutamate transport inhibitors on glutamate toxicity was no longer observed in mature non DA neurons, but surprisingly appeared in mature DA neurons. These data suggest that a moderate glutamate transport is required for the viability of immature non DA and mature DA neurons in mesencephalic cultures, and that both excessive glutamate transport and complete blockade are toxic for these cells. This may be linked to oxidative deamination of glutamate into alphaketoglutarate, a TCA cycle intermediate, by glutamate dehydrogenase as both inhibition and excessive activation of this enzyme have been found to be toxic for DA neurons.

Time-dependent alterations of NMDAR function and synaptic plasticity by corticosterone in the adult hippocampus Tak Pan Wong Department of Psychiatry, McGill University, Douglas Mental Health University Institute, Canada Stress has an important impact on learning and memory, in part, through the actions of adrenal corticosterone (CORT). However, the mechanisms underlying the effects of CORT on synaptic plasticity, a cellular model of learning and memory, remain elusive. Here we provide evidence that CORT can mediate time-dependent changes in 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 synaptic responses mediated by NMDARs within 30 min. Surprisingly, following this fast-onset change, we observed a slow-onset ([1 h after the end of CORT exposure) change in the GluN2 subunit composition of NMDARs, so that the GluN2A/GluN2B ratio was increased. To investigate the effects of these distinct fast- and slowonset changes in NMDARs on synaptic plasticity, we examined the formation of long-term potentiation (LTP) and long-term depression (LTD) within these time windows. In parallel to the increased NMDAR function found during CORT treatment, both LTP and LTD were facilitated. However, after the slow-onset change in NMDAR subunit composition, synaptic plasticity was no longer facilitated. These findings suggest that the delayed NMDAR subunit change after

12th International Congress on Amino Acids, Peptides and Proteins CORT treatment is related to the loss of LTP and LTD facilitation. Our findings indicate that NMDARs in the adult hippocampus show remarkable plasticity in response to CORT. These CORT-mediated alterations of NMDAR function play a critical role in stress regulation of learning and memory.

Urocortin: past, present and a long road to go A. Fatima1, G. Wolf2, M. Engelmann2, and M. G. Spina

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1 Jamia Hamdard, Department of Pharmacology and Pharmacy Practice, Delhi -110062, India 2 Otto-von-Guericke Universita¨t Magdeburg, Institut fu¨r Medizinische Neurobiologie, Leipziger Str. 44, 39120 Magdeburg, Germany

Urocortin 1 (Ucn1) belongs to the mammalian corticotropin-releasing factor family. In brain, Ucn 1 mRNA and peptide distribution is found highest in the non-preganglionic Edinger-Westphal nucleus (npEW). In addition urocortin is also produced in other areas of the rat brain including the lateral superior olivary, hypothalamic supraoptic 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 administration in lab rodents. Further, several studies suggested an involvement of central Ucn1 in neuroprotection, stress adaptation pathway, reproduction, body temperature, alcohol consumption preference as well as learning and memory. Also the roles specific brain regions that produce Ucn1 play were explored to identify the neuronal networks that are involved in the physiological functions of this neuropeptide. Indeed, in these networks Ucn1 was found to colocalise and act in concert with other neuropeptides and neuromodulators. We investigated here the role of Ucn1 in the SON. We found that in the SON both Ucn1 and neuronal nitric oxide synthase are colocalised. Administering low doses of Ucn1 suggest a role of neuropeptide in controlling anxiety-related behaviour within the SON area. Further, our results indicate Ucn1 signalling within this hypothalamic nucleus has little impact on feeding behaviour. In contrast, a reduced immunoreactivity in npEW during pregnancy implies Ucn1 could contribute to an altered feeding behaviour and energy homeostasis observed during pregnancy. Plotted on the background of its selective distribution (versus that of its receptors) in the rodent brain as well as the functional interaction with other neuropeptides and signalling pathways our findings provide a complex picture of how Ucn1 might affect in behavioural regulation by controlling physiological parameters.

Vasopressinergic neurons in the anterior olfactory nucleus of the rat: a role in social odor processing Douglas W. Wacker*, Vicky A. Tobin, Julia Noack, Adrian J. Duszkiewicz, Valerie R. Bishop, Simone L. Meddle, Mario Engelmann and Mike Ludwig *Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK EH8 9XD and Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA We recently identified a large population of vasopressinergic neurons, as well as neurons immunopositive for vasopressin 1a and 1b

S49 receptors in the rat main olfactory bulb. The blockade of bulbar vasopressin 1 receptors causes deficits in conspecific social recognition, and vasopressin administration leads to an inhibition of mitral cell firing rate. We now have evidence for a large population of 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 and higher processing areas. Unlike the glutamatergic vasopressinergic neurons in the olfactory bulb, neurons expressing vasopressin in the AON are GABAergic and co-express the calcium-associated protein calbindin-D-28K. Adult rats exposed to a conspecific juvenile show increased immediate early gene expression (early growth response protein 1; Egr-1) in vasopressinergic neurons in the pars lateralis and pars distalis of the AON when compared with animals 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 do not induce increases in Egr-1 expression in vasopressinergic neurons of the AON. We hypothesize that vasopressin signalling in the MOB, intrinsically or via the AON, acts to filter recognized conspecific social odor information, thereby facilitating the formation of short-term social odor memories.

Nutrition Adipose tissue glutamine synthesis and the development of inflammation and insulin resistance Malcolm Watford, Roshni Patel and Samantha Dori Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ, USA Adipose tissue expresses very high glutamine synthetase (GS) activity that is increased during diet-induced obesity in mice. During the differentiation of 3T3-L1 cells into adipocyte-like cells, expression of GS is increased [50-fold with higher mounts of GS protein seen within 24 h of the initiation of differentiation. Exogenous glutamine is not required for the differentiation of 3T3-L1 cells into adipocytes if they continue to express GS. Knock-down of GS shows that monoclonal expansion, expression of C/EBPb, PPARc and C/EBPa, and maximal rates of lipid storage, all require glutamine. Given the relatively poor blood 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 to inflammation and insulin resistance. Glutamine is the major respiratory fuel of macrophages, but they express very low GS activity and cannot survive without large amounts of exogenous glutamine. However, when macrophages are cultured over mature adipocytes they are able to survive in the absence of exogenous glutamine providing that the adipocytes express glutamine synthetase. Macrophages do not survive in the absence of exogenous glutamine when they are cultured over pre-adipocytes, or over adipocytes where GS has been inhibited. We propose that large adipocytes provide glutamine, not only to maintain adipocyte function but also as a fuel for infiltrating macrophages. Therefore, adipose tissue glutamine synthesis plays a major role in the development of obesity induced inflammation and insulin resistance.

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Chinese Meishan pigs: an important resource for defining genomic regulation of swine reproduction J. J. Ford USDA/ARS/US Meat Animal Research Center, Clay Center, NE 68933 Chinese breeds of pigs are phenotypically diverse from European breeds. Of the many breeds that exist in China, the Meishan breed was imported into other countries for use in research with a primary interest being their large litter size. In a 2009 review, Onteru et al. noted 34 quantitative trait loci (QTL) for female reproductive traits reported in pigs. Of these, 20 were identified in Meishan 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 size were observed on the X chromosome, and thyroid-binding globulin (TBG) emerged as the potential gene associated with these differences. Boars with the Meishan allele for TBG experience pubertal development at a younger age, have smaller mature testes with fewer Sertoli cells, greater expression of inhibin/activin beta-B subunit and FSH beta subunit in their anterior pituitary glands, and more FSH in their blood than boars with the European allele. A model to explain these differences is that fewer Sertoli cells in boars with the Meishan allele for TBG provide lower secretion of inhibin thereby providing less negative feedback to the anterior pituitary allowing for greater synthesis of activin beta-B that stimulates greater synthesis of FSH within the pituitary and its greater secretion into the blood. The unusually high concentrations of FSH observed in Meishan boars were the primary stimulus for this series of studies.

Cooking causes the deterioration of prolamin digestibility in rice Masatoshi Kubota1, Yuhi Saito2, Takehiro Masumura2, Takehisa Kumagai3, Reiko Watanabe4, Shinobu Fujimura1,5, and Motoni Kadowaki1,5 1 Center for Transdisciplinary Research, Niigata University, Niigata, Japan 2 Graduate School of Life and Environmental Science, Kyoto Prefectural University, Kyoto, Japan 3 Kameda Seika Co., Ltd., Niigata, Japan 4 Department of Health and Nutrition, University of Niigata Prefecture, Niigata, Japan 5 Graduate School of Science and Technology, Niigata University, Japan

Background and Objective: Prolamin is one of the major storage proteins in rice and notoriously indigestible. However, there is no direct evidence that cooking changes rice prolamin digestibility and few knowledge about the molecular mechanisms of prolamin indigestibility. Consequently, we attempted to investigate the effect of cooking on the in vivo digestibility of rice prolamin by determining the gastrointestinal (GI) transit of rice protein in rats fed either a rice flour (RF)- or cooked rice (CR)-based diet and the mechanism to cause prolamin indigestible by an in vitro digestion method.

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12th International Congress on Amino Acids, Peptides and Proteins Methods: The digesta in the GI tract and the feces were subjected to western blotting and the morphological observation using immunoelectron microscopy. Furthermore, by using the in vitro digestion method with pepsin (2 h) followed by pancreatin (6 h), the treatment of indigestible starch degraded-rice protein (SD-RP) with dithiothreitol (DTT) and urea was employed to see molecular interactions in protein body-I (PB-I) affecting prolamin digestibility. Results: In the in vivo digestion method, the band intensity of 13 kDa prolamin (13P) in the GI digesta, especially the caecum/colon contents, and the feces of RF group was decreased compared with that of CR group. In the in vitro digestion method, a high dose of DTT (1.5 M) improved prolamin digestibility of SD-RP, while urea (8 M) could have an enough effect only by combining with a low dose of DTT (200 mM). These results suggest that rice prolamin becomes indigestible after cooking and the intermolecular interaction in PB is important to make prolamin indigestible.

Dietary arginine supplementation altered expression of IGFs and IGF receptors in weaning piglets Rongjun Chen1,2*#, Wence Wang2,3#, Yulong Yin2*, Kang Yao2, Yunling Gao2,3, and Tiejun Li2 1

Rice Research Institute of Sichuan Agricultural University, Chengdu, Sichuan 611134, China 2 Key Laboratory of Animal Nutritional Physiology and Metabolic Process and Key Laboratory of subtropical Agro-ecology, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, Hunan 410125, China 3 The Graduate School of The Chinese Academy of Sciences, Beijing 100039, China To whom correspondence should be addressed: Yulong Yin. E-mail: [email protected]. Tel:86-73184619703 fax: 86-731-84612685; [email protected] #Rongjun Chen and Wence Wang contribute equally to this paper [email protected]; [email protected] [email protected]; [email protected] Early weaning may lead to the stress syndrome and increased occurrence of enteric diseases and diarrhea in nursery pig management. Dietary Arginine supplementation may decrease the severity of the weaned stress syndrome in early-weaned piglets. The insulin-like growth factor (IGF) signaling pathway is an important regulatory factor in regulating fetal and placental growth, proliferation, differentiation, migration and aggregation, and inhibit apoptosis of mammalian cells. However, whether insulin-like growth factor system expression is changed in piglets with Dietary Arginine supplementation is unclear. This study was conducted to investigate the effect of dietary arginine supplementation in modulation IGF system of weanling piglets. Twelve, 21-day-old healthy piglets (Landrace 9 Yorkshire) with a mean body weight (BW) were divided into 2 groups randomly. The test group was supplemented with 0.6% L-arginine, the control group was fed with 1.23% L-alanine (isonitrogenous control). At 28 days of age, 12 piglets were killed and longissimus muscle, liver and kidney were collected. IGF-I was increased in three tissues of arginine group (P \ 0.05). IGF-II was increased in muscle of arginine group. Both muscle and liver have a higher level of IGFBP5 with arginine supplementation (P \ 0.05). These results showed that arginine can alleviate weaning stress and improving growth performance in early-weaned piglets through IGFs and IGF receptors.


Dietary L-Arginine supplementation affects protein expression in insulin-sensitive tissues of diet-induced obese rats Jun-Jun Wang1,2, Wenjuan Shi Jobgen2, Cynthia J. Meininger3, and Guoyao Wu1-3 1 State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China 2 Department of Animal Science, Texas A&M University, College Station, TX, 77843, USA 3 Department of Systems Biology and Translational Medicine, Texas A&M Health Science Center, College Station, TX, 77843, USA

This study was conducted to test the hypothesis that dietary arginine supplementation reduces fat mass in diet-induced obese rats. Male Sprague–Dawley rats were fed either low- or high-fat diets for 15 weeks (16 rats/diet). Thereafter, lean or obese rats continued to be fed their same respective diets and received drinking water containing either 1.51% Larginine-HCl or 2.55% alanine (isonitrogenous control) (n = 8/treatment group). Twelve weeks after the initiation of the arginine treatment, rats were euthanized to obtain tissues for biochemical analyses. Results were statistically analyzed as a 2 9 2 factorial experimental design using ANOVA. High-fat diet increased (P \ 0.05) the mass of white adipose tissues at different anatomical locations by 49–96% compared to the lowfat diet. L-Arginine supplementation reduced (P \ 0.05) white adipose tissue mass by 20–40% while increasing brown adipose tissue mass by 15–20% and enhancing glucose and oleic acid oxidation in skeletal 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. Hapatic glutathione peroxidase was more abundant in argininesupplemented rats, compared with nonsupplemented rats. Collectively, these results indicate that arginine regulates energy metabolism and antioxidative responses in a tissue-specific manner. The findings have important implications for treating obesity in humans and companion animals as well as decreasing fat deposition in livestock species. Supported by AHA and the China Thousand-People Talent program.

S51 The purpose of this study was to test the hypothesis that the dietary Larginine supplementation had beneficial effects on edema disease. 156 KunMing mice were randomly assigned to arginine group 1 (0.6% 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 control group 2 (1.22% alanine + basal diet, n = 34). After 3 days of adaptive feeding and a 7 days treatment period with the prepared feed, all mice were challenged by intraperitoneal injection Escherichia coli O139 (E. coli) at LD50 (2.53 9 108 CFU/ml). Arginine group 2 and control group 2 were used to calculate the mortality after 20 h of injection. Serum concentrations of platelet-activating factor (PAF), interleukin (IL)-2, interleukin (IL)-10, secretory immunoglobulin A (sIgA), superoxide dismutase (SOD) activity, total antioxidant capac0 0 ity(T-AOC), cyclic 3 ,5 -adenosine monophosphate (cAMP), cyclic 0 0 3 ,5 -guanosine monophosphate (cGMP) were measured in arginine group 1 and control group 1 in a 10-h interval for three times. The serum concentration of PAF was much lower (P \ 0.01) in arginine group than alanine group in all times. Additionally, T-AOC and SOD activity in the experiment group were increased significantly (P \ 0.05) in the first 10 h after initial injection. Unfortunately, T-AOC and SOD activity in arginine group become quiet (P [ 0.05) compared to the control group after that except T-AOC, was greater (P \ 0.05) in arginine group than the control group in 20 h of initial injection. Meanwhile, arginine supplementation had little effect on the mortality of mice, serum IL-2, sIgA, cAMP, cGMP level. In conclusion, dietary arginine supplementation can partially attenuate the damage caused by edema disease, but have little effect on the clinical result. Keywords: Arginine, Edema disease, Escherichia coli, Plateletactivating factor

Effect of dietary leucine to lysine ratios on performance of growing pigs John K. Htoo1, Hećtor Garcia2, Miguel Cervantes2, Adriana Morales2, and Alfonso B. Araiza2 1

Effect of dietary L-arginine supplementation on edema disease Wenkai Ren1,4,*, Guan Yang2,*, Xiangwei Tang 2, *, Yinghui Li 1,4, Gang Liu1, ,Sisi Cai 3, Yulong Yin 1, , Xinglong Yu5, and Guoyao Wu6 1

Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 2 College of Animal science & Technology, Hunan Agricultural University, 410128 Changsha, Hunan, China 3 Department of veterinary medicine, Orient Science & Technology College of Hunan Agricultural University, 410128 Changsha, Hunan, China 4 Graduate School of Chinese Academy of Sciences, Beijing 100039, China 5 College of Veterinarian, Hunan Agricultural University, Changsha, Hunan 410128, China 6 Department of Animal Science, Texas A&M University, College Station, TX, USA 77843 *[email protected] or [email protected]

Evonik Degussa GmbH, 63457 Hanau-Wolfgang, Germany Instituto de Ciencias Agrıćolas, UABC, Mexicali, Me´xico

2

Leucine (Leu) appears to regulate protein synthesis, but an excess of Leu can reduce feed intake and performance of pigs. To determine the optimal dietary standardized ileal digestible (SID) 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 wheat and wheat bran based diet was formulated, using analyzed ingredient amino acid (AA) contents to meet requirements of SID AA other than Leu (0.68%) and Lys (0.77%). L-Leu was added to the basal diet to create 5 SID Leu:Lys (88, 100, 120, 140 and 160%). Diet 6 was produced to be equivalent to diet 4 but contained 12% more Ile. Samples of intestinal mucosa, liver, and muscles were collected from 8 pigs per each of treatments 1, 3 and 5, respectively. mRNA extraction was performed followed by quantitative expression of cationic AA transporters b0,+ and CAT-1. There was a linear and quadratic response in ADG and a quadratic response in FCR of pigs as the Leu:Lys increased from 88 to 160% (p \ 0.01). Increasing SID Leu:Lys from 88 to 120% increased (p \ 0.05) the expression of b0,+ and decreased (p \ 0.10) CAT-1; no further response was observed when increased to 160%. Two-slope linear regression estimated the SID Leu:Lys of 104 and 108% to optimize ADG and FCR, respectively.

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Effect of immune system stimulation on whole body protein deposition of growing pigs fed tryptophan limiting diets John K. Htoo1, Crystal L. Levesque2, Karl de Ridder2, and Cornelis F.M. de Lange2 1

Evonik Degussa GmbH, 63457 Hanau-Wolfgang, Germany Department of Animal and Poultry Science, University of Guelph, N1G 2W1, Guelph, Canada

2

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 evaluated using 36 pigs (20.0 ± 1 kg; 12 pigs/block). Pigs were assigned to one of 5 diets and fed restrictively (800 g/day). Diets 1 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 (0.89 g/kg), and all other essential AA equivalent to diet 4. Following a 5-day adaptation, pigs were injected every 2 days with E. coli lipopolysaccharide (20 lg/kg BW, increasing 15% each injection). Plasma acute phase protein increased (p \ 0.001) following ISS. There were no interactions of dietary Trp level and ISS. Nitrogen retention decreased linearly (p \ 0.001) with decreasing Trp intake. Whole body N retention was lower (p \ 0.001) compared with Pre-ISS during ISS-1 due to an increase (p \ 0.001) in urinary N excretion. Adding Trp to diet 4 increased N retention (p \ 0.05; diet 4 vs. 5). Regression of response of protein deposition (PD) against Trp intake revealed that Trp requirement increased at 9.1% during ISS-1 compared with prechallenge for achieving the same PD.

Effect of movement training on the amino acids distribution and intestines morphosis in rats Min Gong1,2, Wenkai Ren4, Yulong Yin1,4,* Dehua Wang3, Gang Liu 4,* and Guoyao Wu4,5 1 State Key Laboratory of Food Science and Technology and College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China 2 Jiangxi Science & Technology Normal University, Nanchang 330013, China 3 Nanchang Ctr Dis Control & Prevent, Nanchang 330038, Peoples Republic of China 4 Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 5 Department of Animal Science, Texas A&M University, College Station, TX, USA 77843 *[email protected] or [email protected]

This work was conduct to study the effect of high intensity movement training on the amino acids metabolism and distribution in internal organ and intestinal morphosis. 40 Sprague–Dawley male rats were randomly divided into exercised group and sedentary group. On day 10 after the initiation treatment, average daily feed intake became significant lower, and the average weight became significant lower on day 12, compared with the sedentary

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12th International Congress on Amino Acids, Peptides and Proteins group. There wasn’t significant difference about amino acids in the serum but valine, which was significant lower than sedentary group’s. Movement training increased significantly all amino acids content except cysteine in the intestinal tissue but failed in the liver and muscle, even though all amino acids were higher in the muscle than sedentary group but cysteine. Amino acids’ digestibility was lower in exercised group and it became significant lower, when it comes to lysine and histidine. Additionally, Movement training decreased the numbers of lymphocyte but had no effect on the goblet cell, villi height and crypt depth in middle jejunum.

Effects of dietary arginine or N-carbamylglutamate supplementation on reproductive performance and immunity of sows infected with porcine reproduction and respiratory syndrome virus De Wu*, Lianqiang Che, Ping Yang, Zhengfeng Fang, and Yan Lin Key Laboratory for Animal Disease Resistance Nutrition of the Ministry of Education of China, Animal Nutrition Institute, Sichuan Agricultural University, Ya’an 625014, Peoples Republic China *To whom correspondence should be addressed. Tel: +86-835-2885107 Fax: +86-835-2885065 E-mail: [email protected] This study investigated the effects of dietary supplementation with LArginine (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 (Landrace 9 Large white) with body condition score at 3 received corn and soybean-based control diet during the first 30 days of gestation and were allocated to 5 groups receiving control, 1% L-ArginineHCL (Arg114), 0.1% NCG (NCG114) diet until farrowing, whereas the rest 2 groups received 1% L-ArginineHCL (Arg90), 0.1% NCG (NCG90) diet only until day 90 of gestation followed by control diet until farrowing. Litter performance was recorded at parturition. Blood samples collected at days 30, 90 and 110 of gestation were measured for metabolic and immunological parameters. The RT-PCR detection of serum PRRSV showed sows were infected with PRRSV prior to experiment. At parturition, total litter size was not markedly affected by dietary Arg or NCG supplementation. As a result of less pigs born dead, however, sows in Arg114 group had more pigs born alive than sows in control group (+1.6 pigs, P \ 0.05), total and live litter weights were increased (+1.7*2.3 kg, P \ 0.05) in Arg114 group relative to control and Arg90 groups. However, no significant litter size response to NCG supplementation was observed. Compared with control group, likewise, dietary Arg but not NCG supplementation increased (+12*46%, P \ 0.05) the levels of ornithine, proline and arginine at both days 90 and 110 of gestation. However, both dietary Arg and NCG supplementation decreased (-10*18%, P \ 0.05) the level of plasma urea at days 90 and 110 of gestation. In addition, dietary Arg but not NCG supplementation boosted humoral immunity as indicated by higher serum immunoglobulin and PRRSV antibody level in Arg-supplemented sows. These findings indicate dietary arginine supplementation can improve litter performance and humoral immunity, and intriguingly the effect of arginine supplementation on fetal growth is evident in late gestation. However, the positive effect of arginine supplementation on pregnancy and immunity can not be substituted by the current dose of NCG.


Effects of peptides of soy isoflavones on the male reproductive regulators in male Xiang Pigs Xiaoxue Yuan 1, Lili Li1*, Yulong Yin1*, Juexin Fan2, Chaowu Xiao3, and François Blachier3 1

Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 2 College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, China 3 Hlth Canada, Hlth Prod & Food Branch, Nutr Res Div, Food Directorate, Ottawa, Canada; 3 USC 914 INRA/AgroParisTech, Nutrition Physiology and Alimentary Behavior, 16 rue Claude Bernard, 75005 Paris, France *[email protected] or [email protected] 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 the effects of soy isoflavones on male reproductive regulators in Black Small-eared pigs. The results shown that dietary supplementation with 250 mg/kg of soy isoflavones increased (P \ 0.05) performance, serum testosterone levels and mRNA expression of testosterone biosynthesis regulatory protein StAR. However, a higher doses of 500 mg/kg suppressed (P \ 0.05) the reproductive performance of male pigs. Our finding suggested that soy isoflavones can affect the male reproductive hormone secretion, the growth and development of testis and epididymis, enzyme activity of testosterone synthesis, and expression of reproductive hormones’ gene in the brain, and in dosage-dependant ways. Keywords: Soy isoflavones, Testicle, Reproductive hormone, Testosterone synthesis, Male Xiang pig

Effect of wheat DDGS to grain ratio on changes of protein subfraction profiles in oats, barley and corn Daallkhaijav Damiran1, and Peiqiang Yu1* College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon (*Email: [email protected]) The objective of this study was to investigate the effects of wheat dry distiller grains with solubles (DDGS) to cereal grain ratio on changes of true protein and protein (CP) subfractions. The CP fractions were partitioned according to the Cornell Net Carbohydrate and Protein System and included True protein (TP), PA, PB1, PB2, PB3 and PC for protein subfractions. Each subfraction has different degradation behaviour (degradation rate and extend) which are highly related to component nutrient availability. Three type of grains with wheat DDGS samples from two bioethanol plants were mixed manually to combine in ratios of 4:0, 3:1, 2:2 and 1:3. This has resulted in total of 24 (3 9 2 9 4 combinations of each) mixtures. The results show that the effect of wheat DDGS inclusion had significant effects (P \ 0.05) on CP fractions in oat, barley and corn. Within wheat DDGS inclusion rate increased (0:4, 1:3, 2:2, 3:1), soluble protein PA fraction increased (P \ 0.05) with a range from 227 to 308 (oats), 156–301 (barley), 162–308 (corn) g/kg CP. But rapidly degradable protein PB1 fraction decreased (P \ 0.05) with a range from 181 to 85 (oats), 89–20 (barley), 144–77 (corn)

S53 g/kg CP. Intermediately degradable protein PB2 fraction also 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), 153–300 (barley), 181–308 (corn) g/kg CP. Undegradable protein PC fraction increased (P \ 0.05) with a range from 24 to 132 (oats), 24–92 (barley), 24–132 (corn) g/kg CP. True protein decreased with a range from 349 to 560 (oats), 821–568 (barley), 815–560 (corn) g/kg CP. In conclusion, the protein fraction profiles of grains can be significantly modified when combination with wheat DDGS. These changes will result in significant impact on degradable kinetics and nutrient availability. Keywords: Wheat-based dried distillers’ grains with soluble, Protein and carbohydrate fractions, Nutrient variation and availability

Expression and characterization of bovine lactoferrampin-lactoferricin in Pichia pastoris Xiangshan Tang1,2, Zhiru Tang3 Shengping Wang1,2, Zemeng Feng1,2, Dong Zhou1,2, Tiejun Li1 and Yulong Yin1* 1 Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering, Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 2 Graduate University of the Chinese Academy of Sciences, Beijing 100039, China 3 College of Animal Science and Technology, Southwest University, Chongqing, 400715, China *[email protected]

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N1-domain of bovine lactoferrin. The bactericidal activity of fused peptide LFA-LFC was stronger than that of LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limited the clinical application of antimicrobial peptides. Expression of recombinant peptides in yeast may be an effective approach. At present, the expression of recombinant peptide LFA-LFC in yeast was not reported. Here, we present the expression, purification and antibacterial activity of LFA-LFC using Pichia pastoris expression system. In our study, the gene of bovine lactoferrampin-lactoferricin (LFA268–284-LFC17–30) was obtained by PCR and cloned into the vector pPICZaA. The SacI-linearized plasmid pPICZaA- LFALFC was transformed into P. pastoris KM71 by electroporation and screened by PCR. The expression of LFA-LFC was induced about 72 h with 0.5% methanol at 28°C, One milliliter samples of the expression medium were taken at 24 h intervals for an activity assay. Antibacterial activity of fifty microliters of each expression supernatant was assayed by measuring zones of growth inhibition in thin agar plates with E. coli 0149. Fifty microliters of supernatant from cultures was freeze-dried and dissolved in 2 mL sterile water; Cation-exchange chromatography and size-exclusion chromatography were used for Purification of recombinant LFA-LFC. LFA-LFC was collected and 16.5% Tricine-SDS-PAGE was performed. Molecular weight of recombinant LFA-LFC was analyzed by MALDI-TOF mass spectrometry. The direct sequencing results indicated that the integrity of LFA-LFC recombinant was correct. Zones of growth inhibition of samples taken from the culture were 1.8 ± 0.3, 5.3 ± 0.7, 8.8 ± 0.7 mm at 24, 48 and 72 h,

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S54 respectively. Tricine-SDS-PAGE and mass spectrometry analyses demonstrated that the molecular weight of the purified LFA-LFC was 4.0 kDa, which is consistent with the theoretical molecular weight (3,953.75 Da). Our results showed that Pichia pastoris was a suitable system for secreting LFA-LFC. Bioactivity assay proved that the recombinant LFA-LFC had antimicrobial effects. Recombinant antimicrobial peptide LFA-LFC may serve as an attractive candidate for the development of antimicrobial preparations. Keywords: Bovine lactoferricin, Bovine lactoferampin, Antimicrobial peptides, Pichia pastoris

Functional amino acids in nutrition and health Guoyao Wu Department of Animal Science and Faculty of Nutrition, Texas A&M University, College Station, Texas, USA 77843; State Key laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China Based on nitrogen balance or growth, amino acids (AA) were traditionally classified as nutritionally essential or nonessential for animals and humans. Nutritionally essential AA (EAA) are those AA whose carbon skeletons are not synthesized by animal or human cells and, therefore, must be provided in the diet. Nonessential 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 mammals (including humans, pigs, cats, and dogs), birds, and fish. It had long been tactically assumed that animals or humans could synthesize sufficient amounts of all NEAA and did not need them in diets for optimal nutrition or health. However, a careful examination of the literature does not substantiate this assumption. Growing evidence from animal studies shows that: (1) some of the NEAA (e.g., arginine, glutamine, glutamate, glycine, and proline) and leucine play important roles in multiple signaling pathways, thereby regulating gene expression, intracellular protein turnover, nutrient metabolism, and oxidative defence; (2) young or gestating mammals cannot synthesize sufficient amounts of all NEAA to support maximum neonatal growth or embryonic/fetal survival; and (3) some NEAA (e.g., arginine) can reduce adiposity and ameliorate cardiovascular dysfunction in genetically obese and dietinduced obese animals. Beneficial roles for arginine and glutamine in improving metabolic profiles, cardiovascular function, immune responses, and intestinal health have also been reported for humans. Clearly, cell- and tissue-specific functions of AA beyond protein synthesis should be taken into consideration in recommendation of nutrient requirements for animals and humans. Recognizing that the long-standing classification of AA as EAA or NEAA has major conceptual limitations in protein nutrition, we have recently developed the concept of functional AA (FAA) to capitalize on the new developments of this field. FAA are defined as those AA that regulate key metabolic pathways to improve health, survival, growth, development, lactation, and reproduction of organisms. Recent advances in FAA are the focus of the session on ‘‘Functional Amino Acids in Nutrition and Health’’ at the 12th International Congress on Amino Acids, Peptides and Proteins at Beijing, China between August 1 and 5, 2011. This mini-symposium was supported, in part, by Ajinomoto Inc. (Tokyo, Japan) and National Renderers Association (Alexandria, USA).

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Glutamate: nutrition, metabolism and signaling John T. Brosnan and Margaret E. Brosnan Memorial University of Newfoundland, St. John’s, NL, Canada Glutamate is a central amino acid, both with regard to carbon and nitrogen metabolism. It also occurs at very high concentrations in the brain where it is the major inhibitory neurotransmitter. Glutamate is an important component of the taste of protein-rich foods. As monosodium 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 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 be synthesized within the body. Recent work has centered on glutamate as a signaling agent. The identification of umami receptors (requiring both glutamate and a purine nucleotide such as IMP) in taste buds may provide insights into a dietary protein receptor. Very recently, glutamate receptors have been found throughout the gastrointestinal tract. Glutamate dehydrogenase (GDH) plays a key role in the increased insulin secretion that occurs upon ingestion of a proteinrich meal. Elevated leucine activates GDH in the beta-cells, causing increased ATP production from increased glutamate oxidation. This increased [ATP] results in increased insulin secretion. A genetic disorder, HI/HA Syndrome, in which GDH is constitutively active causes hyperinsulinemia and hypoglycemia in children.

Highly efficient production and product design of VECYGPNRPQF, a potent antioxidative peptide can efficiently quench a variety of free radicals Zemeng Feng1,2, Xiaoli Zhou3, Tiejun Li1, and Yulong Yin

1*

1 Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 2 Graduate University of the Chinese Academy of Sciences, Beijing 100039, China *[email protected]

Oxygen is essential for the survival of aerobic organisms. Through oxidative phosphorylation, organisms obtain large amounts of energy supply for metabolism, with producing reactive oxygen species (ROS) as a price. ROS have roles in cell signaling, bits of ROS are benefit for the health. But overmuch ROS can induce oxidative stress. When in oxidative stress, an uncontrolled fashion causes significant reversible or irreversible damage to a wide range of biological molecules causing a cascade of chain reactions resulting in cellular damage and disease. Tissue damage caused by oxidative stress has a role in a number of pathophysiological conditions including neurodegenerative disorders (Alzheimer’s and Parkinson’s diseases), diabetes mellitus, atherosclerosis, autoimmune disease, and ischaemia/reperfusion injury. A potent anti-oxidative peptide (VECYGPNRPQF, in this paper was named AOP) was isolated from microalgae, Chlorella vulgaris, by Sheih in 2009, which efficiently quench a variety of free radicals, including hydroxyl radical, superoxide radical, peroxyl radical, etc. In our research, AOP and recombinant Tat-AOP were highly efficient expression using Pichia pastoris KM71H fermentation. To avoid generating excrescent amino acid, Red/ET recombination was applied

12th International Congress on Amino Acids, Peptides and Proteins to construct the expression vector pPICZaA-AOP and pPICZaA-TatAOP by insertion a PCR amplified DNA fragment coding AOP or TatAOP into plasmid pPICZaA, at the position right after the cleavage sequence of Kex2. The vector pPICZaA-AOP and pPICZaA-Tat-AOP were transformed into KM71H yeast cells, and positive colonies harbouring genomic integration of multi-copy AOP or Tat-AOP gene were screened out and used for fermentation. Tricine-SDS-PAGE and MALDI-TOF mass spectrometry were used to ensure the peptides were correctly and successfully expressed. AOP and Tat-AOP were purified by two-step column chromatography. The bioactivity of AOP and Tat-AOP were tested in both in vitro an in vivo. Several coated methods containing cyclodextrin, alpha amino boric acid derivatives (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 of AOP and Tat-AOP. Both the two peptide can be used in feed additive industrial and pharmaceutical application.

Identification of the protein composition of five different feed stuffswith Tricine-SDS-PAGE system Li Ai-ke, ZHOU Nai-ji, and Han Fei Academy of State Administration of Grain, 100037, Peoples Republic China Objective: The feasibility of the identification of protein and the analysis of the quality of different feedstuffwith Tricine-SDS-PAGE system was discussed. Method: The protein composition of five feedstuffs, rapeseed meal, cottonseed meal, extract from rapeseed meal cottonseed meal fermented by Monilia tropicalis and cottonseed meal fermented by black mold, was identified with Tricine-SDS-PAGE system. Results: The better isolation efficiency could be produced with 4.0%C and 16.5%T. The protein band of rapeseed meal was showed in the central part (14.4–60.0 kDa) and cottonseed meal, end part (about 90 kDa), which contain was higher among all of experimental materials and the protein in the method was easy to be effectively separated. The result indicated that the effect of the cottonseed meal treated fermented by black mold was better that fermented by Monilia tropicalis. Conclusion: There was significant different in protein image among five feedstuffs and the method of Tricine-SDS-PAGE ought to be the effective method in identification of the protein in feedstuff.

Inhibitory effects of food-derived components on AGE-RAGE signaling Seiichi Munesue, Yasuhiko Yamamoto, So Motoyoshi, Saito Hidehito, Takuo Watanabe, and Hiroshi Yamamoto Department of Biochemistry and Molecular Vascular Biology, Kanazawa University Graduate School of Medical Science Advanced glycation end-products (AGE) have been implicated in aging and the pathogenesis of diabetic complications, inflammation, Alzheimer’s disease, and cancer. AGE engage the cell surface receptor for AGE (RAGE), which in turn elicits intracellular signalling, leading to activation of NFjB to cause deterioration of tissue homeostasis. AGE are absorbed into the body partly through the consumption of foods rich in AGE. However, it is still controversial whether food-related AGE are harmful to health or not. In this study, we tested the effects on RAGE

S55 signalling of food-derived AGE from soy sauce, a Japanese condiment produced by fermenting soybeans. We first fractionated soy sauce into higher ([5,000) and lower (\5,000) molecular-weight fractions. Lower molecular-weight fractions (LMF) was found to inhibit the AGEinduced activation of NFjB in a dose-dependent manner. In contrast, higher molecular-weight fractions (HMF) activated the RAGE signalling. A plate binding assay revealed that the LMF could inhibit and compete AGE-RAGE association. In addition, LMF significantly reduced AGE-dependent MCP-1 secretion from peritoneal macrophages. These results thus indicate that soy sauce-derived small molecular AGE components are rather able to antagonize RAGE signalling and could exhibit beneficial effects on our health.

Integrative application of sequential digestion and elution to the membrane proteome analysis of rat hippocampal cells Xia Xiong1, Jian-ying Shen, Yulong Yin1*, Xian-chun Wang*, and Song-ping Liang* 1 Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 2 College of Life Sciences, Hunan Normal University, Changsha 410081, China *[email protected]

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively plasma membrane (PM) especially the low-abundance plasma membrane (PM) proteins from hippocampal cell, the subsequent study employed multiple analytical strategies, including the sucrose density gradient centrifugation and sequential washing. The remaining membranes were then extracted by Sodium deoxycholate (SDC) and subjected to SDC-assisted on membrane digestion with trypsin. Finally, tube-gel digestion method was treated the remaining membrane. A total of 712 non-redundant proteins were identified including receptor protein, transport protein, signaling protein, structural protein, etc. The result indicated that the integration and application of fraction digestion and elution method could successfully dissipate the membrane and promoted the digestion efficiency. Keywords: Sequential digestion, Proteome, Hippocampus

Intrauterine growth restriction and age alter expression of amino acid transporter b0,+AT in suckling Huanjiang mini-piglets W. C. Wang, D. Z. Fu, X. F. Kong*, M. M. Geng, H. S. Yang, X. Y. Song, and Y. L. Yin* 1

Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China [email protected] or [email protected] Intrauterine growth restriction (IUGR) is a major problem in human medicine and animal production. Efficiency of nutrient utilization is high in neonates with normal birth weights (NBW) but is low in those with

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S56 IUGR. However, the underlying mechanisms are largely unknown. Amino acid transporters take a pivotal role in the small intestine absorption system and growth, especially b0,+AT mediating apical 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 age affect expression of b0,+AT in small intestine, the major organ involved in the digestion and absorption of dietary nutrients. Suckling 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 collected for analysis. Data showed that mRNA abundance and protein 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 abundance and protein amount of the b0,+ AT in piglets with IUGR was lower (P \ 0.05) compared with the piglets with NBW in each timepoint. These findings suggested that the suckling piglets with IUGR did not have a well-developed intestine absorption system due to the transportation of intestine amino acid was not sufficient for their growth. Keywords: Intrauterine growth restriction, Amino acid transporter, Huanjiang mini-pigs Funded by 30901040, 30928018, 2007EA790004.

Metabolism and transporter expression of neutral amino acids in suckling Huanjiang mini-piglets with different body weight Huansheng Yang, Dezhi Fu, Xiangfeng Kong*, Wence Wang, and Yulong Yin* 1

Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 2 Graduate University of the Chinese Academy of Sciences, Beijing 100039, China 3 College of Animal Science and Technology, Southwest University, Chongqing 400715, China *[email protected] or [email protected] Genetic selection strategies towards higher prolificacy resulted in more and more impaired fetal development and increased littler size. The piglets with small body weight (SBW) have long-term alterations in structure, physiology and metabolism, and permanent negative growth performance and sub-optimal carcass quality. The present study was conducted to measure the metabolism and transporter expression of neutral amino acids in piglets with large body weight (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, respectively, and the jejunum was collected for analyzing transporter expression by real-time RT-PCR and Western blot. The levels of neutral amino acids in plasma, liver and skeletal muscle were measured by automatic amino acid analyzer. Results showed that, compared with the LBW littermates, SBW piglets had higher levels of some neutral amino acids in liver and skeletal muscle, while lower plasma levels during the suckling period. Consisted with the level alteration in neutral amino acids, the expression profiles of system ASC amino acid transporter-2 (ASCT2) in jejunum were changed, with lower expression levels in SBW piglets compared with the LBW littermates. These findings suggested that the intestine dysfunction may be one of the reasons in altering physiology and metabolism states of other organs which result in lower growth rate and carcass quality.

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12th International Congress on Amino Acids, Peptides and Proteins Keywords: Neutral amino acids, Body weight, Transporter, Huanjiang mini-piglets Funded by 30901040, 30928018, 2007EA790004.

Molecular cloning and expression profiling of excitatory amino acid carrier 1 in Huanjiang mini-piglets with different body weight Dezhi Fu, Huansheng Yang, Xiangfeng Kong*, Wence Wang, and Yulong Yin* Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China [email protected] or [email protected] Dietary glutamate, as a major oxidative fuel for gut and an important 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 and development of intestine. Excitatory amino acid carrier 1 (EAAC1) is the major transporter of glutamate in the intestine. The present study was conducted to measure the effects of body weight (BW) on the expression of EAAC1 in jejunum during suckling period using Huanjiang mini-piglets as a model. EAAC1 was cloned from Huanjiang mini-pig using PCR method, which encoded a deduced 524-AA protein with 8 putative transmembrane domains. The jejunum expression pattern of EAAC1 in suckling Huanjiang mini-piglets with large BW (LBW) and small BW (SBW) was tested by real-time RT-PCR and Western Blotting, respectively. Comparing with the LBW piglets, SBW piglets had lower expression levels in both mRNA and protein during the early suckling period, and the contents of glutamate in liver and muscle changed at this period. These findings suggested that the dysfunction of intestine will influence the metabolism states of other organs and EAAC1 may be a potential target for improving intestine function in SBW piglets. Keywords: Excitatory amino acid carrier 1 (EAAC1), Body weight, Huanjiang mini-pig

Monosodium glutamate intake and body weight in Vietnamese adults Vu Thi Thu Hien1, Nguyen Thi Lam1, Le Thi Hop1, and Shigeru Yamamoto2 National Institute of Nutrition, Vietnam 2 International Nutrition, Department of Food and Nutritional Sciences, Graduate School of Human Life Sciences, Jumonji University, Japan Background: Vietnamese have been familiar with umami from traditional fermented seasonings, which are rich in the umami amino acid, Glutamate. There have been controversial reports concerning the association between monosodium glutamate (MSG) intake and overweight/obesity. Objective: To determine the association between MSG intake and overweight in Vietnamese adults. Method: A cross-sectional survey was carried out in Vietnamese aged from 20 and over. A total of 1,528 participants living in rural and urban areas of Hanoi, Thua Thien Hue and Ho Chi Minh cities were

12th International Congress on Amino Acids, Peptides and Proteins randomly selected. The majority of participants prepared their foods at home, without using commercially processed foods. Dietary intake was 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 estimated based on the energy intake ratio of the family. Physical activity was assessed by Global Physical Activity Questionnaire. Results: Eighty one percent of participants were MSG users. Average MSG intake was 2.2 ± 1.8 g/day. Pearson correlation coefficients were used to determine associations between MSG intake and BMI, and energy intake. There was no significant association between MSG intake and BMI nor MSG and energy intake. Multiple logistic regression analysis revealed that factors associated with overweight were age, occupation, physical activity and intakes of energy, carbohydrate, saturated fatty acids and animal protein. Conclusion: MSG intake was suggested to have no effect on body weight.

Nutritional significance and molecular basis of intestinal metabolism of DL-methionine and its hydroxyl analogue Zhengfeng Fang*, De Wu, Yan Lin, and Lianqiang Che Key Laboratory for Animal Disease Resistance Nutrition of the Ministry of Education of China, Institute of Animal Nutrition, Sichuan Agricultural University, Ya’an 625014, Peoples Republic China *To whom correspondence should be addressed. Tel: +86-8352885164 Fax: +86-835-2885065 Email: [email protected] Methionine is a nutritionally indispensable amino acid (AA) for vertebrates, but is also toxic to animals and humans due to its transmethylation to produce homocysteine. Furthermore, the extensive 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 metabolism of methionine is regulated by its sources, DL-methionine (DL-MET) and its hydroxyl analogue, DL-2-hydroxy-4-methylthiobutyrate (DL-HMTB) receive growing attention in recent studies. Studies in pigs show that although the directly available L-MET in DLMET diet was about 1.2-fold that in diet containing equal molar methionine source, 30% of which was supplied by DL-HMTB, the net portal appearance of L-MET was not different between the two diets. Noticeably, compared with pigs fed the DL-MET diet, pigs fed the DL-HMTB diet had higher net portal balance and/or appearance of leucine, isoleucine, histidine, arginine and alanine, but had lower portal appearance of glutamate. Plasma ornithine and taurine concentration was higher, but plasma urea concentration was lower in the DL-HMTB fed than in the DL-MET fed pigs. These differences may be explained by the difference in distribution of enzymes for conversion of DL-HMTB and D-MET. The low mRNA abundance and low activity of D-2-hydroxy acid dehydrogenase (D-HADH) and L-2hydroxy acid oxidase (L-HAOX), but high mRNA abundance and high activity of D-AA oxidase (D-AAOX) in the intestine indicate a relatively higher capacity of the intestine to utilize D-MET than to utilize DL-HMTB for L-MET synthesis and its subsequent metabolism. Both D-HADH and L-HAOX activity in the stomach was comparable with those in the liver and/or kidney, indicating the substantial capacity of the stomach to convert DL-HMTB to L-MET. In addition, studies in ducks and chicks show some superiority of DL-HMTB to DL-MET in decreasing homocysteine production and alleviating stress responses, respectively. These novel findings may have important implications for intestinal nutrition and health in animals and perhaps humans.

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Protective effects of arginine supplementation against diquat-induced oxidative stress in piglets Ping Zheng, Bing Yu, Gang Tian, Keying Zhang, and Daiwen Chen** Institute of Animal Nutrition, Sichuan Agricultural University, Key Laboratory of Animal Disease-resistant Nutrition, Ministry of Education, Ya’an, Sichuan, 625014, Peoples Republic China The present study was conducted to test the hypothesis that dietary arginine supplementation may attenuate oxidative stress in piglets. A total of 36 PIC postweaning pigs weighted 8.67 ± 0.43 kg BW were individually penned and assigned to three dietary treatments including basal diet (BD) and BD + 0.8% Arg or 1.6% Arg. On day 8, pigs in each treatment were divided into two groups to be intraperitoneally injected with diquat or sterile saline, respectively. At 96 h postinjection, 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 inflammatory cytokines. All piglets fed ad libitum. The experiment lasted for 11 days. The results showed that arginine supplementation did not affect (P [ 0.05) piglets growth performance but there was a tendency that high dose of arginine can decrease ADG and ADFI of piglets under non-oxidative stress conditions. Oxidative stress induced by diquat significantly decreased ADG (P \ 0.001), ADFI (P \ 0.001) and increased F/G of piglets (P \ 0.05). However, supplementation of 1.6% arginine significantly increased ADFI and the antioxidative capacity in liver of piglets under oxidative stress (P \ 0.05). Supplementing 0.8 or 1.6% arginine to the diet can enhance the activities of GPx, SOD in plasma and decrease the TNF-a mRNA level in jejunum under oxidative stress condition (P \ 0.05). It is concluded that arginine can alleviate the growth depression and stress response induced by oxidative stress to some degree through increasing food intake, increasing the capacity of anti-oxidative stress, and decreasing inflammatory cytokines in piglets.

Protective effects of N-acetylcysteine on intestinal functions of piglets challenged with lipopolysaccharide Yongqing Hou1,*, Lei Wang1, Wei Zhang1, Zhenguo Yang1, Binying Ding1, Huiling Zhu1, Yulan Liu1, Yulong Yin2, and Guoyao Wu3 1

Hubei key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan 430023, China; 2 Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Hunan 410125, China; 3 Department of Animal Science, Texas A&M University, College Station, TX, 77843, USA; *Corresponding author: Dr. Yongqing Hou, E-mail: [email protected] This study was conducted with the lipopolysaccharide (LPS)-challenged piglet model to determine the effects of N-acetylcysteine (NAC) on intestinal functions of piglets. Eighteen crossbred piglets (11.58 ± 0.26 kg BW) were randomly allocated into control, LPS and NAC groups. The control and LPS groups were fed a basal diet, whereas the NAC group was fed the basal diet + 500 mg/kg NAC. On days 10, 13 and 20 of the trial, the LPS and NAC groups received intraperitoneal administration of LPS (100 lg/kg BW), whereas the

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S58 control group received the same volume of saline. On day 20 of the trial, D-xylose (0.1 g/kg BW) was orally administrated to all pigs at 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 intestinal mucosa for analysis. Compared with the control group, LPS 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 jejunal mucosa, RNA/DNA, TP/DNA in jejunal and ileal mucosa, while increased (P \ 0.05) the activities of DAO in plasma, and caspase-3 expression in intestinal mucosa. These adverse effects of LPS were attenuated (P \ 0.05) by NAC supplementation. Moreover, NAC prevented the LPS-induced decrease of claudin-1 and occludin expression in jejunal and ileal mucosa. Collectively, these novel findings indicate that dietary supplementation with 500 mg/kg NAC alleviates the mucosal damage and improves the absorptive function of the small intestine in LPS-challenged piglets.

Recent advances in amino acid nutrition of animals Yulong Yin1*, ChengboYang2, Wenkai Ren1, and Guoyao Wu3 1

Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 2 Department of Food Science, University of Guelph, Guelph, Ontario, Canada 3 Department of Animal Science, Texas A&M University, College Station, TX, 77843, USA *[email protected] Protein nutrition of animals is actually amino acid (AA) nutrition. Recent findings indicate that the gastrointestinal tract (GIT) of nonruminants, like ruminants, has a complex micro-ecosystem consisting of numerous species of bacteria. As an essential ‘‘organ’’, the gut microbiota plays an important role in AA nutrition of the host. In contrast to enterocytes, small-intestinal bacteria can degrade all dietary AA, thereby profoundly affecting their first-pass metabolism 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 explain the health- and growth-promoting effects of dietary interventions by inhibiting the growth of potential pathogenic bacteria in the intestine and enhancing AA absorption by the small intestine. Also, small-intestinal bacteria metabolism results in the losses of endogenous N and AA in ileal digesta and feces, contributing to N pollution in the livestock industry. Dietary non-starch polysaccharides (NSP) are the main exogenous factors that cause endogenous N secretion in pigs. Available evidence shows that NSP enzymes can improve pig growth performance by stimulating the degradation of soluble NSP in the small intestine and reducing the losses of intestinal endogenous N and AA. Furthermore, dietary carbohydrates affect the digestion and absorption of N and AA in the portaldrained visceral tissues via a mechanism that controls glucose release from starch. The most exciting finding is that arginine can attenuate the damages to the GIT, sudden deaths, and neurologic lesions caused by toxins of the bacterium Escherichia coli. Arginine supplementation can enhance immune responses and ameliorate the reproductive failure in mice caused by the porcine circovirus type 2 infections. Thus, AA plays a central role in health, growth, and reproduction of animals. Keywords: Amino acids (AA), Non-ruminants, Small intestine, Bacteria

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Regulatory role for L-glutamine in the utilization of amino acids by pig small-intestinal bacteria Zhao-Lai Dai1,2,3, Xi-Long Li3, Peng-Bin Xi3, Jing Zhang1, Guoyao Wu2,3*, and Wei-Yun Zhu1* 1

Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China 2 State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China 3 Department of Animal Science, Texas A&M University, College Station, TX, 77843, USA *Corresponding author: Dr. Wei-Yun Zhu ([email protected]) 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 (AA) by pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the pig small intestine. Anaerobic AA basal media containing 0–5 mmol/L of glutamine were used for the experiment. After 3 h of incubation, samples were taken for the determination of AA concentrations for the calculation of AA utilization. Results showed that the dose-dependent increase in the bacterial utilization of glutamine and altered flux of glutamine into glutamate in the bacteria. The addition of glutamine affected the arginine utilization and the flux of arginine into ornithine in pig small-intestinal bacteria. Complete utilization of asparagine, aspartate and serine were observed in pure bacterial strains after 3 h of incubation. The addition of glutamine reduced (P \ 0.05) the net utilization of asparagine by both jejunal and ileal mixed bacteria. Net utilization of lysine, leucine, valine, ornithine and serine by jejunal or ileal mixed bacteria decreased with the addition of glutamine. Overall, the addition of glutamine affected the metabolism of the argininefamily of AA and serine- and aspartate-family of AA in small-intestinal bacteria and reduced the utilization of most AA in jejunal or ileal mixed bacteria. These findings suggest that the beneficial effects of glutamine to the swine nutrition may partially by regulating the microbial utilization and metabolism of AA in the small intestine.

Relationship between dietary lysine levels and serum concentration of amino acids and performance of growing pigs Miguel Cervantes, Adriana Morales, Alfonso Araiza, Jose´ Landero, John K. Htoo, Hećtor Garcia, and Ernesto Avelar Typical feed ingredients are first limiting in Lysine (Lys) for pigs, but usually contain excess of arginine and neutral amino acids (AA), which interact with Lys for its intestinal absorption. The availability of AA significantly affects the performance in pigs. An experiment was conducted to analyze the effect of three dietary Lys levels (0.40%Deficient, DEF; 1.18%-Intermediate, INT; and 1.56%-Excess, EXC) on the performance and serum concentration (SC) of indispensable AA. Twenty-four crossbred pigs (14.4 ± 1.7 kg) with 8 replicates per treatment were used. A wheat based diet was formulated, using analyzed ingredient amino acid (AA) contents to meet the requirements of AA other than Lys. Treatments (T) were: T1, wheat-based diet, 0.40% Lys (DEF); T2, basal plus 0.78% L-Lys (INT); T3, basal plus 1.56% L-Lys (EXC). At the end of a 28-day trial, all pigs were sacrificed and blood was collected to analyze the SC of AA. Two contrasts were constructed to analyze the effect of the Lys levels (DEF vs. INT; and

12th International Congress on Amino Acids, Peptides and Proteins INT vs. EXC), Increasing the Lys level from 0.40 to 1.18% improved the ADG, ADFI, and FCR (p \ 0.01). No further response was observed with the EXC level. Serum concentration of arginine, threonine, and valine reduced (p \ 0.01) when Lys increased from DEF to INT; no further response occurred with the EXC diet. Lys SC linearly increased (p \ 0.01). Methionine, leucine and phenylalanine tended to reduce with the increase in Lys. Performance and SC of selected AA are well correlated each other, in response to dietary Lys levels.

Significant reverse correlation between dietary intakes of three branched essential amino acids (Leu, IIe and Val) and percent body fat in the Newfoundland population Daniel Wadden, Farrell Cahill, Feiyu Han, Yagang Xie, Wayne Gulliver, Hongwei Zhang, and Guang Sun Recent studies show that the amount of dietary intake of branched essential amino acids (BCAA) may affect the short and long term metabolism. Our lab has previously shown that dietary protein intake is negatively associated with body adiposity. In the present study we further investigate the role of BCAA in this beneficial effect in the Newfoundland population in Canada. A total of 2,334 adults (577 men and 1,757 women) from the CODING study participated. Daily BCAA intake was computed from the Willett Food Frequency Questionnaire. Body fat percentage was measured using a dual energy 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 obesity status (percent body fat, Bray criteria) were compared among normal, overweight and obese groups. All three BCAAs were found reversely correlated with all obesity phenotypes with WC having the strongest correlation coefficient in both women (r varies from -0.27 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 had the highest intake, and the obese had the lowest intake of all three BCAAs with the overweight in the middle. This strong dose effect relationship holds based on any obesity criteria used in the study. Our findings provide solid evidence of the beneficial effect of BCAA on obesity at the free living population level.

The dynamic changes of blood flow, oxygen consumption and metabolites response to acute arginine supplementation in growing-finishing pigs Bie Tan1, Yulong Yin1*, Xinguo Li2, Xiangfeng Kong1, Zhiqiang Liu1, Tiejun Li1, Ruilin Huang1, and Guoyao Wu1,3 1 Research Center for Healthy Breeding of Livestock and Poultry, Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, 410125 Hunan, China 2 Hunan Institute of Animal Husbandry and Veterinary Medicine, Changsha, Hunan 410131, China 3 Department of Animal Science, Texas A&M University, College Station, TX 77843, USA *[email protected]

Arginine plays an important role regulating nutrient metabolism. This study was conducted to determine the dynamic changes of blood metabolites response to acute arginine supplementation in growingfinishing pigs using blood catheter technique. Eight barrows

S59 (Duroc 9 Large White 9 Landrace) with an average initial BW of 70 kg were surgically fitted with chronic catheters in the portal vein, ileal vein and carotid artery and were randomly allocated to two groups to receive alanine (103 mg/kg body wt, isonitrogenous control) or Larginine-HCl (61 mg/kg body wt) by the portal vein. Blood flows were measured with infusion of p-aminohipuric acid (PAH) into the ileal vein, and simultaneously obtain blood samples very 0.5 h for 4 h for 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 90 min after infusion but decreased PVDO at 60, 90, 120 and 150 min after infusion (P \ 0.05). Plasma concentrations of glutamate at infusion 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, arginine infusion decreased (P \ 0.05) plasma cystine concentrations at infusion time of 60, 120, 180 and 240 min and valine at infusion time of 60 and 120 min compared with alanine treatment. Plasma concentrations of insulin and glucagon at infusion time of 30, 60 and 90 min were higher and free fatty acid at infusion time of 60, 90 and 120 min were lower than those of pigs both in alanine treatment on the same infusion time and at arginine pre-infusion time. These results suggest that acute arginine supplementation improves blood flow and reduces the PVDO oxygen consumption, thereby enhancing the amino acids availabilities for utilization. And insulin and glucagons may play important roles in arginine infusion transiently regulating nutrient metabolism. Keywords: L-Arginine, Blood flow, Oxygen consumption

The endothelial arginine-nitric oxide pathway in diabetes and obesity Cynthia J. Meininger1 and Guoyao Wu1,2 1 Department of Systems Biology and Translational Medicine, Texas A&M Health Science Center, Temple, TX, USA; and 2 Department of Animal Science, Texas A&M University, College Station, TX, USA

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 in regulating endothelial cell function in blood vessels. We demonstrated that in diabetes the bioavailability of NO is reduced because of decreased endothelial tetrahydrobiopterin, a critical co-factor for eNOS. Dietary L-arginine increases tetrahydrobiopterin—normalizing NO synthesis and vascular function—by stimulating GTP cyclohydrolase I, the first and rate-controlling enzyme for de novo synthesis of tetrahydrobiopterin. L-arginine also decreases glucosamine production, helping to reverse impaired endothelium-dependent relaxation, oxidative injury and vascular insulin resistance in diabetic and obese patients. The arginine-NO pathway has widespread effects on the metabolism of energy substrates. Arginine supplementation increases plasma arginine while decreasing plasma glucose in diabetic rats. L-arginine reduces body weight loss in type 1 diabetic rats but, interestingly, decreases adiposity in genetically obese rats, diet-induced obese rats and humans with type 2 diabetes. In Zucker diabetic fatty rats, a genetically obese animal model of type 2 diabetes, arginine treatment reduces white adipose tissue while increasing NO synthesis. This arginine-induced increase in NO stimulates oxidation of energy substrates (including fatty acids and glucose). L-arginine supplementation stimulates brown adipose tissue development possibly by enhancing synthesis of cell signaling molecules and increasing expression of genes that promote mitochondrial biogenesis. Modulation of the arginine-NO pathway may

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S60 be beneficial for vascular function, metabolic homeostasis, and weight management in diabetes and obesity. Supported by NIH, JDRF and AHA.

The metabolic characteristics of the proteins in Canadian hulless barley: comparison of the zero-amylose waxy, waxy, and high-amylose cultivars with the normal starch cultivar D. Damiran, L. Yang, and P. Yu* College of Agriculture and Bioresources, The University of Saskatchewan 51 Campus Drive, Saskatoon, SK, S7 N 5A8, Canada *Corresponding author: Peiqiang Yu, Ph.D. SAF Research Chair College of Agriculture and Bioresources University of Saskatchewan, Room 6D10 Agriculture Building, 51 Campus Drive, Saskatoon, SK, Canada, S7 N 5A8 Tel: +1-306-9664132 Fax: +1-306-9664151 [email protected] The hulless barley (HB) with normal starch structure is considered to be a less favourable feed grain for ruminant animals in comparison to the hulled counterpart. This is largely due to the rapid degradation of its starch in the rumen. Recently, several new Canadian hulless barley cultivars with altered starch characteristics (waxy and high-amylose), have been developed. The variations in the molecular composition and characteristics 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/ 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, 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 intestine (DVE; 123 vs. 111 g/kg DM) and the metabolizable protein (MP; 112 vs. 92 g/kg DM), while the high starch HB had the lowest DVE (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, indicating the N shortage for the microbial biosynthesis in the rumen. In general, HB with modified starch structure provided a relatively balanced source of energy and protein for microbial synthesis in the rumen. Consequently, the hulless barley with modified starch might be a better feed grain for dairy cattle than the normal starch HB. However, larger feed samples with a wider range of chemical compositions needs to be evaluated in order to be more conclusive. Keywords: Hulless barley, Protein evaluation, Starch structure, Protein metabolic characteristic

The muscle proteome of growing zebrafish is affected by dietary lysine Mahaut de Vareilles1,2, Luis E.C. Conceiçaõ2, Pedro Go´mez-Requeni1, Katerina Kousoulaki3, Pedro M. Rodrigues2, Nade`ge Richard2, Kari E. Fladmark4, Odete Cordeiro2, Tome´ S. Silva2, and Ivar Rønnestad1 1

Department of Biology, University of Bergen, Norway Centro de Cieˆncias do Mar do Algarve (CCMAR), Faro, Portugal 3 Nofima Ingredients, Fyllingsdalen, Norway 4 Department of Molecular Biology, University of Bergen, Norway 2

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12th International Congress on Amino Acids, Peptides and Proteins Many amino acids (AA) regulate key metabolic pathways crucial to maintenance, growth, reproduction and immune responses, making 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 lysine limitation affects growth and muscle proteome expression in juvenile zebrafish (Danio rerio). From 33 to 49 days post-fertilisation (dpf), quadruplicate groups of 8 fish were fed adequate (Lys(+)) or low-lysine (Lys(-)) diets. Fork length was monitored and white trunk 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 527 ± 11 (mean ± SEM) protein spots observed (10–150 kDa and 4–7 pI values), 30 were over-expressed and 22 under-expressed in skeletal muscle of Lys (-) fish (|fold-change| [1.2). Sixteen proteins of the 20 identified spots were involved in the cytoskeletal network and contractile apparatus of skeletal muscle, 11 of which were more abundant in the Lys (-) fish (isoforms of myosin-binding protein H-like, fast skeletal myosin heavy chain (fsMHC), fast skeletal myosin light chains and skeletal muscle actin; alpha tropomyosin) and 5 less abundant (isoform of fsMHC, actin fragments and F-actincapping protein). Proteins involved in energy metabolism (beta enolase and mitochondrial ATP synthase) were more abundant in Lys (-) fish. Finally, the proteins apolipoprotein A-I precursor (lipid transport) and Pdlim7 (PDZ-LIM protein family) showed increased abundance in Lys (+) fish. Overall, these results demonstrate the key role of lysine as regulator of muscle development, metabolism and growth.

Thyroglobulin and iodine nutrition Radovan Bı´lek, and Vaćlav Zamrazil Institute of Endocrinology, Prague, Czech Republic Na´rodnı´ 8, 116 94 Prague 1, Czech Republic Tel: +420 224905111, Fax: +420 224905325, E-mail: [email protected] Our results from population studies indicate that serum thyroglobulin (Tg) seems to be a valuable indicator of iodine nutrition. Thyroglobulin is the major iodoglycoprotein of the thyroid gland and one of the largest proteins in the human body (molecular weight of soluble dimer about 660 kDa). The immunoanalytical determination of serum Tg is difficult because of the inhomogeneity of the Tg molecule, where various isoforms of Tg exist with differences both in primary structure and iodine or carbohydrate content. In addition, the presence of circulating autoantibodies against Tg may substantially interfere with determination of serum Tg. The main problem is the specification of Tg cut-off value corresponding to various levels of iodine intake, which is limited by large interassay variability and lack of reference data for Tg in healthy, iodine-sufficient individuals. Urinary iodine (laboratory spectrophotometric method based on alkaline ashing and Sandell–Kolthoff reaction) and serum Tg (electrochemiluminiscence immunoassay, Roche Diagn., Germany) were determined in the general healthy population of 3,014 randomly selected individuals aged 6-98 years (1,272 males, 1,742 females), or in the hospital population (n = 5,516), which consisted of individuals aged 0–91 years (527 males, 4,989 females) attending the Institute of Endocrinology, Prague. The serum Tg concentration was elevated in iodine-deficient subjects and serum Tg decreased progressively as the urinary iodine concentration rose. We can conclude that thyroglobulin is a valuable indicator of iodine nutrition in a population, if thyroid disorders are not frequent.


Peptides Aryl hydrazine resin as a tool in the synthesis of C-terminal modified peptides: optimization of oxidation conditions A. Walewska, I. Małuch, D. Sobolewski, and A. Prahl Faculty of Chemistry, University of Gdan´sk, Sobieskiego 18, 80-952 Gdan´sk, Poland Recently, Y. Kwan et. al. presented a novel and straightforward strategy to synthesize peptides with p-nitroanilides and other anilide analogues using an aryl hydrazine resin. The resin is also a multipurpose 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 (NBS) or copper(II) acetate in pyridine. The resulting acyl diazene resin is then cleaved by peptide displacement at the C-terminus with amine. The fully deprotected peptide amide is finally obtained by treatment with trifluoroacetic acid (TFA). In our approach, we used a 4-Fmoc-hydrazinobenzoyl AM NovaGel resin to synthesize a peptide-substituted amide in the C-terminus. First, the oxidative cleavage was carried out with NBS in pyridine and a nucleophile [a protected 4-(aminomethyl)benzimidamide (Amba)]. However, the yield of the reaction was very poor. In the next step, we applied copper(II) acetate in the presence of pyridine and Amba. Following optimization, the efficiency of the process was significantly improved. Herein we discuss the conditions needed to obtain a reasonably high efficiency of the oxidative cleavage in the synthesis of the C-terminal modified peptides using the aryl hydrazine resin linker.

Cathepsin B-sensitive peptide-oligonucleotide conjugates for cancer therapy L. A. Yarinich1,2, A. S. Chubarov1,2, L. S. Koroleva1,2, I. Yu. Serpokrylova1, V. N. Silnikov1, and T. S. Godovikova1,2 1

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Lavrentyev Ave., 630090 Novosibirsk, Russia 2 Novosibirsk State University, 2 Pirogova St., 630090 Novosibirsk, Russia Induction of apoptosis in endothelial cells is considered an attractive strategy to therapeutically interfere with a solid tumor’s blood supply. We constructed cytotoxic conjugates that specifically target angiogenic endothelial cells, thus preventing typical side effects of apoptosis-inducing drugs. For this purpose, we conjugated the antisense oligonucleotide via a lysosomal cleavable linker to Nhomocysteinylated human serum albumin and further equipped this drug-albumin conjugate with RGD-peptides for multivalent interaction with integrin. Peptides: substrates for cathepsins taking part in cancer progression has been described in literature. Application of such peptides as biodegradable linkers let to release the therapeutic in transformed cells. Synthesis of peptides (AlaLeu)2Gly, b-Ala(AlaLeu)2 were carried out in solution using Boc-strategy. After that flexible amino linker with maleimide fragment was coupled to C-terminal and antisense oligonucleotide was connected to N-terminal of peptides by phosphamide bond. Modification of

S61 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 intravascular contrast agent for magnetic resonance imaging. 19F NMR offers a unique quantitative means of imaging infused agents, as there is essentially no 19F background signal in tissue. In addition, 19F spectroscopy is a powerful alternative to 1H NMR spectroscopy because of its high sensitivity (83% with respect to 1H) due to a high gyromagnetic ratio, 100% natural abundance of the (spin = 1/2) 19F isotope, narrow lines, and short longitudinal relaxation times (T1) which permit rapid pulsing with a corresponding improvement in the signal-to-noise ratio per unit time. The research is supported by Integration Grant of SB RAS 88, RFBR Grant 09-04-01483-a, Grant of Novosibirsk region government.

Cell penetrating peptides: a nanocarrier for delivery of ssDNA and protein complexes in plant cell Francois Eudes Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, Lethbridge, AB, T1J 4B1, Canada Cell-penetrating peptides (CPPs) are a class of short peptides with property to translocate across cell membranes and target subcellular localisation, such as the nucleus. CPPs also have the capacity to noncovalently bind to cargo molecules, such as nucleic acid and protein, and transport them to its final subcellular destination. Despite fundamental differences between animal cell and plant cell composition, 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 of dsDNA, ssDNA and proteins. Synthetic or in vivo produced nucleic acid, proteins and CPPs are blocks that conjugate to form nano-complexes in a relatively predictable manner. Single stranded DNA binding proteins such as RecA, Rad51 and VirD2 were used to form various cargo complexes with ssDNA, and increased the integrity of ssDNA integrated in the haploid plant genome of microspore. This novel process fit very well with cell culture systems, as demonstrated with isolated microspore, a unique haploid cell amenable to the production of haploid and doubled haploid plant lines. Stable DNA integration has been achieved following CPP mediated delivery of dsDNA and ssDNA in wheat and triticale microspores, and inheritance has been documented in subsequent generations. CPP mediated transfection in plant microspore opened new possibilities for precision genetic engineering of this unique cell type and crops of commercial importance.

Designing and efficient synthesis of anticancer peptides contained novel linkers and investigation of their activities Fatemeh Tahoori, Reza Sheikhnejad, and Saeed Balalaie Peptide Chemistry Research Center, K. N. Toosi University of Technology, Tehran, Iran Peptides can be used for treatment of different diseases. Although numerous anticancers drugs with peptide skeletons exist, most of

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S62 them have some drawbacks. The development and designing of new peptides is a highly demand and considerable challenge for synthetic 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 methodologies. Synthesis of desired heptapeptides was done according to the known SPPS Strategy. Synthesis was carried out using 2-CTC resin according to the standard Fmoc strategy. Before final deprotection and cleavage from the surface of resin, new linker is formed via the reaction of free amine with anhydrides. The purification was done using preparative HPLC. The structure of the synthesized peptides were confirmed using HR-Mass(ESI). The anti-cancer activity of the peptide was studied. Since the lipophilicity is a key factor in determining the rate at which a drug crosses the blood–brain barrier (BBB), we inserted alkyne and alkyl linkers. The desired products are purified and their biological activity are studied. We believe that the existence of lipophilic moieties cause more lipophilicity and their cell permeability. The investigation of anti-cancer activity of the synthesized peptides are in progress. The details will be discussed in the conference.

Design of peptides with nuclear binding and potent broad-spectrum antimicrobial activities Lirong Li, Guowei Le, and Yonghui Shi Center for Food Nutrition and Function Factor, College of Food, Jiangnan University, WuXi, 214122, China Objective: Emergence of multi-resistant strains has prompted search for new antimicrobial drugs. Antimicrobial peptides (AMPs) are of greatest potential sa a new class of antibiotics. We designed a series of AMPs that could translocate across bacterial membranes and bind to DNA. Aiming to explore their antibacterial mechanism and analyse 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 microdilution assay. We investigated the secondary structure of the peptides using circular dichroism (CD) spectroscopy. Using microbial adhesion to solvents (MATS) method measured strains surface hydrophobicity. The ability of permeabilization of inner membrane (IM) was evaluated by cytoplasmic L-galactosidase release. Binding of the peptides to pathogenic bacterium genomic DNA was evaluated by gel retardation assays. Result: These antibacterial peptides containing of 20 amino acids, net charge from +3 to +7, with 45–70% of hydrophobic residues showed classic amphipathic characteristic and stability. They fold into an amphipathic a-helical conformation and showed random coil stereo structures. Decreasing peptides mean hydrophobicity and maintaining the original charge could enhance antimicrobial activity. Peptides exhibited less than 50% hemolytic activity at 600 lg/ml. The increase in hemolytic activity was correlated with mean hydrophobicity increase. All peptides except P7 had no defined secondary structure in aqueous buffer, but in membrane mimetic condition peptides fold into an a-helical conformation. Treating with peptides, strains surface hydrophobicity decreased and cytoplasmic membrane permeabilization increased. Peptides with net charge from +5 to +7 and low mean hydrophobicity inhibited migration of DNA above a weight ration of DNA to peptide of 1:1. Conclusion: these design peptides may serve as useful templates which had potent.

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Epigenetic mechanisms of evolution: peptides regulate gene expression and increase resource of the organism V. Kh. Khavinson, V. V. Malinin, and B.F. Vanyushin* Saint Petersburg Institute of Bioregulation and Gerontology, Russian Academy of Medical Sci., 3, Dynamo Pr., Saint Petersburg, 197110, Russia; E-mail: [email protected] *Belozersky Institute of Physical and Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia Peptides increase lifespan of rats by about 30–40% and inhibit growth of tumors. Peptides introduced in vivo influence the gene expression profile. Peptides introduced to transgenic mice suppressed expression of the HER-2/neu mammary gland cancer gene two to fourfold. The peptides introduced increase transcription of IL-2 and c-fos genes in lymphocytes and various structures of hypothalamus. The mechanisms of the peptide action associated with chromatin activation in blood lymphocytes of aged patients. Human fibroblasts treated with peptides showed the telomerase activity induction and a 2.5-fold 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 physiological functions and decreased the lethality by 44–49% in a 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. Peptides modulate specifically the action of eukaryotic endonucleases depending on DNA methylation status. The peptide modulating action on DNA hydrolysis seems to be due to site-specific peptide binding with DNA that may protect DNA against enzymatic hydrolysis. Peptide binding to CNG or CG sites in gene promoters should prevent their methylation with respective DNA-methyltransferases and leave promoters to be unmethylated that is crucial for activation of most genes. Thus, specific peptide-DNA interactions control epigenetically the cell genetic functions and can be responsible for homeostasis recovery and life-span prolongation.

Metabolic stability of long acting LHRH antagonists Jinfeng Yao1, Ning Zhou2, Keliang Liu2, and Ming Xue1,* 1

Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China 2 Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China *Corresponding author Tel: +86-10-83911520; Fax: +86-10-83911520. E-mail address: [email protected] Purpose: Long acting luteinizing hormone-releasing hormone (LHRH) antagonists are a series of novel LHRH antagonist analogues, which synthesized on the base of the new concept of the long acting peptides design for protease-resistant. The bioactivities of them were evaluated in rats by the testosterone test model. The designed peptides showed a long action of inhibiting testosterone secretion comparing with parent peptide. In the present study, a method was established to assess metabolic stability of the LHRH antagonists in vitro rapidly. Methods: The cassette dosing (N in one) was used, 12 compounds were divided into 3 groups. For cassette dosing, 4 compounds were mixed with leuprorelin (as a control), and added to solution of pancreatin, then the mixture were incubated at 37°C for 4 h. Samples

12th International Congress on Amino Acids, Peptides and Proteins (100 ll) were withdrawn at different time intervals and determined by HPLC method. Results: Calibration curves of all compounds were linear in the concentration range of 1.0–400 lg/ml, correlation coefficients were more than 0.9991. The low limits of quantification of these twelve compounds were 1.0–10 lg/ml. The inter-day and intra-day precisions were within ±10.0% at three concentration levels. The accuracy percent error was 2.80–6.0%. The half-life of leuprorelin was 4.2 min, the half-lives of the other compounds were 0.73–5.5 h. Conclusion: The half-lives of the 12 LHRH antagonists were not different from those determined, respectively (p [ 0.05). Among them LY617B had a long half-life. These results showed that this method had been successfully applied to predict the metabolic stability of LHRH antagonists in vitro pancreatin system.

Peptides regulate proliferative activity of stem cells V. Kh. Khavinson, N.S. Linkova, A.V. Dudkov, A.V. Trofimov, and S.V. Trofimova Saint Petersburg Institute of Bioregulation and Gerontology, Russian Academy of Medical Sci., 3, Dynamo Pr., Saint Petersburg, 197110, Russia; E-mail: [email protected] The objective of the experiment was the evaluation of the proliferative activity of the MSC under the influence of peptides H-Ala-Glu-AspGly-OH, H-Lys-GLu-Asp-OH and H-Lys-Glu-OH. MSC was cultivated (37°C, 5% CO2) in the a-MEM media (Biolot, Russia) with the cattle fetal serum (Gibco, USA), 2 mm of glutamine (Gibco, USA) and 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 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 culture growth the cells were counted in the Gorayev chamber. In the control on the 3rd and 5th day the quantity of the cells was 50,000 ± 5,600 and 65,000 ± 6,200 correspondingly. Under the influence of the tetrapeptide on the 3rd and 5th day the quantity of 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 influence of the dipeptide and tripeptide on the 3rd day the quantity of the MSC showed a tendency to reduction and was valuated at 50,000 ± 3,200 and 40,000 ± 3,400 cells. On the 5th day of the introduction of the dipeptide and tripeptide to the culture of the MSC the quantity of cells reduced reliably by 1.9- and 1.6-fold and was valuated at 35,000 ± 3,000 and 40,000 ± 2,900 cells. Depending on the structure of the peptide different proliferative ability of the MSC was noticed. Tripeptide stimulated growth of the MSC. Di- and tripeptide inhibited the proliferation of the MSC.

Preparation and characterization of peptide conjugated magnetoliposomes to biomedical applications Roberta V. Ferreira*, Rodrigo M. Verly, Victor Hugo Munhoz, Dorila Pilo´-Veloso and Rosana Z. Domingues Departamento de Quı´mica/ICEx/UFMGl Avenida Antoˆnio Carlos 6627, Cidade Universita´ria Pampulha 30270-901, Belo Horizonte, MG, Brazil *[email protected] Keywords: Antimicrobial peptide, Ferrofluids, Magnetic hyperthermia, Magnetoliposome

S63 This work presents the development of controlled drug delivery vehicles based on magnetic nanoparticles. The system based on magnetoliposomes with incorporated peptide on surface was tested as drug delivery device. Iron oxide nanoparticles were synthesized by co-precipitation of ferrous and ferric salts in alkali medium. The black powder obtained was functionalizated with phosphatidylcholine to form biocompatible magnetoliposome. The surface modification of iron oxide nanoparticle after functionalization with phospholipid was accompanied with zeta potential measures and DLS. Magnetoliposome exhibited values of zeta potential (f = 30 mV) higher than those values obtained for suspension of iron oxide nanoparticle in water (f = 2.5 mV) showing a better colloidal stability. The hydrodynamic diameter obtained by DLS measurements confirmed this behavior. The peptide 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 released peptide was monitored by measuring the absorbance using UV spectrophotometry. The magnetic heating characteristics were obtained to samples in an alternating magnetic field of strength 502 Oe and frequency 230 kHz. Samples were entered into the centrifuge tubes which were then placed in the center of the induction coil. The alternating magnetic field was applied for a given time 30 s until 5 min, in 30 s intervals. The results suggested that the new system prepared in this study have a great potential to biomedical applications.

Preparation and identification of Zn (II) chelating peptides and their complexes from sesame protein hydrolysates Chan Wang, and Bo Li* College of Food Science and Nutritional Engineering, China Agricultural University, Qinghua East Road 17, Haidian District, Beijing 100083, People’s Republic of China *Corresponding author Tel: 86-10-6273-6243; Fax: 86-10-6234-7334 E-mail: [email protected] The sesame protein hydrolysates (SPH) obtained with trypsin, which exhibited high metal-chelating abilities were used for separation of metal-chelating peptides. The metal chelating peptides were isolated from the hydrolysates using affinity chromatography with zinc immobilized on solid supports (chitosan). Further, six zinc-chelating peptides were identified with reversed phase (RP)-HPLC and electrospray ionization (ESI) -MS/MS, two of these metal-chelating peptides, Ser-Met (SM), Asn-Cys-Ser (NCS), were then synthesized and the zinc-chelating abilities were measured by EDTA titration, and radical scavenging 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 radical scavenging activity, while peptide NCS and zinc-NCS complex had significant radical scavenging activities with 0.514 ± 0.008 (mM Trolox) for peptide and 0.404 ± 0.008 (mM Trolox) for the complex. Studies of zinc complexes of two peptides by pH- potentiometric titration and mass spectrometry (MS) demonstrated that the complexations 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) spectrometry of peptides and complexes, the carboxyl of C-terminus amino acid, peptide bond and side chain such as sulfhydryl group of Cys, amino and acyl of Asn, play important roles in the complexation between peptide and zinc. From Circular dichroism (CD) spectrometry of peptide SM, a-helix disappeared from 25.7% and b-sheet was increased from 74.3 to 100% after complexation with zinc. However,

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S64 CD spectrometry of peptide NCS indicated that a-helix increased from 12.9 to 42.5% and b-sheet was increased from 0 to 57.5%. These structural changes lead to a significant increase in the peptide’s stiffness and it is just the way the peptides found to adjust its structure to the presence of the metal ions at minimum energy expense. Keywords: Sesame protein, Metal-chelating peptides, Hydrolysis, Antioxidant activity, Zinc

Simultaneous determination of long acting LHRH antagonists and its application to high-throughput pharmacokinetics Jinfeng Yao1, Ning Zhou2, Keliang Liu2, and Ming Xue1,* 1 Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China 2 Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China *Corresponding author. Tel: +86-10-8391 1520; Fax: +86-10-8391 1520. E-mail address: [email protected]

Purpose: Long acting luteinizing hormone-releasing hormone (LHRH) antagonists are novel LHRH antagonist analogues. The bioactivities of the long acting peptides were evaluated by the rat testosterone test model. The designed peptides showed a long action of inhibiting testosterone secretion compared with the parent peptide. The metabolic stabilities of the peptides were assessed in pancreatin system. It showed there were good protease-resistant activities for the peptides. In the present study, rapid liquid chromatography-electrospray tandem mass spectrometry (LC-ESI–MS/MS) detection was established for the simultaneous determination of the five LHRH antagonists in rat plasma for the purpose of high-throughput pharmacokinetic screening. Method: The method was operated under selected reaction monitoring (SRM) mode in the positive ion mode. The analytes were extracted from 50 ll rat plasma by liquid–liquid extraction with acetonitrile. Chromatographic separation of the analytes was successfully achieved on a Hypersil Gold (100 9 2.1 mm, 3 lm) using a mobile phase composed of acetonitrile–water (30:70) containing 0.1% (v/v) formic acid. Results: The method showed good linearity and no endogenous material interfered with the marked compounds. The limits of quantification of five peptides were 10–4,000 ng/ml. The average extract recoveries of the five compounds in the rat plasma were all over 70%. Intra-day and inter-day precisions (R.S.D.%) were all within 15% and the method assessed a quite good accuracy (R.E.%). Conclusion: This method has been successfully applied in the simultaneous quantification and the pharmacokinetic studies of these five long acting peptides after sublingual intravenous administration in rats.

Size dependent uptake efficiency of cell penetrating peptide mediated delivery systems: mechanisms and therapeutic application Heesok Kim, So Ra Kang, Byung Keun Oh, and Kwanwoo Shin* Department of Chemistry and Interdisciplinary program of integrated Biotechnology, Institute of Biological Interfaces, Sogang University, Seoul 121-742, South Korea *E-mail: [email protected]

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12th International Congress on Amino Acids, Peptides and Proteins Positively charged cell-penetrating peptides (CPPs), which are a class of short peptide sequences that can traverse cell membranes efficiently, are often used as transporters for various biological drugs such as peptides, proteins, and genes, which are attached as cargo. The cellular translocation properties of the silica nanoparticles coated with transcription-activating-factor-derived peptide (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, and translocation efficiency into the various living mammalian cells. We found that there is an optimized cargo size for the efficient cellular uptake, which has to closely related to the translocation mechanism due to the presence of CPPs. We will present the size-dependant peptide mediated delivery systems, and plausible mechanism. Based on the efficient Tat-mediated delivery system, we could show that the Tat-associated particles can be used as a highly cell imaging agent (with fluorescence incorporated cargos), and a gene therapeutic agent (with si-RNA incorporation).

Structural and orientational study of peptide phenylseptin in micellar environment V. H. O. Munhoz*1,2, M. T. Q. de Magalha˜es3,4, R. M. Verly1, S. F. C. de Paula1, J. M. Resende1, C. Aisenbery2, D. Pilo´-Veloso1, C. Bloch Jr.3, and B. Bechinger2 1

Departamento de Quı´mica, Instituto de Cieˆncias Exatas, Universidade Federal de Minas Gerais, Av. Pres. Antoˆnio Carlos, 6627, 31270090, Belo Horizonte-MG, Brazil (*[email protected]) 2 Universite´ de Strasbourg/CNRS UMR7177, Institut de Chimie, 4, rue Blaise Pascal, 67070 Strasbourg, France 3 Embrapa Recursos Gene´ticos e Biotecnologia, PqEB- Final W5, Brası´lia DF, Brazil 4 Po´s-graduaçaõ em Biologia Molecular, Instituto de Biologia, Universidade de Brası´lia, Campus Universita´rio Darcy Ribeiro, Brası´lia DF, Brazil E-mail:[email protected] Phenylseptin (Phes) is a new class of antimicrobial peptides isolated from the anuran Hypsiboas punctatus. They are known to be phenylalanine-rich peptides on their N-terminus. There are two knowing forms belonging to this class which share, to some degree, the same amino acid sequence, except for an enantiomerization at one phenylalanine residue. Both peptides show highly a-helical and amphipathic structures when in contact micelles and bilayers. In vitro assays have shown that both peptides have mild antimicrobial activity against Gram-positive and Gram-negative bacteria, being the Phenylseptin containing D-amino considerably more active than its homologue. Nonetheless, the exact mechanism which describes the antimicrobial activity of these peptides is still not fully determined. To elucidate some aspects of this mechanism, static cross-polarization 15N and 2H solid-echo solid-state NMR were performed in samples with the peptide in contact with mechanically oriented lipid bilayers. By combining these two experiments, it was possible to determine the orientational constraints which were used on these peptides’ structures determined through solution NMR in the presence of DPC micelles, leading to the most probable orientation of the peptide when it interacts with membrane-like media. With these results, it was possible to observe that both peptides, although similar in amino acid composition, interact very differently with membrane-like environments and thus exert different activity.

12th International Congress on Amino Acids, Peptides and Proteins The cause of the difference between their activities probably is the differential anchoring, showed by the variation on the 2H quadrupolar splitting, which is quite sensitive to strong interactions between the side chains and the bilayer. Keywords: Antimicrobial peptides, Solid-state NMR

Structural and thermodynamic studies of the dimeric peptide homotarsinin in aqueous and mimetic membrane environments Rodrigo M. Verlya*, Jarbas M. Resendea, Fa´bio C. L. Almeidaa Marcelo P. Bemquererb, and Dorila Pilo´-Velosoa a

Departamento de Quı´mica/ICEx/UFMG, Embrapa-Recursos Gene´ticos e Biotecnologia/Brası´lia-DF, c CNRMN/UFRJ *E-mail: [email protected] b

The homodimeric peptide homotarsinin was isolated from Phyllomedusa tarsius, a tree-frog that inhabits the Brazilian Amazonian basin. This peptide is composed of two identical linear chains each with 24 amino acid residues carrying a natural amidation at the C-termini and connected by a single disulfide bond. Antimicrobial assays demonstrate that homotarsinin dimmer as well as its monomer shows antimicrobial activity against both Gram-positive and Gram-negative bacteria. From aqueous buffer and DPC micelles NMR data, the ten lowest 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 contrast, the absence of inter-chain NOEs when homotarsinin is in contact with the membrane suggests the existence of a more open structure. These results imply that hydrophobic peptide–peptide interchain 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 interactions in order to allow a better partitioning of the amphipathic helices into the membrane interface. Isothermal titration calorimetric study shows a great homotarsinin interaction with both POPC and POPC:POPG (3:1) LUVs. Dynamic light scattering measurements indicated that homotarsinin associates to phospholipid vesicles leading to a supramolecular structure, which is characterized by an increased hydrodynamic diameter, up to certain a point when the disruption occurs. Keywords: Peptide antibiotics, Amphipathic a-helix, Coiled-coil structure, nmr

Study of a bradykinin potentiating peptide, bpp7a, and its b-cyclodextrin complex ˆ ngelo M. L. Denadai, Ivana Lula,1 Frederico B. De Sousa, A Danielle Ianzer, Antoˆnio C. M. de Camargo, Robson A. S. Santos, and Rubeń D. Sinisterra 1

Universidade Federal de Minas Gerais, Instituto de Cieˆncias ExatasDepartamento de Quı´mica. Av. Antonio Carlos 6627, Belo Horizonte MG 31270-901, Brazil Serpent venom, especially from Bothrops jararaca, contains small molecule peptides which greatly enhance the smooth-muscle

S65 contraction induced by bradykinin. These peptides, called bradykinin potentiating peptides (BPPs), have also demonstrated the ability to inhibit angiotensin-I converting enzyme (ACE). Typically, BPPs from Bothrops jararaca contain 5–14 amino acid residues and have similar 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 with drug degradation and, moreover, are able to modify the administered dose. Therefore, cyclodextrins could be used once this class of molecules is shown as a potential colon-specific drug carrier system. The heptapeptide BPP7a, p-Glu1Asp2Gly3Pro4Ile5Pro6Pro7, forms an association complex with b-cyclodextrin. The peptide and its complex were characterized by circular dichroism (CD) and isothermal titration calorimetry (ITC), which showed a very weak interaction between b-cyclodextrin and the peptide. Assignments of all hydrogen resonances of the peptide alone and as a complex were made using 1H nuclear magnetic resonance (NMR) experiments at 400 and 600 MHz. High resolution diffusion ordered spectroscopy (HR-DOSY) experiments were carried out to establish the selfaggregation state of BPP7a. In addition, the anti-hypertensive activity of the BPP7a/b-cyclodextrin complex was evaluated in spontaneous hypertensive rats (SHR), showing increased activity than pure BPP7a. In vivo experiments in SHR showed that the complex reduced blood pressure in the rats, which may be attributed to increased bioavailability of BPP7a after its complexation with b-cyclodextrin.

Synthesis and study of the solvent effect on selfassembly in a novel tripeptide molecule contained unusual amino acid using molecular dynamics simulations Bahareh Talaei, Hengameh Fallah, and Saeed Balalaie* Peptide Chemistry Research Center, K. N. Toosi University of Technology, Tehran, Iran, Corresponding author: [email protected] The diphenylalanine dipeptide is a suitable building block for molecular self-assembly. We were encouraged to synthesis some novel peptides which contained c-amino acids in their backbones. 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-BaclofenOH was selected due to the existence of aromatic moiety in the structure of baclofen which is responsible for self-assembly via p–p stacking. The realm of applications of computational chemistry is considerably expanding owing to steady advances in computer power. An ab initio quantum mechanical calculation was performed on H-Phe-Phe-Baclofen-OH tripeptide using Gaussian 03 package. The gas phase energy of the tripeptide was optimized at the HF/3-21G level. Molecular Dynamics Simulations were carried out with explicit solvent (water) using Gromacs 4.0.7 package. The details of simulations will be reported. Different possible ways of selfassembly such as H-bonding, p–p stacking and T-stacking can be seen in the presentation. The details about the self assembly of these compounds will discuss in the conference.

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Synthesis of novel bisphosphonate containing peptide and unusual amino acids Sorour Ramezanpour, Saeed Balalaie*, Armin Arabanian, and Hamid Reza Bijanzadeh Peptide Chemistry Research Center, K.N. Toosi University of Technology, Tehran, Iran Biophosphonate (BPs) are an important class of drugs with different biological activities. They can be used for treatment of diseases characterized by abnormal calcium metabolism; such as osteoprosis and tumor-associated osteolysis. It was shown that the existence of heterocycle skeleton has an efficient role for the biological activity of the bisphosphonate derivatives such as imidazole moiety in zoledronic acid as the anti-cancer bisphosphonate compound. In continuation of our research for the synthesis of novel peptides, some novel peptides containing free carboxylic acid functional group were synthesized via solution phase peptide synthesis (SIS) or solid phase peptide synthesis (SPPS) approaches. We synthesized novel peptides with histidine in their structure. Meanwhile, some unusual amino acids such as gabapentine and baclofen, were used as the starting materials. The synthesis of bisphosphonate moiety was done using reaction of phosphorus trichloride and phosphorous acid with free carboxylic acid moiety. The details for the synthesis of desired bisphosphonates will be discussed in the conference.

Synthesis of pseudopeptides contained indole skeletons and their applications in synthesis of lipophilic peptides Sorour Ramezanpour, Nahid Sadeghi Alavijeh, Saeed Balalaie*, and Hamid Reza Bijanzadeh Peptide Chemistry Research Center, K.N. Toosi University of Technology, P.O.Box 15875-4416, Tehran, Iran Pseudopeptides contained indole skeletons, suitable for synthesis of lipophilic peptides which can probably affect the ability of these bioactive compounds to penetrate blood–brain barrier (BBB) as well as transport across cell membranes, can be synthesized by using suitable indole carboxylic acid derivatives. Indole nucleus is a substructure found in numerous natural products and pharmaceuticals possessing anti-inflammatory, antimalarial, antidepressant, antitumor and various other activities. Therefore, we have been 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 amide bonds. Among the protocols for the synthesis of pseudopeptides and peptides or amino acids, the important method is the Ugi four component reaction (Ugi-4CR) as it offers significant advantages over stepwise procedures, especially with respect to diversity, complexity, green chemistry, and atom economy. In this approach, we have designed an Ugi-4CR strategy via the reaction of benzaldehyde derivatives, Indole carboxylic acids, primary amines and isocyanides to construct these valuable pseudopeptides which have a huge potential for further reactions such as

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12th International Congress on Amino Acids, Peptides and Proteins esterification, methanolysis, Heck and Buchwald. The influence of the different starting materials is evaluated and numerous studies are underway at this time to explore their biological activities.

Topological determination of the antimicrobial peptide hylaseptin P2 in oriented bilayers V. H. O. Munhoz1,2, B. A. Assunçaõ*1, S. F. C. de Paula1, J. M. Resende1, D. Pilo´-Veloso1, and B. Bechinger2 1

Departamento de Quı´mica, Instituto de Cieˆncias Exatas, Universidade Federal de Minas Gerais, Av. Pres. Antoˆnio Carlos, 6627, 31270090, Belo Horizonte MG, Brazil (*[email protected]) 2 Universite´ de Strasbourg/CNRS UMR7177, Institut de Chimie, 4, rue Blaise Pascal, 67070 Strasbourg, France *[email protected] Keywords: Antimicrobial peptides, Solid-state NMR The Hylaseptin P2 (HSP-2) is an antimicrobial, a-helical peptide present on Hypsiboas punctatus anurans, found at the Amazon rainforest. It has activity against both Gram-positive and Gram-negative bacteria and fungi. It is believed that its mechanism of action is guided by its affinity with the bacterial membrane. Although the outlines of the mechanism are proposed, its full pathway is not yet completely unveiled, hence the need to perform studies to elucidate it better. One of the techniques that have been useful to study peptidephospholipid 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 through 31P, 2H, and 15N cross-polarization NMR experiments. The 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 over the other, and hydrated at 93% of relative humidity. The 15N 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 triple-resonance probe. The peptides were synthesized with 15N label at L18 and 2H label at A16. The resulting 31P spectrum indicates a good orientation of the bilayer. The 15N spectrum shows a peak at d74 ppm and the 2H spectrum, a quadrupolar splitting of 27 kHz. By calculating the tilt and rotational pitch angle it was possible to determine the orientation of the peptide.

Plant amino acids An omics approach to elucidating amino acid metabolism in plant Kansuporn Sriyudthsak, Eiji Okamura, Yuji Sawada, and Masami Yokota Hirai RIKEN Plant Science Center/CREST, JST These days it is becoming more and more important to understand plant metabolism, because it is closely related to plant production,

12th International Congress on Amino Acids, Peptides and Proteins namely, food and biomass production. However, genes involved in metabolism and its regulation are not yet fully identified even in Arabidopsis. We are aiming at discovery of novel genes responsible for amino acid metabolism and secondary metabolism by means of transcriptomics and metabolomics. Based on coexpression analysis using a large-scale transcriptome dataset of Arabidopsis, we have identified/predicted novel gene functions in glucosinolate (secondary metabolite specific to Capparales) and amino acid metabolism. In order to obtain a large-scale metabolome dataset, we have established an ultra high-throughput methodology named ‘widely targeted metabolomics’ (Sawada et al. 2009, Plant Cell Physiol. 50:37–47). We obtained timeseries metabolome data after a certain metabolic perturbation. Furthermore, we constructed a mathematical model of amino acid metabolism by our novel simulation algorithm. The simulation results agreed well with the experimental data (time-series metabolome data), showing the algorithm is useful to topologically predict dynamic behaviors of metabolite levels in the metabolic system. In this presentation, our recent progress will be introduced.

Bio-available diamino amino acids in soil of differently managed mountain meadows and forests Valerie Vranova1, Dalibor Janous3, Klement Rejsek1, Jindrich Kynicky1, Martin Brtnicky2 and Pavel Formanek1 1

Mendel University in Brno, Faculty of Forestry and Wood Technology, Department of Geology and Soil Science, Zemedelska 3, 613 00, the Czech Republic, e-mail: [email protected], 2 Mendel University in Brno, Faculty of Agriculture, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemedelska 1, 613 00, The Czech Republic, 3 CzechGlobe, Global Change Research Centre AS CR, v.v.i., Porici 3b, 603 00 Brno, The Czech Republic Bio-available or so-called ‘‘free’’ amino acids occurring either in soil solution or exchangeably bound on soil colloids may be directly taken up by plant roots without previous mineralization. This may be especially relevant in alpine, arctic and boreal areas, where the availability of mineral nitrogen is low, and thus, amino acids are considered to be important for plant nutrition. In this work we have concentrated on selected bio-available diamino amino acids including (1) nitrogen rich and more strongly exchangeably bound arginine as well as strongly exchangeably bound lysine, and (2) cystine. This work has shown that in the top 7 cm of Ap horizon of differently managed mountain meadows, arginine, lysine and cystine formed (on average) 14, 2.5 and 11, respectively, of the totally seventeen amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, alanine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, proline plus the three studied diamino amino acids) extracted from soil using 0.5 M ammonium acetate. In Oe horizon of differently managed mountain Norway spruce stands, arginine, lysine and cystine formed (on average) 2.7, 3.1 and 7.1, respectively, of the seventeen extracted amino acids. Each of the three studied diamino amino acids formed ca. 7–8% of the total bio-available nitrogen including nitrogen of all seventeen amino acids, ammonium and nitrates in soil of all studied ecosystems. Acknowledgments: This work was financially supported by IGA MENDELU 2010-2012 and NAAR - QI91C054.

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Characterization of type 1 ribosome inactivating proteins in edible plants Munish Puri1*, Inderdeep Kaur2 and C. J. Barrow3 1,3 Centre for Biotechnology and Interdisciplinary Biosciences, Institute for Technology Research and Innovation (ITRI), Geelong Technology Precinct (GTP), Deakin University, Victoria 3217, Australia, 2 Fermentation and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, India

The ribosome inactivating proteins (RIPs) from plants possess RNA N-glycosidase activity that depurinates the major rRNA, thus damaging ribosome in an irreversible manner and arresting protein synthesis. RIPs occur in fungi, bacteria and plants and are abundant in angiosperms, where they appear to have defensive role. RIPs are presently classified as rRNA N-glycosidase in the enzyme nomenclature (EC 3.2.2.22) and do exhibit other enzymatic activities such as ribonuclease and deoxyribonuclease 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 attention because of their anti-viral, anti-tumor, and anti-microbial activities, which is useful in medical research and development. Here, we describe isolation of a novel protein from Momordica sp, a highclimbing vine from family Cucurbitaceae which is native to the tropical regions of Africa, Asia, Arabia and Caribbean. The purified protein has been verified by SDS-PAGE and mass spectrometry to contain only single chain Type-1 ribosome inactivating proteins (RIPs). With present experiments, we determined the presence of RIPs in edible plant materials, including some that are eaten raw by human beings. The novel protein is further characterized to validate its therapeutic potential.

Contributions of individual amino acids to CLV3 peptide in shoot apical meristem maintenance in Arabidopsis Xiufen Song, Dali Yu, Peng Guo, Tingting Xu, Shichao Ren, and Chun-Ming Liu* Center for Signal Transduction and Metabolomics, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Fragrance Hill, Beijing 100093, China Compared to traditional phytohormones, peptide hormones in theory have more flexibility in changing their primary structures and subsequently specificities in evolution through amino acid (AA) substitutions. Previous genetic and biochemical studies reveal that CLV3 acts as a small peptide to interact with the CLV1/CLV2/SOL2 receptor complexes for shoot apical meristem (SAM) maintenance. To elucidate how peptide specificities are established in AA sequence, we introduced point mutations to the peptide-coding and its flanking regions of CLV3 to substitute AAs one by one by alanine and introduced these constructs to clv3-2 for in vivo complementation. This study yielded a complete contribution map of individual AAs for the CLV3 function, showed that glycine-6, aspartate-8 and histidine11 are among the most important residues, while previously assigned modification residues of proline-7 and -9 and flanking sequences showed very little contributions. Further, in vitro SAM treatments using chemically synthesized alanine-substituted CLV3 peptides revealed an evidently different contribution pattern as the in vivo assay and the previously reported root assay, suggesting different

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S68 perception machineries are involved. The contribution of individual AA in CLV3 established in this work may help to understand peptide hormones in general.

Effect of alkali-extracted rice protein on the renal function in type 2 diabetic rats Reiko Watanabe1, Masatoshi Kubota2, Akihiko Saito3, Takehisa Kumagai4 and Motoni Kadowaki2 1

Department of Health and Nutrition, University of Niigata Prefecture, Japan, 2 Graduate School of Science and Technology, Niigata University, Japan, 3 Graduate School of Medical and Dental Sciences, Niigata University, Japan, 4 Kameda Seika Co., Ltd., Japan Backgrounds and objectives: Type 2 diabetes mellitus (T2DM) is rapidly increasing and is becoming a serious problem all over the world. T2DM caused by life style is associated with several long-term complications, including diabetic nephropathy, and the important role of dietary protein in kidney function has been recognized. Rice is a stable food and important as energy and protein sources in many countries. We had clearly proved that alkali-extracted rice protein (AE-RP) lowered total cholesterol and triglycerides in the plasma and liver. Therefore, we focused on effects of AE-RP on T2DM, especially diabetic nephropathy, as new beneficial functions. Materials and methods: AE-RP used included high CP content ([90%) which have high digestibility and bioavailability. Male nonobese type 2 diabetic Goto-Kakizaki (GK) rats were fed high sucrose diets for 10 weeks with 20% casein (C) or AE-RP. Fasting blood glucose levels were measured every other week. Three-day urine collection was performed to determine albuminuria at the end of experiment. Kidneys are removed and the tissue sections obtained for histological analysis. Results: AE-RP had no effects on fasting blood glucose levels. However, the level of urinary albumin in GK rats fed AE-RP was significantly lower (36%) than that of C group. Degrees of renal glomerular damages were significantly higher in C group, but those in AE-RP group were similar to those in Wistar rats fed high sucrose C diets. These results suggest that AE-RP has a suppressive effect on progression of diabetic nephropathy.

Mineralization of enantiomers of glutamic acid in soil Peter Dundek1, Valerie Vranova1, Klement Rejsek1, Marian Pavelka3, Jindrich Kynicky1, Martin Brtnicky2 and Pavel Formanek1 1

Mendel University in Brno, Faculty of Forestry and Wood Technology, Department of Geology and Soil Science, Zemedelska 3, 613 00, the Czech Republic, e-mail: [email protected], 2 Mendel University in Brno, Faculty of Agriculture, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemedelska 1, 613 00, the Czech Republic 3 CzechGlobe Research Center, Porici 3 b, Brno, the Czech Republic D-Amino

acids artificially added to soils are mineralized at lower rates compared to their corresponding L-enantiomers; nevertheless, the mechanism of this effect is not known. The L- to D-amino acid respiration ratio is commonly reported in the range from 1.0 to 8.7. The differences between L- and D-amino acid respiration were found to be affected by stress conditions (acidity, heavy metals). In this work we have attempted to determine respiration of L- and D-glutamic acid as

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12th International Congress on Amino Acids, Peptides and Proteins well as their ratio within soil profiles in young spruce stands (soil type Typic Haplohumod) and meadows (soil type Oxyaquic Hapludalf) of mountain regions, and young oak stands in lower altitudes (soil type Oxyaquic Haplustept). Results of this work showed that the L- to Dglutamic acid respiration ratio ranged from 1.2 to 5.1 in different horizons of soil profiles, being the lowest in organic horizons and decreasing with depth. Acknowledgments: This work was financially supported by IGA MENDELU 2010-2012 and NAAR, QI91C054.

Mineralization of c-aminobutyric acid, citrulline, ornithine and b-alanine in soil Peter Dundek1, Valerie Vranova1, Klement Rejsek1, Martin Brtnicky2, Marian Pavelka3, Jindrich Kynicky 1 and Pavel Formanek1 1 Mendel University in Brno, Faculty of Forestry and Wood Technology, Department of Geology and Soil Science, Zemedelska 3, 613 00, The Czech Republic; e-mail: [email protected], 2 Mendel University in Brno, Faculty of Agriculture, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemedelska 1, 613 00, The Czech Republic, 3 CzechGlobe, Global Change Research Centre AS CR, v.v.i., Porici 3b, 603 00 Brno, The Czech Republic

Gamma-aminobutyric acid (GABA), citrulline, ornithine and b-alanine play different roles in plants including protection against stress. These amino acids were also found in soils of various ecosystems including subtropical heathlands, tundra communities, forests, coastal salt marshes, arable land and grasslands. In soil, these amino acids may occur incorporated in soil organic matter, as ‘‘free’’ in soil solution or exchangeably bound to soil colloids. GABA was found in the highest concentrations mainly in waterlogged soils forming up to 50% of the total ‘‘free’’ amino acid-N pool. Ornithine was found to be among dominant amino acids in leachates from some forest soils. Besides the ‘‘free’’ amino acid pool, occurrence of b-alanine was reported in hydrolysates of soils or humic substances. Data on kinetics of GABA, citrulline, ornithine and b-alanine mineralization in soils of different properties are presented and discussed in this work. Acknowledgments: This work was financially supported by IGA MENDELU 2011.

Nascent peptide-mediated control of gene expression in Arabidopsis: feedback regulation of methionine biosynthesis and beyond Hitoshi Onouchi1,2, Isao Ebina3, Mariko Takemoto1, Shun Watanabe3, Noriyuki Onoue3, Yui Yamashita3, Yayoi Endo4, Hiroaki Koyama1, Yoko Yamashita-Nagami1, Hiro Takahashi5, and Satoshi Naito3 1

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan, 2 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan, 3 Division of Life Science, Graduate School of Life Science, Hokkaido University, Sapporo 060-8589, Japan, 4 Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan, 5 College of Bioscience and Biotechnology, Chubu University, Matsumoto-cho 1200, Kasugai, Aichi 487-8501, Japan Expression of the Arabidopsis CGS1 gene, which encodes the first committed enzyme of methionine biosynthesis, is feedback

12th International Congress on Amino Acids, Peptides and Proteins regulated at posttranscriptional level. A 14-residue stretch of amino acid sequence, termed the MTO1 region, encoded within the first exon of CGS1 itself is involved in this regulation. Our in vitro studies have revealed that, during translation of CGS1 mRNA, the MTO1 region within the nascent CGS1 polypeptide acts inside of the ribosome before emerging from it to cause translation elongation arrest in response to S-adenosyl-L-methionine (AdoMet). The translation arrest then triggers CGS1 mRNA degradation. In addition, we recently found that AdoMet induces compaction of the nascent peptide chain inside the ribosomal exit tunnel upon the translation arrest. Apart from the CGS1 system, most of eukaryotic examples of nascent peptide-mediated ribosomal regulation involve peptides encoded by upstream open reading frames (uORFs) located in 50 untranslated regions. In order to further identify regulatory nascent peptides, we searched for Arabidopsis uORFs whose amino acid sequences are conserved in a wide range of dicot plants by using a comparative genomic approach, and identified 14 novel conserved uORFs. Mutational analyses revealed that 4 of the conserved uORFs control the expression of the main coding sequence in an amino acid sequence-dependent manner.

Proline accumulation is inhibitory to Arabidopsis seedlings during heat stress Wei-Tao Lv, Bin Lin, Min Zhang, and Xue-Jun Hua* Research Center for Molecular and Developmental Biology, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, People’s Republic of China, Graduate School of Chinese Academy of Sciences, Beijing 100049, China 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 gene promoter. During heat stress, the heat-inducible expression of AtP5CS1 transgene was capable of enhancing Pro biosynthesis. 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 recovery at 22°C for 96 h, the growth of Pro-overproducing plants was significantly more inhibited than were control plants that do not accumulate Pro, manifested by lower survival rate, higher ion leakage, higher ROS and MDA levels and increased activity of Pro/P5C cycle. The activity of antioxidant enzyme SOD, GPX, and CAT, but not that of GR and APX, increased in all lines after heat treatment, but the increase was more significant in Pro-overproducing seedlings. Staining with MitoSox-Red, reported for being able to specifically detect O2- formed in mitochondria, showed that Pro accumulation during heat stress resulted in elevated level of ROS in mitochondria. Interestingly, exogenous abscisic acid and ethylene were found to partially rescue the heat sensitive phenotype of Pro-overproducing seedlings. Measurement of ethylene and ABA levels further confirmed that these two hormones are negatively affected in Pro-overproducing seedlings during heat stress. Our results indicated that Pro accumulation under heat stress decreases the thermotolerance, probably by increased ROS production via Pro/P5C cycle and inhibition of ABA and ethylene biosynthesis.

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Proteome and free amino acid of Polygonum minus leaf Roohaida Othman1,2, Hana-Marlin Mahfodz2, Chang Li Yen3, Nur Afiqah Sukiran1, Syarul Nataqain Baharum1, and Normah Mohd Noor1 1 Institute of Systems Biology, Universiti Kebangsaan Malaysia, Malaysia, 2 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Malaysia, 3 Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Malaysia, Correspondence: [email protected]

The aromatic plant, Polygonum minus, produces essential oil of high economic value with high demands in the food, flavor and fragrance industry. The essential oil contains a variety of secondary metabolites, including aliphatic aldehydes and terpenes that have been associated with this plant unique smell. To understand the secondary metabolite biosynthesis, a proteomic approach was undertaken to identify the 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 leaf polypeptide profile was generated based on the consistently 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. Enzymes involved in carbohydrate metabolism and photosynthesis were among the main proteins identified. Subsequent amino acid analysis using LC–TOF–MS showed that from 19 amino acids detected, leucine was found to be the most abundant amino acid. The enzyme involved in the precursor biosynthesis of leucine was identified from the proteomic study. The proteome map obtained provides the basis for further study on P. minus physiology and will contribute to improve our understanding of plant metabolism in aromatic plant thus aiding in crop improvement effort.

The defense function and secondary structure analysis of HpaXm from Xanthomonas citri subsp. malvacearum Weiguo Miao1*, Congfeng Song2, Fucong Zheng1, and Jinsheng Wang2* 1 College of Environment and Plant Protection, Hainan University, Haikou 570228 China, 2 Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095 China, *Corresponding author Weiguo Miao, e-mail: [email protected], Jinsheng Wang, e-mail: [email protected]

HpaXm from, HpaXm (harpin) is a type III secreted protein of the cotton blight bacterial pathogen Xanthomonas citri subsp. malvacearum that elicits a hypersensitive response (HR) in nonhost tobacco. Two a-helical domains were predicted in HpaXm by the HNN program; a hydrophobic h-region was found between the N- and C-terminal flanking regions. The former contains the 1st to 18th residues, in which ab-strand resides. The latter contains the 101st to 133rd residues. H-regions were also found in other harpin-like proteins. A signal peptide was predicted to exist in the 15 leading amino

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S70 acids of the N-terminus. The identical heptads, 39-LDQLLTQ– LIMALLQ-52, were found in the N-terminal a-helix region of HpaXm. Italicized letters indicate different residues in HpaXm. Three DNAbinding residues were found in N and C-terminal regions in HpaXm, especially C-terminal flanking regions. HpaXm is predicted to contain two potential coiled-coil (CC) regions, one at the N-terminus with a high probability of formation, and one at the C-terminus with a lower probability of formation. To investigate the role of the CC regions in HpaXm function, two peptides and two mutated peptides containing 14 residues of the N- and C-terminal CC regions were designed and synthesized. Peptide purities of the synthetic peptides were all higher than 84%. Tobacco leaves infiltrated with aqueous solutions of these peptides showed an HR within 24 h. The result indicated that the peptide, containing 14 amino acids from the N-terminal CC region of HpaXm, was sufficient to cause an HR after infiltrating the intercellular spaces of tobacco leaves. Specifically, the CC domain of the N-terminal a-helix contributed to HR activity. Acknowledgments: This work was supported by grants from the NKBRPC (2003CB114204 and 2006CB101902), NKPC (2004BA901A36), RFDP(20104601110004), CARS-34-GW8 and KYQD1006(China).

The effect of abandonment of mountain meadows on uptake of 14C-labelled glutamic acid and alanine by soil microbial community Klement Rejsek1, Valerie Vranova1, Dalibor Janous3, Michael Po¨schl4, Jindrich Kynicky1, Martin Brtnicky2 and Pavel Formanek1 1

Mendel University in Brno, Faculty of Forestry and Wood Technology, Department of Geology and Soil Science, Zemedelska 3, 613 00, the Czech Republic; e-mail: [email protected], 2 Mendel University in Brno, Faculty of Agriculture, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemedelska 1, 613 00, The Czech Republic, 3 CzechGlobe Research Center, Porici 3 b, Brno, The Czech Republic, 4 Mendel University in Brno, Faculty of Agriculture, Department of Molecular Biology and Radiobiology, Zemedelska 1, 613 00, The Czech Republic The effect of a 13-year abandonment of previously long-term mown mountain meadows on uptake of L-glutamic acid (14CO2H[14CH2]2 14 [NH2]14CO2H) and L-alanine (14CH14 3 CH[NH2] CO2H) by microbial community of Ap horizon (3–13 cm) (soil type Oxyaquic Hapludalf) was determined in this work. Soil of Ap horizon of mown and abandoned meadows showed the same total carbon content and similar total nitrogen content (0.29 vs. 0.33%). One hundred fifty microlitres of alanine solution (15.7–717 lM) or glutamic acid solution (9.2–588.3 lM) was briefly mixed with 0.3 g wet soil in 15-ml polypropylene tubes. One ml of 1 M NaOH was added to each sample tube to capture the respired 14CO2 and the sample tubes were sealed with a gas-tight stopper and incubated at 14.5°C for 3 h. Subsequently, the soils were extracted with 0.5 M K2SO4 to remove the not-utilized amino acids (10 min, 200 rpm) followed by centrifugation (3,550 rpm, 20 min) and counting of the 14C label remaining in the supernatant solution. The abandonment of the meadow had no significant (P [ 0.05) effect on L-alanine or L-glutamic acid uptake. Uptake rate of L-alanine at 717 lM was 135-159 nmol g-1 dry soil h-1; glutamic acid uptake rate at 588.3 lM was 83-85 nmol g-1 dry soil h-1. Acknowledgments: This work was financially supported by IGA MENDELU 2010-2012 and NAAR, QI91C054.

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The effect of amino acids on activity of extracellular acid phosphomonoesterase in soil Valerie Vranova1, Silvia Chersich2, Dalibor Janous4, Klement Rejsek1, Jindrich Kynicky1, Martin Brtnicky3 and Pavel Formanek1 1

Mendel University in Brno, Faculty of Forestry and Wood Technology, Department of Geology and Soil Science, Zemedelska 3, 613 00, The Czech Republic, e-mail: [email protected], 2 Pavia University, Department of Earth Sciences, Via Ferrata, 1 27100, Pavia, Italy, 3 Mendel University in Brno, Faculty of Agriculture, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemedelska 1, 613 00, The Czech Republic, 4 CzechGlobe, Global Change Research Centre AS CR, v.v.i., Porici 3b, 603 00 Brno, The Czech Republic Extracellular acid phosphomonoesterase (orthophosphoric monoester 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 codworm (Phocanema decipiens) or muscle of codfish (Gadus morhua) was found to be inhibited by different compounds including amino 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 phosphomonoesterase in soil and thus participate in regulation of phosphorus cycling in terrestrial ecosystems. Amino acids were applied to soil in concentration of 5 lg g-1 dry soil. Results of this work showed that L-arginine has no effect, whereas D-alanine, Lglutamic acid and L-cysteine tend to slightly increase (by up to 10%) activity of extracellular acid phosphomonoesterase in tested soils.

Polyamines Characterization of the hypusine biosynthetic pathway from Leishmania donovani Rentala Madhubala School of Life Sciences Jawaharlal Nehru University, New Delhi, India Hypusine (N€-(4-amino-2-hydroxybutyl) lysine), an unusual amino acid derived from the polyamine spermidine, is present in all the eukaryotes. It is synthesized as a result of post-translational modification occurring exclusively on one cellular protein, eukaryotic initiation factor 5A (eIF5A). It is formed in two enzymatic steps. The first step is catalyzed by the enzyme deoxyhypusine synthase (DHS) (EC 2.5.1.46) which catalyses the NAD+ dependent transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue of the eIF5A precursor protein to form an intermediate, deoxyhypusine. This intermediate is subsequently hydroxylated by the enzyme deoxyhypusine hydroxylase (DOHH) (EC 1.14.99.29) which completes the synthesis of hypusine and maturation of eIF5A. Leishmania has two genes containing DHS domains viz., DHS-like gene (DHSL20) and DHS34 gene, of which only DHS34 protein was found to contain functional activity in vitro. DHS34 is much longer

12th International Congress on Amino Acids, Peptides and Proteins with 601 amino acids as compared to the human enzyme (369 amino acids) and contains several unique insertions. Gene replacement studies for DHS34 showed that the enzyme deoxyhypusine synthase and eIF5A modification play an essential role in cell viability of L. donovani. The second enzyme of the hypusine biosynthetic pathway, DOHH, is a HEAT-repeat protein with eight tandem repeats of a-helical pair. Four conserved histidine-glutamate sequences have been identified which may act as metal coordination sites. A *42 kDa recombinant protein was obtained by heterologous expression of DOHH in Escherichia coli. Circular dichroism spectroscopy revealed that the purified recombinant L. donovani DOHH is largely a-helical. L. donovani DOHH showed low sequence identity (40.6%) with the human homolog. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibit the growth of L. donovani and DOHH activity in vitro. These inhibitors were found to be less effective against the human enzyme. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159–162 (four amino acid residues) and 174–183 (ten amino acid residues), the latter being present near the substrate binding site. The structural differences between the L. donovani enzymes and the corresponding human homologs may be exploited for structure based design of selective inhibitors against the parasite.

Effect of L-arginine on the catabolism of polyamine and oxidative stress in testes of rats treated by ethanol Pavlovic D1, Stojanovic I1. Nikolic J1, Kocic G1, Cvetkovic T1, Jevtovic-Stoimenov T1, and Visˇnjic´ M2 1

Institute of Biochemistry, Medical Faculty University of Nis, Serbia, Surgery Clinic of the Clinical Center, Medical Faculty University of Nis, Serbia, [email protected]

2

Despite the low oxygen tensions characterizing the testicular microenvironment, this tissue remains vulnerable to oxidative stress. Previous studies have reported that excessive alcohol consumption 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 testosterone synthesis, DNA and RNA, being necessary for normal mammalian spermatogenesis, cellular proliferation and tissue integrity. On the other hand degradation products of polyamines exert cytotoxic effects both in vitro and in vivo. Since L-arginine is the precursor in the synthesis of polyamines, the aim of this study was to determine the parameters of oxidative stress and the activity of polyamine oxidase (PAO) and diamine oxidase (DAO)— enzymes responsible for polyamine catabolism in the testes of rats treated by ethanol, and the effect of L-arginine administration. The animals were allocated into four experimental groups: ethanol treated (15% solution in drinking water), arginine treated (0.5% solution of arginine in drinking water), ethanol plus arginine, and controls. Animals were treated for 3 weeks. The obtained results demonstrated that ethanol administration inhibit the antioxidant enzymes in the testes—catalase, SOD, GST, diminishes the testicular content of GSH, while MDA concentration, as well as PAO and DAO activity are increased. L-arginine ameliorates ethanol induced oxidative stress in the testes, suppressing lipid peroxidation and restoring catalase, SOD, GST, PAO, DAO and GSH to normal levels. These results raise the possibility of novel nutrition strategy for preventing and treating ethanol-dependent testicular dysfunction.

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Establishment of growth arrest by polyamine depletion Guy Landau, Ester Feldmesser, Zippy Bercovich, Avichai Ran, and Chaim Kahana Department of Molecular Genetics, the Weizmann Institute of Science, Rehovot 76100, Israel, e-mail: [email protected] The polyamines spermidine, spermine and their diamine precursor putrescine are naturally occurring polycations that are essential for cellular growth and proliferation. Depletion of cellular polyamines results in growth cessation that will be resumed upon re-addition of polyamines to the growth medium of the arrested cells. The tight association between polyamines and cellular proliferation made the polyamine metabolic pathway an appealing therapeutic target for treating hyperproliferative pathologies including cancer. However, the mechanism by which polyamine depletion inhibits cellular proliferation is mostly unknown. We set out to investigate the molecular mechanism by which polyamine depletion causes growth inhibition by investigating changes in translational and transcriptional activity in the depleted cells. 35 S-methionine incorporation experiments demonstrated that both DFMO and GC7 treatments inhibit protein synthesis activity. Polysomal 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 is a result of its immediate competition with the physiological polyamines on cellular sites of action. On the mechanistic side we have demonstrated that DFMO and GC7 treatment affected the phosphorylation state of two translation factors that regulate the initiation step. Both treatments increased phosphorylation of eIF2a and decreased phosphorylation of 4E-BP1, two changes that each by itself can provoke the initiation block. Kinetic studies have demonstrated that the phosphorylation of eIF2a that might be part of the establishment of stress response preceded all other changes. Characterization of transcriptional changes occurring during polyamine depletion further supported the notion that the growth of polyaminedepleted cells is inhibited due to establishments of stress response program in the treated cells.

Monitoring cancer by urinary polyamine analyses Uriel Bachrach Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel, e-mail: [email protected] The naturally occurring polyamines, spermine, spermidine and the diamine putrescine are widespread in nature. Due to their cationic nature, they interact with negatively charged macromolecules, such as DNA and transfer RNA and stabilize them. Therefore, during growth processes polyamines are synthesized along with nucleic acids replications. Over 75,000 research papers have been written on this subject since 1900 and more than half (54%) were published between 1990 and 2009. As cancer cells grow rapidly, polyamines accumulate in cancerous tissues and their concentration is elevated in body fluids of cancer patients. Various analytical methods were used to determine the amounts of polyamines in biological fluids. We developed an enzymatic-based chemiluminescence method to determine urinary polyamines and analyzed more than 2,000 urine samples from breast, colon, lung and rectal cancer patients. Polyamine levels are high in the urine of cancer patients. Upon successful treatment, urinary polyamines return to their normal values. When therapy fails,

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S72 polyamine levels remain high. Longitudinal studies of polyamine levels can permit the assessment of the relapse or remission of the disease. Elevated urinary polyamines, indicate the presence of cancerous cells, sometimes even before the appearance of clinical symptoms. Polyamines are present also in red blood cells and in the hair of cancer patients. It may be concluded that the determination of polyamines in biological fluids, may serve as a useful tool for cancer diagnosis and treatment.

Polyamine and nitric oxide metabolism interaction markers in colorectal cancer, surrounding and healthy tissue samples: possible prognostic value I. Stojanovic1, A. Veljkovic1, B. Brankovic2, D. Pavlovic1, G. Stanojevic2, G. Kocic1, D. Sokolovic1, P. Janosevic3, M. Nestorovic2, D. Petrovic2, and M. Visnjic2 1 Institute of Biochemistry, University of Nis, Faculty of Medicine, Serbia, 2 Clinic for Surgery, Clinical Centre Nis, University of Nis, Faculty of Medicine Serbia, 3 Clinic for Stomatology, University of Nis, Faculty of Medicine, Serbia

Although L-arginine-derived polyamines and nitric oxide are known as the promoters of tumorigenesis, the contradictory literature data point out anti-proliferative activity of nitric oxide, opposed to polyamine role in cancer cell proliferation and differentiation. Considering the literature data that dietary arginine can enhance the risk for colorectal cancer development by a mechanism including NOS2 and polyamine synthesis, we have examined the markers of L-arginine metabolism in the samples of cancer tissue, as well as adjacent surrounding and healthy tissue at the incision margin in 50 patients with colorectal cancer. The obtained results suggest that the present L-arginine seems to be competent for L-ornithine production in context of high putrescine production and, consequently, spermidine and spermine synthesis. Together with diminished putrescine catabolism through diamine oxidase pathway, this could be the way to estimate the proliferative potential of surrounding adjacent and healthy colon tissue in these patients. Increased nitric oxide level in relation to enhanced polyamine synthesis and high arginase activity in cancer tissue points out the possibility of Larginine synthesis from citrulline, rising arginine bioavailability for both polyamine and nitric oxide synthesis, leading to tumor progression. The differences in polyamine and nitric oxide content, as well as other markers of L-arginine metabolism, in healthy and adjacent tissue compared to cancer one offer a possibility to estimate the risk of tumor recidivity and the survival of these patients at the time of surgical intervention.

Polyamine oxidases in cyanobacterium Synechocystis sp. PCC 6803 under osmotic stress Aran Incharoensakdi, and Khanittha Samasil Chulalongkorn University, Bangkok, Thailand Polyamines namely, putrescine, spermidine and spermine, are polycationic compounds found in both prokaryotic and eukaryotic cells.

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12th International Congress on Amino Acids, Peptides and Proteins They are involved in cell growth, developmental processes as well as responses to environmental stress. The effect of osmotic stress imposed by sorbitol on total polyamine contents and polyamine oxidases were investigated in unicellular cyanobacterium Synechocystis sp. PCC 6803. The growth rate of the cells decreased when BG11 medium contained 300 mM sorbitol. The cells could not grow in the presence of 700 mM sorbitol. The changes in polyamine oxidase 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, Synechocystis crude extracts were found to oxidize putrescine, spermidine and spermine. Overall results suggest that at least two amine oxidases are present in Synechocystis cells.

Spermine enzymatic oxidation products in cancer therapy: Lysosomotropic compounds and Docetaxel potentiate their cytotoxicity Enzo Agostinellia, Giampiero Temperaa, Nikenza Vicecontea, Stefania Saccoccioa, Eris Bidollaria, Maria Condellob, Giuseppina Bozzutob, Giuseppe Aranciab and Agnese Molinarib a Department of Biochemical Sciences, SAPIENZA University of Rome and CNR, Institute of Molecular Biology and Pathology, P.le A. Moro 5, 00185 Rome b Department of Technology and Health, Istituto Superiore di Sanita` Viale Regina Elena 299, 00161 Rome, Italy, [email protected]

The in situ formation of cytotoxic metabolites by an enzyme-catalyzed reaction is a recent approach in cancer chemotherapy. It was demonstrated that bovine serum amine-oxidase (BSAO) and spermine (SPM) addition to human cancer cells induces cell growth inhibition and over-run the multidrug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites H2O2 and aldehydes produced by the oxidative reaction. The results demonstrate that cytotoxicity induced by SPM metabolites was greater in multidrug resistant cells (MDR) than in the corresponding wildtype ones (WT), due to an increased mitochondrial activity. It was observed that the combination of BSAO/SPM enzymatic system with docetaxel (DTX) had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent. The results suggest that DTX could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM. Preliminary studies also show that the cytotoxicity of BSAO/SPM was enhanced on human melanoma M14 cells by pre-treatment of tumour cells with the antimalarial 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 treatment than by BSAO/SPM alone and was higher in MDR cells than their wild-type counterparts. The release of lysosomal enzymes into the cytoplasm by CQ, as shown by confocal microscopy, is the major reason for its sensitizing effect. The findings suggest that the association DTX with BSAO/SPM, the lysosomotropic effects caused by CQ and mitochondrial alterations induced by SPM oxidation products, allow the design of a new therapeutic strategy based on the use of these combinations in human neoplasms, making this approach mainly attractive in treating MDR cancer patients.


Tracing the evolutionary origin of polyamine interconversion pathway by characterizing zebrafish spermidine/spermine N1-acetyltransferase 1 isoenzymes Yi-Chin Lien, Ting-Yu Ou, Po-Chih Kuo, Yu-Tzu Lin and Han-Jia Lin

S73 glucose uptake in the obese. Muscle biopsies showed increased postabsorptive inhibitory IRS-1 serine phosphorylation in T2D and lesser Hyper3 increases in T2D and obese, vs lean. Signaling via Akt and downstream of mTORC1 was also less than lean. Thus, in insulinresistant states hyperaminoacidemia: (1) can overcome resistance of protein anabolism and (2) does not exacerbate impaired glucose 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] Spermidine/spermine N1-acetyltransferase 1 (SSAT1) catalyzes the rate-determining step in polyamine interconversion pathway, which maintains the homeostasis of polyamines. In addition, mammalian SSAT1 is also involved in many physiological and pathological events, such as hypoxia, cell migration and carcinogenesis. By using cross-genomic bioinformatic analysis, we found that bony fish of vertebrates seems to be the origin of polyamine interconversion pathway. It might be helpful to further clarify the physiological functions of human SSAT1 by comparing it with the original SSAT1, such as zebrafish SSAT1. There are three potential SSAT1 isozymes, tentatively denoted as zSSAT1a (NM_001093748), zSSAT1b (NM_001030199) and zSSAT1c (NM_001002169) in the zebrafish genome. The RNA messengers of the three zSSAT1 isogenes were all detectable in the zebrafish tissue that suggests none of these isogenes are psudogenes. The recombinant enzymes of zSSAT1 isogenes were prepared to characterize their enzymatic kinetic properties. Moreover, the regulation and protein–protein interaction relationship of these isoenzymes were also studied, and such properties were compared with that of human SSAT1.

Proteomics Amino acids can overcome the insulin resistance of protein metabolism in humans Re´jeanne Gougeon, Maya Bassil, Ste´phanie Chevalier, Sergio Burgos, Jose´ A. Morais, and Errol B. Marliss McGill Nutrition and Food Science Centre, McGill University, Montreal, QC, Canada Amino acids (AA) stimulate protein synthesis both independently and interactively with insulin. Insulin resistance of protein accompanied 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 and isoaminoacidemia maintained by variable infusions of glucose and 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 steady-state, with serum insulin, glucose and AA at peak postprandial levels (‘‘Hyper3’’). Marked increases in anabolism, and attenuation of insulin-stimulated glucose uptake occurred in lean men. Next, severely insulin-resistant men with Type 2 diabetes (T2D) underwent Hyper3 clamps. Unexpectedly, their protein synthetic and net anabolic responses (/kg FFM) matched those of the lean subjects, despite lesser hyperinsulinemia. Furthermore, their resistance of glucose uptake was not worsened. We then tested less insulin-resistant obese men. Identical hyperinsulinemia in obese and lean required pancreatic clamps (octreotide, glucagon, and hGH). HyperAA again induced equal net protein anabolism in both groups, without worsening the attenuated

Analysis of amino acids in plant extracts using thin layer chromatography, amino acid analyzer and matrix free material enhanced laser desorption ionization mass spectrometry Muhammad Nasimullah Qureshi1,2, Guenther Stecher2,3, Gudrun Abel4, and Guenther K. Bonn2 1

Medicinal Botanic Centre, PCSIR Laboratories Complex Peshawar, Pakistan, 2 Institute of Analytical Chemistry and Radiochemistry, LeopoldFranzens-University Innsbruck, Innrain 52 a, 6020 Innsbruck, Austria, 3 Bionorica Research GmbH, Mitterweg 24, 6020 Innsbruck, Austria, 4 Bionorica AG, Kerschensteinerstr. 11-15, 92318 Neumarkt, Germany The standardization of natural product drugs needs reliable methods for the analysis of raw materials and final products. In this study, 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 chromatography (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-MELDIMS). Samples employed in the study were standards and water extracts from Althaea officinalis, Taraxacum officinale, and Matricaria chamomilla. TLC analysis not only proved the presence of different amino acids in the biological sample, but also hinted at the existence of other unknown compounds. The application of mf-MELDI-MS further confirmed the presence of different amino acids. The quantification of amino acids in the plant extracts was performed using an automatic amino acid analyzer. Evaluation of all three techniques employed clearly proved the adequate performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requests only a few minutes. Furthermore, amino acid analyzer is suitable for quantitative analysis.

Global analysis of synaptic protein complexes by interaction proteomics Ning Chen, August B. Smit, and Ka Wan Li Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands Glutamatergic synapses represent the primary fast excitatory connections that link principal neurons in all brain areas into circuits. Presynaptic terminals are responsible for converting electrical signals from axons into released chemicals packaged in synaptic vesicles (SV). The exocytosis and endocytosis of SV involve coordinated dynamic molecular events within networks of proteins in the pre-

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synaptic active zone. In the postsynaptic spine, AMPA-type glutamate receptors (GluA/AMPAR) function as primary carrier of synaptic current, and mediate the majority of excitatory synaptic transmission. Both synaptic transmission and plasticity are governed by changes in spatio-temporal patterns of AMPAR complexes via differential subunit expression and dynamic protein interactions. While a few selected synaptic proteins have been extensively examined, the global synaptic protein interactome and their dynamics that underlie synapse function and plasticity remain largely unknown. In this study we use a proteomics approach to characterize the synaptic protein interactome. We have characterized [50 synaptic protein complexes including those involved in (1) SV docking, priming, vesicle fusion, endocytosis and vesicle recycling, (2) AMPAR accessory proteins, (3) proteins involved in actin dynamic, and (4) synaptic cell adhesion molecules. In 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.

have implications on bryozoan anti-fouling methods. Despite many studies on metamorphosis of this species, little is known about the molecular mechanism of these processes. Here, we report a comparative study of swimming larvae and metamorphosing individuals at 4 h and 24 h post-attachment using label-free quantitative proteomics. We identified more than 1,100 proteins at each stage, 61 of which were differentially expressed. Specifically, proteins involved in energy metabolism and structural molecules were generally down-regulated, whereas proteins involved in transcription and translation, the extracellular matrix, and calcification were strongly up-regulated during metamorphosis. Many tightly regulated novel proteins were also identified. Subsequent analysis of the temporal and spatial expressions of some of the proteins, and assay of their functions indicated that they may have key roles in metamorphosis of B. neritina.

Proteomic analysis of the anticancer mechanism of resveratrol against neuroblastoma

Haruhiko Sakiyama, Noriko Fujiwara, Takahiro Noguchi, Hironobu Eguchi, Daisaku Yoshihara, and Keiichiro Suzuki

Xiaoqing Ren, Qingsong Wang, Xuyang Zhao, Mingrui An and Jianguo Ji*

Department of Biochemistry, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan

State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China

The carbohydrate response element-binding protein (ChREBP) functions as a transcription factor in mediating the glucose-activated gene expression of multiple liver enzymes, which are responsible for converting excess carbohydrate to storage fat. ChREBP is translocated into the nucleus in response to high glucose levels, and then up-regulates transcriptional activity. Although this glucose activation of ChREBP is generally observed only in liver cells, overexpression of wild type maxlike protein X (Mlx), but not an inactive mutant Mlx, resulted in the exhibition of the ChREBP functions also in a human kidney cell line. Because high glucose conditions induce the glycosylation of cellular proteins, the effect of O-linked GlcNAc modification on ChREBP functions was examined. Treatment with an O-GlcNAcase inhibitor (PUGNAc), which increases the O-linked GlcNAc modification of cellular proteins, caused an increase in the glucose response of ChREBP. In contrast, treatment with a glutamine fructose amidotransferase inhibitor (DON), which decreases O-GlcNAcylation by inhibiting the hexosamine biosynthetic pathway, completely blocked the glucose response of ChREBP. These results suggest that the O-linked glycosylation of ChREBP itself or other proteins that regulate ChREBP is essential for the production of functional ChREBP.

Resveratrol (Res), a dietary polyphenolic compound, has strong anticancer properties on various tumors. However, the mechanism of the chemopreventive effect of Res against neuroblastoma remains unclear. Treatment of neuroblastoma cell SH-SY5Y with Res resulted in a decrease in cell viability and an induction of apoptosis in a dosedependent manner. Comparative proteomic approaches were applied to explore the profile of protein expression changes. Applying two dimensional gel electrophoresis and MALDI TOF/TOF mass spectrometry techniques, we analyzed the proteome of 20 lM Res treated SH-SY5Y and untreated, 20 proteins were found significantly changed. Seven proteins were up-regulated and four down-regulated. These proteins are involved in oxidation reduction, microtubule polymerization, cell cycle, signal transduction, electron transport chain. Further experiments demonstrate that Res can suppress the proliferation of SH-SY5Y through reducing oxidation, inhibiting microtubule polymerization, participating protein signal transduction. The results suggest that Res has good potential as effective chemopreventive agent against neuroblastoma.

The role of O-linked GlcNAc modification on the glucose response of ChREBP

Redox Quantitative proteomics identify molecular targets that are crucial in metamorphosis of the marine bryozoan Bugula neritina Tim Yue Him Wong The Hong Kong University of Science and Technology, Division of Life Science Metamorphosis of the marine bryozoan Bugula neritina transforms a ball-shaped swimming larva into a tubular sessile juvenile within 48 h. The metamorphosis consists of various complex processes such as the morphogenetic rearrangement of larval tissues and the development of juvenile tissues from primordial cells. Understanding metamorphosis of B. neritina can provide insights into their colonization, as well as

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Characterization of antioxidative peptide in free radical system: electronic properties in key positions C4, C1, N1 for ORAC and superoxide databases; C2 for TEAC database Yao-Wang Li, and Bo Li* College of Food Science and Nutritional Engineering, China Agricultural University, Qinghua East Road 17, Haidian District, Beijing 100083, People’s Republic of China Antioxidative peptide attracts more and more attention of researchers since it possesses special functions. However, the relationship

12th International Congress on Amino Acids, Peptides and Proteins between the structure and activity is not clear, especially the antioxidative peptide in the free radical system. Therefore, many antioxidative peptide which come from different sources measured with different measuring methods are formed three databases with our effort: TEAC, ORAC and superoxide databases respectively. Before these three databases are dealed with PLSR method with amino acid descriptors to build QSAR models, they have been processed by TTPN methods since the length of peptide are different. Then each of databases have found suitable descriptors for describing their structures (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 some significant positions in the sequence for antioxidant activity, they are C4, C2, C1 and N1. Additionally, low electronic properties which is the most important properties for activity and high hydrophobic properties for these position are suitable, especially position C4, N2 need acidic or basic amino acid and position C1 welcome amino acid which possess electronic, hydrophobic properties and steric or bulky property for obtaining high activity. This research shows the features and key positions of antioxidative peptide in the free radical system which will benefit the further research and the mechanism.

Oxidation of Cys111 residue in loop VI of human copper/zinc superoxide dismutase Noriko Fujiwara1, Kentaro Ihara2, Shinsuke Kato3, Daisaku Yoshihara1, Hironobu Eguchi1, Haruhiko Sakiyama1, Soichi Wakatsuki2, Naoyuki Taniguchi4 and Keiichiro Suzuki1 1

Department of Biochemistry, Hyogo College of Medicine, Japan, Structural Biology Research Center, Institute of Material Structure Science, High Energy Accelerator Research Organization, Japan, 3 Department of Neuropathology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Japan, 4 Systems Glycobiology Group, Disease Glycomics Team, Advanced Science Institute, RIKEN, Japan 2

Modifications of cysteine residues in proteins involve two types of oxidation. One is reversible oxidation such as disulphide bonding, sulfenation (Cys-SO) and S-nitrosation (Cys-SNO), and the other is irreversible oxidation such as sulfination (Cys-SO2H) and sulfonation (Cys-SO3H). Copper/zinc superoxide dismutase (SOD1) catalyzes the conversion of toxic superoxide anion radical into molecular oxygen and hydrogen peroxide, thereby protecting cells against oxidative stress. In contrast, mutations in SOD1 have been found from patients of familial amyotrophic lateral sclerosis (FALS). Only human and great ape SOD1 s among mammals have two free cysteine residues, Cys6 and Cys111. We have shown that Cys111 located in the loop VI at the surface of the SOD1 molecule is selectively sulfonated even by air oxidation. The polyclonal antibody raised against a synthesized peptide containing Cys111-SO3H reacted with oxidized SOD1 but not reduced-form SOD1 by Western blot analysis and immunostained inclusions in the spinal cord of FALS patients and ALS model mice. On the other hand, the recombinant human SOD1 oxidized with 2-mercaptoethanol (2-ME) only at Cys111 by disulphide bonding (2ME-SOD1, developed by Ube Industries Ltd.) was resistant to oxidation. We will present a crystal structure of the dimer of 2-MESOD1 showing some interactions between 2-ME molecules in the both subunits. These results indicate that Cys111 is a primary target for oxidative modification with oxygen or other thiols and plays an important role in oxidative damage to human SOD1 including FALS mutants.

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Redox reactions of hydroperoxides formed on amino acids and proteins: key intermediates in oxidative damage to proteins Michael J. Davies The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW 2042, Australia, e-mail: [email protected] Proteins are major targets for oxidative damage due to their high abundance and rapid rates of reaction with many radicals and excited states (e.g. 1O2). Exposure of proteins to radicals generated by radiation, metal ion/hydroperoxide systems, peroxyl radicals, peroxynitrite, and activated white cells, results in the formation of protein-derived radicals. Subsequent reaction with O2 gives rise to new reactive groups including hydroperoxides and 3,4-dihydroxyphenylalanine. These species are long-lived, can diffuse from their site of generation due to poor removal by enzymes, and can be detected in intact cells. Reaction of these hydroperoxides with thiol (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, glutathione reductase, caspases, cathepsins, Ca2+-ATPases, and protein 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 concomitant hydroperoxide consumption and thiol oxidation; in some cases sulfenic acid intermediates are detected. Some protein hydroperoxides are more effective than H2O2, probably as a result of the longer lifetime of protein hydroperoxides in cells. Overall, these data support the hypothesis that hydroperoxides formed on oxidized proteins may contribute to cellular dysfunction and altered redox signalling in systems subject to oxidative stress by inducing strand breaks and mutagenic lesions in DNA, inhibiting key cellular enzymes, altering cellular redox status and signalling, and depleting antioxidants.

Study on P16 damage induced by peroxynitrite Yunjing Luo*, Jingjing Li, Yang Ding, and Rugang Zhong College of Life Science and Bioengineering, Beijing University of Technology, 100124 People’s Republic of China, *Corresponding Author: e-mail: [email protected] P16, as a CDK4 inhibitor, is redox regulated in the course of cell cycle control. Over-expression of CDK4 induced by the deactivation and abnormality of p16 can cause uncontrollable cell proliferation and division, increasing the possibility of cell carcinogenesis. Oxidation injury of p16 has been involved in the pathogenesis of cancer. In this paper, with the aim to determine nitration sites, we used liquid chromatography-mass spectrometry technology. Tyr129 and Tyr44, which is more vulnerable to ONOO-, were detected to be two nitration sites of p16. Since they are both essential for CDK4 binding, we focus on the need for the nitration damage of ONOO- on the combination of p16 and CDK4 by SDS-PAGE of the reaction compound. The result revealed that under the conditions of low accumulated doses of peroxynitrite, the gradual decrease of P16 compound is proportional to the increase of monomers over time, along with small molecular weight polymers over time, suggesting the potent nitration modification by ONOO-on P16 has played a critical role in activity of CDK4, even implicating the cell progression.

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S76 Acknowledgements: This work was supported by National Natural Science Foundation of China (No. 20875006) and Beijing Natural Science Foundation (No. 2102005).

Sports & Exercise Chronic effects of leucine supplementation of whey protein on mTOR activation in the skeletal muscle of sedentary and exercised Wistar rats P. C. B. Lollo1; T. M. Batista2; E. M. Carneiro2; and J. Amaya-Farfan.1* 1

School of Food Engineering and Institute of Biology, University of Campinas, SP, Brazil. *[email protected]

2

The milk-whey proteins are rich sources of L-leucine and L-leucine can promote animal growth by activating m-TOR-mediated skeletal 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 would reduce the hyperinsulinemic effect of L-leucine, as ascertained by m-TOR activation and the determination of common blood biomarkers. Twenty-four sedentary weanling male Wistar rats were divided into four diet groups for 30 days as follows: (a) Control (AIN 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 groups were submitted to physical exercise. Serum insulin, uric acid, glucose, AST, ALT, CK, LD and gastrocnemius mass, total and body mass-adjusted protein were determined by standard methods, and mTOR by Westernblot analysis. The data were analyzed by ANOVA and pos-hoc Duncan. mTOR concentration increased 3.91-fold at 6% L-leucine in the sedentary, against 6.34-fold in the exercised animals. Exercise damped the hyperinsulinemic effect of higher serum L-leucine. Other blood parameters and muscle mass/protein and markers of muscle damage did not change with supplementation. mTOR expression in the gastrocnemius of Wistar rats was increased by supplementing MWP with L-leucine, up to the 6% level, with a reduction of hyperinsulinemia and no other apparent detrimental effects. Keywords: L-Leucine, Milk whey proteins, Dose–response; mTOR, Skeletal muscle tissue

Effect of exercise and protein feeding on the interstitial amino acid profile of human skeletal muscle sampled by microdialysis William McCormack Faculty of Education and Health Sciences, University of Limerick, Limerick, Ireland In vivo microdialysis is a minimally invasive, membrane based sampling technique, capable of determining the solute concentration

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12th International Congress on Amino Acids, Peptides and Proteins in the interstitial space. In a contralateral limb design (Rest vs. Exercise), this study used in vivo microdialysis to study the change in 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 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) solution or placebo (flavoured water). Amino acid concentration was determined using reverse phase HPLC. 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 (192 vs. -17 lM). Analysed over the same time point analysis of the amino acids recovered in the lD revealed a 30% greater increase in the interstitial amino acid concentration in the exercise compared to the resting muscle; BCAA (912 vs. 545 lM), Leu (362 vs. 226 lM) and GLU (176 vs. -154 lM). Nutrient uptake into tissues from the circulation is influenced both by concentration and flow. The observed difference in the interstitial concentration of amino acids in the exercised muscle is probably related to an exercise-induced increase in nutritive blood flow.

Human skeletal muscle interstitial cytokine response to protein feeding measured by in vivo microdialysis Philip M Jakeman Faculty of Education and Health Sciences, University of Limerick, Limerick, Ireland In vivo microdialysis is a minimally invasive, membrane based sampling technique, capable of determining the solute concentration in the interstitial space of a target tissue. Insertion of a microdialysis probe leads to a sterile inflammatory response characterised by an innate immune reaction and cytokine signalling. Whey proteins are promoted as anti-inflammatory. This study investigated the effect of whey protein feeding upon the cytokine response to in vivo microdialysis. Microdialysis catheters (CMA 70) with a 100 kDa molecular mass cut-off were inserted into the vastus lateralis of subjects and perfused at a flow rate of 1.0 ll min-1 and serial sampled for 7 h. Following a 2 h equilibration period subjects consumed either a 9% w:v whey 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 (SectorÒ, Meso Scale Discovery). Following the 2 h equilibration the mean concentration of all cytokines increased in both the PLA and WPI to a peak 6 h after 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 (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 p = 0.15]. TNFa was below the LLOD in the majority of lD samples and was therefore omitted from the analysis. Though the sample size is small, the reduction in the recovery of the pro-inflammatory cytokine IL-8 is indicative of a potential immunomodulatory function of whey protein isolates.


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Influence of increased blood ammonia by long duration running on the performance of calculation task

Protein S-guanylation and its unique regulation mechanisms involving cysteine metabolism

Hajime Ohmori1, Daisuke Aoki2, Keisuke Ishikura3, Song-Gyu Ra1, Takafumi Suzuki1, Shoichi Komine1, Michio Okido1, and Teruo Miyazaki4

Takaaki Akaike Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan

1

Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan 2 Master’s Program in Health and Physical Education, University of Tsukuba, Tsukuba, Japan 3 Sports Research and Development Core, University of Tsukuba, Tsukuba, Japan 4 Department of Development for Community Medicine, Tokyo Medical University, Ami, Japan

The purpose of this study was (1) to investigate the influence of the interval exercise simulated for soccer game (INT) and the steady rate exercise with same average running speed as the interval exercise (SR) in calculation task performance, and (2) to investigate the relationship between blood ammonia level and calculation task performance. Subjects were 5 soccer players (age 21.5 ± 1.6 years, VO2max 56.7 ± 4.3 ml/kg/min).They were required to perform the three conditions (INT, SR and REST). Three times of calculation tasks were required in each condition, and they were evaluated in two different ways, the work speed expressed as work rate, and the work accuracy expressed as error rate. For the error rate, there was significant difference between INT and REST in 3rd calculation task. Significant correlation between error rate and blood ammonia level (r = 0.65). This study suggests that the rise in blood ammonia induced by long duration running would influence the decrease of work accuracy.

Sulfur containing amino acids

We recently clarified the physiological formation of 8-nitroguanosine 30 ,50 -cyclic monophosphate (8-nitro-cGMP) in cells in culture, which is the first demonstration of a new second messenger derived from cGMP in mammals since the discovery of cGMP more than 40 years ago. 8-Nitro-cGMP is formed via nitric oxide (NO) and has a unique electrophilic property. It can thus react with nucleophilic sulfhydryls of cysteine and protein, causing protein S-guanylation. However, regulation mechanisms of formation of 8-nitro-cGMP and S-guanylation are still unclear. Here, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. More than micromolar concentration of 8-nitro-cGMP was evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. 8-Nitro-cGMP caused S-guanylation of Keap1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes including heme oxygenase-1, and thus 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. We revealed that 8-nitro-cGMP S-guanylated the Cys434 of Keap1 in cells. Protein S-guanylation induced by 8-nitrocGMP may thus have important implications in NO-related physiology 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 critically involved not only in the 8-nitro-cGMP signaling but also in other electrophilic cellular signaling in general.

Mercaptopyruvate sulfurtransferase knockout mouse production: what is the physiological role of the enzyme?

Structure and function of selenocysteine lyase and cysteine desulfurase

Noriyuki Nagahara1, and Takaaki Ito2

Hisaaki Mihara

1

Dept. of Environ. Med., Nippon Med. Sch., Dept. Pathol. Exp. Med., Grad. Sch. Med., Kumamoto Univ

2

In cysteine catabolism, mercaptopyruvate sulfurtransferase (MST: EC 2.8.1.2) catalyzes the reaction from mercaptopyruvate to pyruvate. MST is localized in the cytoplasm and mitochondria in animal cells. We found higher MST expression during mouse embryonic development than in the postnatal period although the MST gene is a housekeeping gene. Our studies of the structure–function relationship of MST revealed that (1) a disulfide bond between the dimeric MST and a catalytic site cysteine contributes to redox-dependent regulation of MST activity; (2) the disulfide bond serves as a redox-sensing molecular switch, and (3) a catalytic site cysteine is oxidized to form low redox potential sulfenate, which is reduced not by glutathione but by thioredoxin. These findings suggest that MST contributes to maintain cellular redox homeostasis. To confirm the physiological function and tissue specificity of a gene repression of MST, we generated MST knockout mice (C57BL/6) by gene targeting in embryonic stem cells using the Cre/loxP system. Functional expression of a Cre/LoxP sitespecific recombination system is applied in generating tissue-specific knockout mice. A comprehensive analysis of pathologic and morphologic changes caused by MST deficiency is in progress.

Department of Biotechnology, Institute of Science and Engineering, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan Selenocysteine lyase (SCL) is a pyridoxal 50 -phosphate (PLP)dependent enzyme that specifically acts on L-selenocysteine to yield L-alanine and selenium. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. We have revealed that the catalytic reaction of SCL proceeds via the formation of an enzyme-bound selenopersulfide intermediate on Cys375. Cys375 on the flexible loop directed L-selenocysteine, but not Lcysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. This provides the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system. L-Cysteine is the sulfur source for the biosynthesis of a variety of cofactors such as thiamin, iron-sulfur clusters, molybdopterin (MPT), and tRNA thionucleosides. Cysteine desulfurase, a PLP-containing homodimer, decomposes L-cysteine to L-alanine and sulfane sulfur via the formation of an enzyme-bound persulfide intermediate. The

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S78 persulfide sulfur is subsequently incorporated into the biosynthetic pathways of several sulfur-containing biofactors, which provides an elegant mechanism for making sulfur atoms available without releasing them in solution. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of MPT synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. We revealed that IscS, among three cysteine desulfurases present in E. coli, is the primary physiological sulfur-donating enzyme in MPT biosynthesis.

Sulfur metabolism in the cysteine dioxygenase knockout mouse: impairment in taurine synthesis and increased formation of acid labile sulfur

12th International Congress on Amino Acids, Peptides and Proteins most MCR chemistry performed with isocyanides relates to the classical reactions of Passerini and Ugi. Passerini reactions involve an oxo component, an isocyanide and a carboxylic acid. The success of multicomponent condensations in organic synthesis during the last few years has increased the interest for doing novel reactions or modifying the old ones. Such modifications include the use of polyfunctional building blocks, the employment of non-classical starting units, or new solvents according to green chemistry. 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 component in the Passerini reaction in 1-butyl-3-methylimidazolinium bromide ([bmim]Br) as ionic liquid (IL) at room temperature.

Stipanuk, Martha H, Krista Fieselmann, Lawrence L. Hirschberger, Ueki, Iori, Jimmy Lam, Rachel Peters, Heather B. Roman, and Alessandro Valli

A solvent-free synthesis of highly functionalized benzothiazolediamides (mimic natural peptide) via Ugi four component reaction

Division of Nutritional Sciences, Cornell University, Ithaca, NY, 14853, USA

Fatemeh Sheikholeslami Farahani*1, and Ashraf S. Shahvelayati2

Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. Cysteine dioxygenase oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate + sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO+/- mice) were crossed to generate CDO-/-, CDO+/-, and CDO+/+ mice. CDO-/- mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO-/- mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO-/- mice than in CDO+/- or CDO+/+ mice and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. H2S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H2S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H2S levels and facilitate the use of H2S as a signaling molecule. Acknowledgments: Supported by grant DK056649 from the National Institute of Diabetes and Digestive and Kidney Diseases.

1 Department of Chemistry, Firoozkooh Branch, Islamic Azad University, Firoozkooh, Iran, 2 Department of Chemistry, Islamic Azad University Shahr-e Rey Branch, Tehran, Iran

Multicomponent reactions (MCRs) are one-pot processes that combine three or more substrates simultaneously. Such processes are of great interest in diversity-oriented synthesis, especially to generate compound libraries for screening purposes. The Ugi four-component reaction (Ugi 4CR) is one of the milestones in this field and great efforts have been devoted to the exploration of the potential of this transformation. A primary amine, a carbonyl compound, a carboxylic acid, and an isocyanide react together to give a-amidoamides in this remarkable reaction. In recent years several modifications of the classical Ugi 4CR have been described; these include variations of one of the components or the introduction of a linkage between two of them. In particular, the groups of Zhu and Do¨mling have contributed significantly to the advancement of this transformations. As part of our continuing interest in multicomponent reactions, herein we describe an efficient synthesis of unsaturated thioamidodipeptides (pseudopeptides) 4 from 4-benzothiazol-2-ylamino-4oxo-2-butenoic acid 1, prepared from 2-aminobenzothiazol and furan2,5-dione, as a new acid component in the Ugi reaction under solventfree condition.

Synthesis Comparative syntheses of peptide thioesters derived from mouse and human prion proteins A one-pot synthesis of functionalized a-acyloxythioamides from N-protected a-amino acids as an acid component in the Passerini reaction in an ionic liquid

Jaroslav Sˇebestı´k, Zbigniew Zawada, Martin Sˇafarˇ´ık, and Jan Hlavaćˇek Institute of Organic Chemistry and Biochemistry, AS CR, 16610 Prague, Czech Republic

Ashraf S. Shahvelayati*1, Issa Yavari2, and Maryam Ghazvini 1

Department Chemistry, Islamic Azad University Shahr-e Rey Branch, Tehran, Iran, 2 Department Chemistry, Islamic Azad University Science and Research Branch, Ponak, Tehran, Iran Multicomponent reactions (MCRs) are generally defined as reactions where more than two starting materials react to form a product. Today

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Prions are suspected as causative agents of several neuropathogenic diseases. However, they mode of action is still not clear. Combination of chemical and recombinant synthesis can provide suitable probes for determination of prion role in pathogenesis of related diseases. Peptide thioesters are key building blocks for chemical syntheses of proteins by native chemical ligation. Prions are sources of difficult sequences for synthesis by Fmoc/tBu approach. Therefore, a scan of the prion domain 93–231 was carried out in order to discover which

12th International Congress on Amino Acids, Peptides and Proteins thioesters are easily available as suitable building blocks for total chemical synthesis of prion protein based probes. First, the synthesis on chlorotritylchloride resin was employed and after deprotection of small quantity the segments were purified and characterized. If the difficulties were observed during synthesis, the segment was re-synthesized with either special dipeptides or by splitting to 2 or 3 smaller segments. When the sequences were synthesized correctly without main impurities, the protected segments were coupled with EtSH using DIC/DMAP activation. If the technique did not provide a suitable peptide thioester, the protected segment was coupled using PyBOP/p-Ac-NH–Ph-SH. In some special cases, the other techniques of thioester formation were carried out. Peptide thioesters from C-domain of prion protein were synthesized and characterized. Acknowledgments: This work was supported by grant of Czech Science Foundation (GA CR) No. 203/07/1517 and Research Project Z40550506.

EAAC1-mediated neuronal glutathione synthesis

S79 non-neuronal cells suggesting that they could be implicated in carcinogenesis. mGlu receptors are G-protein-coupled receptors and eight subtypes (mGluR 1–8) have been identified and classified into three groups (I–III) based upon sequence homology, transduction mechanism and pharmacological profile. Because of their modulating properties, mGlu receptors are recognized as promising therapeutic targets and many ligands (agonists and antagonists) have been prepared to better understand the pharmacology of mGlu receptors in order to selectively activate the different groups and subtypes of receptors. An a-amino acid moiety can be found in all mGlu receptors competitive ligands and most of the side chains hold an acidic function. Examination of the glutamate binding site in the mGlu receptors and pharmacological data of some ligands shows that sterically constrained 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 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 Glutathione (GSH) is a tripeptide comprised of glutamate, cysteine and glycine. Cysteine is the rate-limiting substrate for GSH synthesis in neurons. Most neuronal cysteine uptake is mediated by a sodium dependent excitatory amino acid transporter, known as excitatory amino acid carrier 1 (EAAC1). EAAC1-null mice have reduced neuronal GSH levels and showed increased susceptibility to oxidant injury. GSH depletion in the brain has been considered to be an early event in some neurodegenerative diseases. Endogenous mechanisms to increase the neuronal GSH level in the brain might be a potential strategy to protect against neurodegeneration. Our ongoing studies focus on the mechanisms to regulate neuronal GSH synthesis via EAAC1.

New developments in the synthesis of acetylenic analogues of glutamate P. Meffre*1,2, Z. Benfodda1,2, D. Benime´lis1,2, V. Rolland1 and F. C. Acher3

Novel efficient and stereoselective synthesis of the bamino-a-hydroxy acid units in bestatin and amastatin Youngran Seo, Hyeonjeong Kim, Young Gyu Kim* School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-744, Republic of Korea Development of efficient synthetic methods for b-amino-a-hydroxy acids has been of interest because the b-amino-a-hydroxy acid units are frequently found in many biologically active natural products such as bestatin and amastatin as well as paclitaxel. Bestatin is an immune response modifier and a dipeptide containing (2S,3R)-3-amino-2hydroxy-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 activities. Both efficient and stereoselective construction of the same 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 commercially available a -amino acids, phenylalanine and leucine.

1

Institut des Biomolećules Max Mousseron-UMR 5247 -CNRSUniversite´s Montpellier 1 et 2, Place E. Bataillon, 34095 Montpellier Cedex 5, France, 2 UNIVERSITE DE NIMES, Laboratoire de ChimieBioOrganiqueLCBO, Site des Carmes, Place Gabriel Pe´ri, 30000 Nıˆmes, 3 Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601, CNRS Universite´ Paris Descartes, 45 rue des saints Pe`res, 75006 Paris, France Glutamate ((S)-Glu) is the major excitatory amino acid in the central nervous system. It acts by stimulating ionotropic and metabotropic glutamate receptors (iGluR and mGluR, respectively). Glutamate has been shown to be involved not only in many neuropathologies such as anxiety, pain, ischemia, Parkinson’s disease, epilepsy and schizophrenia. More recently, mGlu receptors have also been detected in

Novel synthetic methodologies to prepare unnatural acids, aminoacids and peptidomimetics Mauro F. A. Adamo We have developed an original synthon, 3-methyl-4-nitro-5-styrylisoxazole 1 (Scheme 1), 1 and novel organocatalyses (Schemes 1, 2). Compounds 1 are stable crystalline compounds which could be generated in multigram scale from isoxazole 2 and aldehyde 3. Compounds 1 contain two electrophilic centres: (1) soft E1 which reacted selectively with stabilised nucleophiles; (2) hard E2 which reacted selectively with hard nucleophiles such as –OH. The addition of –OH to E2 is the bases of a synthetically useful reaction which transformed the 4-nitroisoxazol-5-yl core into a carboxylate,

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demonstrating compounds 1 as synthetic equivalent to cinnamates 4. The 4-nitroisoxazole-5-yl core also activated the alkene as the resulting anion, delocalised across a large number of conjugated atoms, was more stable: compared to cinnamic esters and aldehydes, isoxazoles 1 reacted faster and more efficiently. Compounds 1 were intermediate in multi component processes (over 20 peer-reviewed papers published 2005–2010) and more recently substrates for enantioselective phase transfer catalyses (Scheme 1). c-Aminoacids 8,2 derived from 5, a-aminoacids 6 and their derivatives 9, heavily substituted cyclopropanes 10, and chiral isonitriles 11 were obtained from 1 in high yields and ees. My group has also developed a unique sulfonylation process which allowed obtaining multigram amounts of enantiopure sulfonic acids 13 (Scheme 2). 3 Sulfonic acids are extensively employed as resolving agent and possess several interesting biological properties, exerting key metabolic roles in the central nervous system (CNS). The sulfonic acid functionality is also present in natural products, for example in 6-gingesulfonic acid, extracted from ginger (Zingiberis Rhizoma) and used as an anti-ulcer drug in Chinese and Japanese medicine. My independent work resulted in 40 peer reviewed publications, five patents and three application pending. The paper describing the synthesis of five was selected as Hot paper in Angewandte Chemie Int. Ed., and was highlighted twice in Synfacts for the novelty of one being synthetic equivalent to cinnamates and for the use of five in the preparation of Baclophen. The synthesis of compounds 6–7 is still unpublished. The patent describing the sulfonylation of alkenes (Scheme 2) won the IDEA price 2009 hosted by Enterprise-Ireland.

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One-pot efficient synthesis of a-aminophosphonates derivatives via multicomponent reactions of triphenyl phosphite, aldehydes and amines in ionic liquid Rahimeh Hajinasiri* Chemistry Department, Qaemshahr Branch, Islamic Azad University, P. O. Box 163, Qaemshahr, Iran Ionic liquids (ILs) have gained great attention in recent years. Ionic liquids have a high polarity and low vapor pressure. These characteristics combined with immiscibility with most less polar organic solvents led to their use as solvent or co-solvent in catalysis. a-aminophosphonic acids are probably the most important substitutes for the corresponding amino acids in biological systems. A number of potent antibiotics, enzyme inhibitors, and pharmacological agents are a-aminophosphonic acids as well as their derivatives, particularly peptides. In this study, we describe an efficient synthesis of a-aminophosphonates through the reaction of aldehydes 1, amines 2 and phosphites 3 in ethyl methyl imidazolium bromide. For optimizing the reaction conditions, a sample reaction between benzaldehyde, benzyl amine and triphenylphosphite was carried out in different ILs as the solvent. The results indicated that ionic liquid ethyl methyl imidazolium bromide is excellent solvent for these reactions.


Selective synthesis of fluorinated cyclic b-amino acids Lorańd Kiss1, Enik}o Forro´1, Santos Fustero2, and Ferenc Fu¨lo¨p1 1 Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Eo¨tvo¨s u. 6, Hungary, 2 Departamento de Quı´mica Orgańica, Facultad de Farmacia, Universidad de Valencia, Valencia, Spain, E-mail: [email protected]

b-Amino acids are key components of natural products and precursors of bioactive b-lactams. A number of cyclic b-amino acids possess antifungal or antibacterial activities. The alicyclic and O- or N-containing 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, fluorinated amino acids and peptides present a high importance in 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 spite of the great potential of cyclic amino acids, only few cyclic fluorinated derivatives have been reported so far, among either aamino acids or c-amino acids. Furthermore, although cyclic b-amino acids have recently generated increasing interest, an extremely few fluorinated cyclic analogues have been synthetized so far. The regio- and stereoselective approach to fluorinated b-aminocyclohexane or cyclohexane esters has been developed, from a bicyclic b-lactam. The procedure involves six or seven steps, and consists in regio- and stereoselective iodolactonization, lactone opening and hydroxy-fluorine exchange. The method has been extended to the synthesis of fluorinated amino ester enantiomers.

One-pot synthesis of functionalized 2,3-dihydrothiazoles Maryam Ghazvini, Issa Yavari, and Nasir Iravani Payam noor university, Iran Thiazoles and their derivatives exhibit various biological activities such as antimicrobial, anti-inflammatory, antiviral, antituberculosis and cytotoxic activities. For example, the thiazolium ring present in vitamin B1 serves as an electron sink, and its coenzyme form is 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 functionalized dihydrothiazole derivatives in good yields. A simple one-pot synthesis of ethyl 3-(alkyl)-4-methyl-2-(phenylimino)-2,3-dihydrothiazole-5-carboxylate or 1-(3-cycloexyl-4methyl-2-(phenylimino)-2,3-dihydrothiazole-5-yl)ethanone from the reaction of phenylisothiocyanate, primary amines and ethyl 2-chloroacetoacetate or 3-chloroacetylaceton is described. Keywords: 2,3-Dihydro-chloroacetylaceton drothiazole; Phenylisothiocyanate; Primary alkylamines, Ethyl 2-chloroacetoacetate

Parallel synthesis of kahalalide A analogues through on-resin cyclization Tingting Wei, Jin Ren, and Chuanguang Qin* Faculty of Life Sciences, Northernwestern Polytechnical University, Xi’an 710072, China

S81 Kahalalide A is one of a family of peptide natural products isolated from the marine mollusk Elysia rufescens and its algal diet Bryopsis sp. Among these, the structurally simpler kahalalide A is one of the few marine-derived cyclic peptides with antimycobacterial activity, and it is not cytotoxic to various tumor cell lines. Recently, Line Bourel et al. has reported a chemical route to generate kahalalide A and its analogues. Besides the motivation of discovering biologically active compounds, there was a purely chemical impetus for the total synthesis. Based on our previous enormous work on synthesis of cyclic peptides, a parallel synthesis of kahalalide A derivatives were designed and carried out in our laboratory by a total solid-phase route with Fmoc chemistry. Later, we will investigate their biological activities in detail.

Syntheses and selective functionalisations of carbocyclic b-amino acids Ferenc Fu¨lo¨p Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Eo¨tvo¨s utca 6, Hungary, [email protected] The alicyclic b-amino acids have acquired great interest in recent years in view of their pharmacological potential. Cispentacin, an antifungal antibiotic with a cyclopentane skeleton, is one of the most important derivatives. (1R,2S)-2-amino-4-methylenecyclo-pentanecarboxylic acid (Icofungipen) is a known antifungal agent. Cyclic, conformationally rigid b-amino acids such as cis and trans-2-aminocyclopentanecarboxylic 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 acids. The Scheme illustrates one example where conformationally restricted, orthogonally protected 2,4-diaminocarboxylates with a cyclopentane skeleton were efficiently synthetized. The syntheses involve strategies of diastereoselective epoxidation of the b-lactam and the corresponding protected amino esters with opposite selectivities, followed by regioselective opening of the oxirane ring with sodium azide. This new class of compounds can be regarded not only as conformationally constrained b,c-diamino acid derivatives, but also as potential functionalized carbocyclic nucleoside precursors.

Synthesis of constrained carbocyclic and heterocyclic b-amino acid derivatives Sven Mangelinckx1, Karen Mollet1, Tamara Meiresonne1, Lorand Kiss2, Matthias D’hooghe1, Ferenc Fu¨lo¨p2, and Norbert De Kimpe1 1

Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium, 2 Institute of Pharmaceutical Chemistry, University of Szeged, H-6701 Szeged,PO Box 427, Hungary The synthesis of conformationally constrained carbocyclic and heterocyclic b-amino acids is becoming increasingly important in synthetic, agricultural and pharmaceutical chemistry. This attention results from the specific properties of these non-proteinogenic constrained amino acids with respect to biological activity (antifungal,

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S82 gametocidal, peptidase-inhibition) and structural features (building blocks for structurally and functionally unique b-peptides). In this presentation, the synthesis of new conformationally constrained b-amino acid derivatives via elaboration of different strategies starting from electrophilic allylic halides and halogenated imines is disclosed. Diverse targeted carbocycles and heterocycles, such as cyclopropanes, cyclobutanes, aziridines, azetidines and tetrahydrofurans, all with the b-amino acid (or ester) motif have been successfully prepared. The presence of a constrained ring and/or functional groups in the synthesized b-amino acid derivatives allowed further study of ring opening reactions, ring transformations and functional group transformations.

Taurine

12th International Congress on Amino Acids, Peptides and Proteins is a very abundant amino acid, whereas its concentration is significantly lower in the adult brain. It also was the most prominent amino acid released in 7-day-old mice in ischemia from all brain regions studied, cerebral cortex, striatum, cerebellum, hippocampus and brain stem. In 3-month-old mice glutamate, aspartate and GABA were released at relatively high amounts. Significantly less taurine was released in adult than in developing mice. However, ischemia caused a markedly larger increase in taurine release than in the release of other neuroactive amino acids even in the adult brain. In all brain regions taurine was the amino acid whose release was increased most 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 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 Hideo Satsu, Yusuke Gondo, and Makoto Shimizu Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan Taurine, present in high concentration in several mammalian tissues, plays an important role in essential biological processes such as antioxidation, anti-inflammation, and osmoregulation. However, the mechanisms involved in these physiological roles at the genetic level remain to be elucidated. In the present study, we investigated the effect of taurine on gene expression profile in human intestinal Caco-2 cells to analyze which genes or signaling pathways are involved in the function of taurine. Caco-2 cells were treated without or with 50 mM taurine for 24 h. Then global analysis of gene expression change was performed by DNA microarray. The mRNA and protein expression level of selected genes was measured by real-time PCR and by western blotting, respectively. As for the transcriptional activity, the reporter vector including its promoter region was constructed and the promoter activity was measured by luciferase assay. The result of DNA microarray and real-time PCR analysis showed that taurine increased the expression level of thioredoxin interacting protein (TXNIP) mRNA in a time- and dose-dependent manner. On the other hand, taurine decreased glucose transporter 1 (GLUT1) and GLUT3 mRNA levels. The increase in TXNIP mRNA was not observed by b-alanine or GABA treatment, suggesting that this induction was specific for taurine. We further revealed that taurine increased the protein level and also the promoter activity of TXNIP. These findings demonstrated the novel insight of taurine at the molecular level.

Effect of ischemia on the release of taurine and other neuroactive amino acids from the brain Simo S. Oja1 and Pirjo Saransaari Department of Paediatrics, Tampere University Hospital, Tampere, Finland Ischemia is a condition which dramatically hampers the functions of brain cells which derived most of their energy from oxidative metabolism. We studied under ischemic conditions the release of endogenous taurine and other neuroactive amino acids from slices prepared from different brain regions from developing 7-day-old and young adult 3-month-old mice. In the developing mouse brain taurine

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Effects of lotus and taurine supplementation in diet-induced obesity rat model Kyung Ja Chang, Jeong Soon You, and Zhao Xu Department of Food and Nutrition, College of Human Ecology, Inha University, Incheon 402-751, Korea Taurine is the most abundant free amino acid in the body and may serve as an anti-obesity agent. Lotus (Nelumbo nucifera) has been used to treat obesity. The adipokines secreted by adipose tissue including leptin and adiponectin are known to play an important role in the pathogenesis of obesity. In order to investigate the effect of lotus and taurine on obesity, 5-week-old male Sprague-Dawley rats were randomly divided into five groups for a period of 8 weeks (normal diet, N group; high fat diet, HF group; high fat diet + taurine, HFT group; high fat diet + lotus leaf, HFL group; high fat diet + lotus root, HFR group). Taurine was supplemented by dissolving in feed water (3% w/v). Lotus leaf and root hot water extract was orally administrated (400 mg/kg/day) to HFL and HFR groups and the same amount of distilled water was orally administrated to N and HF groups. Final body weight and relative weights of retroperitoneal adipose tissue were significantly lower in HFR groups compared to HF group. In serum, total cholesterol concentration was lower in HFT, HFL and HFR groups compared to HF group. Serum triglyceride concentration was lower in HFL and HFR groups compared to HF group. Serum adiponectin concentration was higher in HFT and HFR groups compared to HF group. These results suggest that lotus root hot water extract has the effect of reducing the body weight and adipose tissue weight, and taurine and lotus root have preferable effects in adiponectin levels.

Effects of taurine and complex magnesium taurate on calcium oxalate crystallization and antioxidant activities in vitro Zhang Chaoyan1,2, Wu Wenhui

1,2

, Shu Yunying1, and Wang Jue1

1

College of Food Science and Technology, Shanghai Ocean University, Shanghai, People’s Republic of China, 2 Institutes of Marine Sciences, Shanghai Ocean University, Shanghai, People’s Republic of China The purpose of this study was to evaluate the inhibitory effect of taurine and complex magnesium taurate on the crystallization of

12th International Congress on Amino Acids, Peptides and Proteins calcium oxalate (CaC2O4) with measurement of electrical conductivity and its morphology. The inhibition effects of taurine and complex magnesium taurate with different concentrations on CaC2O4 crystallization were measured by electrical conductivity; in addition, crystals generated in the mixing solutions were harvested and analyzed by inverted microscope. The antioxidant activities of taurine and complex magnesium taurate were evaluated for scavenging of 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) activities in vitro. The results of electrical conductivity research suggest that both taurine and complex magnesium taurate can inhibit the growth of calcium oxalate crystals, but the latter has much stronger capability to inhibit that growth. The morphological of calcium oxalate crystals were traced with inverted microscope, to induce calcium oxalate dehydrate (COD) formation than did taurine under same condition. With the increasing of concentrations, the scavenging activity of taurine and complex magnesium taurate against DPPH radical increased; furthermore, the antioxidant ability of complex magnesium taurate better than that of taurine. So it concludes that the inhibition of complex magnesium taurate is clearly superior to that of taurine. Acknowledgments: Financial support: National 863 plans projects 2011AA09070109.

Functional consequences of taurine interaction with the GABAergic system Abdeslem El Idrissi City University of New York/College of Staten Island The goal of this study is to characterize the functional consequences of taurine interaction with the GABAA receptors. Taurine is a sulfur-containing, conditionally-essential amino acid amino acid, found in high concentrations in the brain and act as a GABAA receptor. We found that acute injection of taurine to mice alters a variety of neurobehaviors that are mediated by GABAA receptors. These include suppression of locomotor activity, anxiety and heightened fear potentiated freezing responses. Outside the CNS-mediated behaviors, acute taurine injection resulted in a hypoglycemia and hypotension. All these affects (both central and peripheral) are mediated through activation of the GABAA receptors and could be blocked by specific GABAA receptors antagonists. On the other hand, taurine supplementation in drinking water induced a state of brain excitability characterized by increased susceptibility to kainic acid-induced seizures. Taurine-fed mice had elevated brain levels of glutamate and GABA. This increase in neurotransmitter levels was accompanied by an increase in the expression of GABA synthesizing enzyme, glutamic acid decarboxylase. Furthermore, taurine-fed mice have reduced expression of GABAA receptors. The down-regulation of GABAA receptors is due to the sustained interaction of taurine with GABAA receptors which decreases the efficacy of the inhibitory synapses at postsynaptic membrane. As a compensatory mechanism to this increased excitability, there is increased GAD expression as demonstrated biochemically and pharmacologically. Peripheral effects of taurine supplementation included glucose tolerance and hypertension. Thus, chronic supplementation of taurine induces several biochemical changes in the GABAergic system.

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Mechanism underlying the antioxidant activity of taurine Chian Ju Jong1, Junichi Azuma2 and Stephen W. Schaffer1 1 University of South Alabama College of Medicine Department of Pharmacology, Mobile, AL 36688; 2 Hyogo University of Health Sciences School of Pharmacy, Kobe, Japan

Taurine is a potent antioxidant, however, the mechanism underlying its antioxidant activity is unclear. Because taurine is neither a free radical scavenger nor a promoter of the antioxidant defenses, the possibility that taurine might be a determinant of mitochondrial superoxide production was examined. To test this hypothesis, intracellular taurine levels were reduced *50% by exposing isolated neonatal cardiomyocytes to medium containing the taurine transport inhibitor, b-alanine. Associated with the decline in intracellular taurine levels was a decrease in the expression of specific mitochondria encoded proteins, such as ND5 and ND6, a pattern indicative of inadequate decoding of UUG. The decline in 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 oxygen, forming in the process superoxide. Therefore, three measures of oxidative stress (elevations in the glutathione redox state, MitoSox fluorescence and aconitase activity) were examined in the b-alanine treated cells. All three measures revealed that taurine deficiency is associated with an elevation in oxidative stress. However, co-administration of taurine with b-alanine abolished the rise in oxidative stress, suggesting that b-alanine-mediated taurine depletion promotes the generation of superoxide by the mitochondria. Hence, taurine is required for normal mitochondrial function; severe taurine depletion reduces mitochondrial function and enhances the generation of reactive oxygen species by the electron transport chain.

Metabolic changes mediated by taurine depletion: role in mitochondrial function Stephen Schaffer1, Chian Ju Jong1 and Junichi Azuma2 1 University of South Alabama College of Medicine Department of Pharmacology, Mobile, AL 36688; 2 Hyogo University of Health Sciences School of Pharmacy, Kobe, Japan

Rats treated with the taurine transport inhibitors, guanidinoethylsulfonate or b-alanine, lose about 50% of their myocardial taurine content. Associated with the decline in taurine levels is a modest change in myocardial relaxation (-dP/dt) but no apparent change in other measures of myocardial function. However, the taurine depleted hearts experienced significant changes in energy metabolism, which are characterized by a shift away from aerobic metabolism in favor of anaerobic metabolism. The taurine deficient hearts experienced a twofold increase in glucose utilization, a twofold increase in pyruvate production and a three- to fourfold increase in lactate production. These effects were exaggerated in hearts perfused with buffer containing insulin. Crossover plots revealed that the stimulation in glycolysis by the taurine depleted heart was largely caused by the activation of

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S84 phosphofructokinase, in part because of a decrease in citrate levels. The size of the creatine phosphate and adenine nucleotide pools of the taurine deficient heart fell by 26 and 6%, respectively. These data are consistent with evidence in isolated cardiomyocytes that b-alaninemediated taurine depletion slows flux through the electron transport chain secondary to impaired assembly of electron transport chain complexes.

Perinatal taurine exposure alters neural control of arterial pressure via the renin-angiotensin system but not estrogen in rats Sanya Roysommuti1, Atcharaporn Thaeomor1, Wichaporn Lerdweeraphon1, Sawita Khimsuksri1, Dusit Jirakulsomchok1, and Stephen W. Schaffer2 Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand and 2 Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, Alabama 36688, USA Perinatal taurine exposure alters neural and renal controls of arterial pressure in adult rats. This study tests the hypothesis that perinatal taurine status influences arterial pressure control by altering the reninangiotensin 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 tap water (TDG, TSG, CG) or tap water alone (TDW, TSW, CW) throughout the experiment. Acute inhibition of the renin-angiotensin system with captopril (CW + Cap, CG + Cap, TDW + Cap, TDG + Cap, TSW + Cap, TSG + Cap) or estrogen receptors with tamoxifen (CW + Tam, CG + Tam, TDW + Tam, TDG + Tam, TSW + Tam, TSG + Tam) were studied. At 7–8 weeks of age, mean arterial pressures (MAP) and heart rates were not significantly different among control treated groups and only MAP decreased in all rats with the captopril but not tamoxifen. Baroreflex sensitivity controls of heart rate and renal nerve activity significantly decreased only in TDG and these were restored to CW groups by the captopril but not tamoxifen. In addition, sympathetic nerve activity significantly and markedly elevated and parasympathetic nerve activity decreased only in TDG and these were also restored to the CW groups by the captopril but not tamoxifen treatment. Altogether, the present data suggest that high sugar intake alters the autonomic nervous system control of arterial pressure via the renin-angiotensin system in perinatal taurine depleted adult female rats and this effect is not attenuated by estrogen.

Role of taurine and taurine transporter (TauT) in cell death Xiaobin Han University of Tennessee Health Science Center, Department of Pediatrics, 50 N. Dunlap St. CRFC 336, Memphis, TN, USA, Email: [email protected] Cisplatin is a commonly used chemotherapeutic agent that has a major limitation because of its nephrotoxicity. We have demonstrated that cisplatin down-regulates the expression of taurine transporter gene (TauT) in LLC-PK1 proximal tubular renal cells and forced overexpression of TauT protects against cisplatin-induced apoptosis in renal cells in vitro. Since cisplatin-induced renal injury is mainly

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12th International Congress on Amino Acids, Peptides and Proteins confined to the S3 segment of renal proximal tubules—the primary site for renal adaptive regulation of the taurine transporter, we hypothesized that TauT is a target of p53 and c-Jun during cisplatininduced nephrotoxicity. A recent study showed that oxidative stressinduced JNK signaling pathway together with c-Jun and AP1 activity contribute to drug resistance. In the present study, we have shown that TauT is a direct target of p53 and c-Jun. Expression of TauT is downregulated by p53 and up-regulated by c-Jun in human embryonic 293 renal cells as determined by promoter analysis, reporter assay, DNA binding, and western blot analysis. Forced overexpression of TauT protects cisplatin-induced apoptosis. Inhibition of TauT by RNA interference resulted in a significant reduction of 293 cell growth and enhanced the sensitivity of 293 cells to cisplatin in dose- and timedependent manners. Furthermore, we have demonstrated that overexpression of TauT prevented the progression of cisplatin-induced AKI in TauT transgenic mice, as measured by the levels of BUN and serum creatinine, and TUNEL assay. Cisplatin activated p53 and PUMA (a p53-responsive proapoptotic Bcl-2 family protein) in the kidneys of both wild-type and TauT transgenic mice. However, AKI was only clearly observed in the wild-type animals, suggesting that functional TauT plays a critical role in protecting against cisplatininduced AKI, possibly through attenuating a p53-dependent pathway. In conclusions, functional TauT plays an important role in cisplatin-induced renal injury. Expression of TauT is regulated by p53 and c-Jun and the balance of such regulation will determine the levels of TauT that may decide the fate of the renal cells.

Taurine and Calculus Bovis: benefits of ethnopharmacological knowledge Kyoko Takahashi1,2, Yuko Azuma2, Junichi Azuma3 and Stephen W. Schaffer4 1

The museum of Osaka University; Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; 3 School of Pharmacy, Hyogo University of Health Sciences, Hyogo, Japan; 4 School of Medicine, University of South Alabama, Mobile, AL, USA 2

The use of animal products in healing is an ancient and widespread cross-cultural practice. Calculus Bovis (the gallstone of Bos Taurus domesticus Gmmelin) is one of the most precious and commonly-used medicinal materials in Japan and China. Taurine, discovered from ox 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 inflammation and/or normalizing function of the gallbladder, its detailed mechanism remains largely unknown. To ensure sustainable 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 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 the efficacy of C. Bovis by using ICP-MS and cultured cardiac cells, respectively. First, principal component analysis (PCA) and multielemental focus were effective in discriminating C. Bovis samples derive from different habitats. Second, C. Bovis was found to exert cardioprotection against antiarrhythmias by: (1) diminishing arrhythmias produced by low and high medium Ca2+; (2) antagonizing the pro-arrhythmogenic actions of beta-alanine (an inhibitor of taurine transport); (3) attenuating the harmful actions of bile acids. It is plausible that the relationship between taurine, Ca2+ and the bile acids contribute to the therapeutic effect of C. Bovis.


Taurine and metabolic syndrome Masato Imae1 and Shigeru Murakami2 1

R&D Laboratories and R&D Headquarters of Taisho pharmaceutical Co. Ltd., Saitama-shi, Saitama 331-9530 and Toshima-ku, Tokyo 170-8633, Japan

2

Metabolic syndrome is a cluster of cardiovascular risk factors including abnormal obesity, hyperglycemia, dyslipidemia and elevated blood pressure. The prevalence of metabolic syndrome is increasing in modern societies and is becoming a significant problem around the world. Many studies in animal models and humans have revealed that taurine prevents these components of metabolic syndrome. The beneficial effects of taurine are thought to be attributable to its basic physiological actions, including osmoregulation, antioxidation, immunomodulation, Ca2+ modulation, angiotensin II antagonism, and bile acid conjugation. Taurine stimulates bile acid synthesis from cholesterol in the liver, and thereby decreases the cholesterol levels in the plasma and liver, thus leading to improvements in hyperlipidemia, atherosclerosis and fatty liver. The antidiabetic effect of taurine is related to its improvement of insulin resistance through amelioration of lipid and glucose metabolism, and islet b-cell protection. Taurine has also been shown to enhance the energy production in adipose tissue. Anti-hypertensive effects of taurine have also been demonstrated in hypertensive rats and humans. The suppression of the sympathetic nerve activity may be responsible for the effects of taurine on blood pressure. In addition, the protective effect on endothelial cells may be important for the prevention of hypertension as well as atherosclerosis. Worldwide epidemiological studies have showed that urinary taurine excretion as a marker of taurine intake is inversely related to the body mass index, blood pressure, serum total cholesterol and coronary heart disease mortality. As a result, taurine may therefore prevent metabolic syndrome via multiple mechanisms.

Taurine haloamines as anti-microbial, anti-inflammatory and anti-oxidant agents: new perspectives for clinical use Janusz Marcinkiewicz Department of Immunology, Jagiellonian University Medical College, Krakow, Poland Taurine haloamines, taurine bromamine (TauBr) and taurine chloramine (TauCl), the physiological products of peroxidase halide system, 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 oxygenase-1 (HO-1). On the other hand, only TauBr was found to be highly membrane-permeable, showing stronger microbicidal activity than TauCl. In addition, TauBr shows in vitro ability to reduce formation of bacterial biofilm. Therefore, TauBr is promising candidate for a local treatment of chronic infectious/inflammatory disorders associated with the presence of biofilm, namely in acne vulgaris and chronic rhinosinusitis. To supported the idea to use TauBr for topical anti-acne therapy we have compared the effectiveness of TauBr to that of Clindamycin, one of the most common topical agent used in the treatment of acne.

S85 In our pilot clinical study both treatments produced comparable beneficial results. Therefore, the results from clinical studies are in agreement with previous in vitro data and strongly suggest that TauBr could be considered a new therapeutic agent in acne vulgaris. Finally, we speculate on a therapeutic potential of taurine/TauCl in a prevention of the induction of autoimmunity. This idea is supported by our recent studies showing that scavenging of HOCl by taurine (formation of TauCl) neutralizes oxidative modification of self proteins by HOCl. Therefore, this reaction protects proteins against enhancement of their immunogenicity and reduces probability of autoantibodies production.

The combined effect of taurine with BCAA on the delayed-onset-muscle-soreness and damages after eccentric exercise—a double-blind study Song-Gyu Ra1, Teruo Miyazaki2, Keisuke Ishikura3, Takafumi Suzuki1, Seiji Maeda1, Shumpei Miyakawa1, Yasushi Matsuzaki2, and Hajime Ohmori1 1

Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan; 2 Department of Development for Community Medicine, Tokyo Medical University, Ami, Japan; 3 Sports Research and Development Core, University of Tsukuba Aim: A high-intensity-exercise causes delayed-onset-muscle-soreness (DOMS) and damages that disturb continuous and practice of exercise. Although previous studies have evaluated the effectiveness of BCAA on the DOMS, consistent finding has not still convinced. Therefore, the present study investigated the combined effect of taurine that has many physiological and pharmacological roles with BCAA on the DOMS and damages. 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 ingested these supplementations before 2 weeks, on the day, and after 4 days of exercise. Muscle damages were caused by repeating of forced eccentric elbow flexors. All experiments were carried out under double-blind method. Result: In the period by 4 days after exercise, the Combination significantly improved the DOMS evaluated by VAS, upper arm circumferences, range of elbow’s motion, and serum markers of muscle damages, although there were some effects in both the taurine and BCAA alone compared to those in the Control. Conclusion: Combination of taurine with BCAA would be useful way to attenuate DOMS and muscle damages after high-intensityexercise.

The effect of taurine treatment on the differentiation of cultured skeletal muscle cell Teruo Miyazaki1, Akira Honda1, Tadashi Ikegami1, Mutsumi Shirai1, Shumpei Miyakawa2, and Yasushi Matsuzaki1 1

Tokyo Medical University Ibaraki Medical Center, and University of Tsukuba, Ibaraki, Japan

2

Background and aim: Taurine has been considered as one of essential factors on the differentiation/growth of skeletal muscles

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