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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #2)

Effect of beta-alanine supplementation on muscle carnosine concentrations and exercise performance by Craig Sale; Bryan Saunders; Roger C. Harris (pp. 321-333).

High-intensity exercise results in reduced substrate levels and accumulation of metabolites in the skeletal muscle. The accumulation of these metabolites (e.g. ADP, Pi and H⁺) can have deleterious effects on skeletal muscle function and force generation, thus contributing to fatigue. Clearly this is a challenge to sport and exercise performance and, as such, any intervention capable of reducing the negative impact of these metabolites would be of use. Carnosine (β-alanyl-l-histidine) is a cytoplasmic dipeptide found in high concentrations in the skeletal muscle of both vertebrates and non-vertebrates and is formed by bonding histidine and β-alanine in a reaction catalysed by carnosine synthase. Due to the pKa of its imidazole ring (6.83) and its location within skeletal muscle, carnosine has a key role to play in intracellular pH buffering over the physiological pH range, although other physiological roles for carnosine have also been suggested. The concentration of histidine in muscle and plasma is high relative to its K _m with muscle carnosine synthase, whereas β-alanine exists in low concentration in muscle and has a higher K _m with muscle carnosine synthase, which indicates that it is the availability of β-alanine that is limiting to the synthesis of carnosine in skeletal muscle. Thus, the elevation of muscle carnosine concentrations through the dietary intake of carnosine, or chemically related dipeptides that release β-alanine on absorption, or supplementation with β-alanine directly could provide a method of increasing intracellular buffering capacity during exercise, which could provide a means of increasing high-intensity exercise capacity and performance. This paper reviews the available evidence relating to the effects of β-alanine supplementation on muscle carnosine synthesis and the subsequent effects on exercise performance. In addition, the effects of training, with or without β-alanine supplementation, on muscle carnosine concentrations are also reviewed.

Keywords: Beta-alanine; Carnosine; Exercise performance and capacity; Muscle buffering

Luminal sulfide and large intestine mucosa: friend or foe? by François Blachier; Anne-Marie Davila; Sabria Mimoun; Pierre-Henri Benetti; Calina Atanasiu; Mireille Andriamihaja; Robert Benamouzig; Frédéric Bouillaud; Daniel Tomé (pp. 335-347).

Hydrogen sulfide (H₂S) is present in the lumen of the human large intestine at millimolar concentrations. However, the concentration of free (unbound) sulfide is in the micromolar range due to a large capacity of fecal components to bind the sulfide. H₂S can be produced by the intestinal microbiota from alimentary and endogenous sulfur-containing compounds including amino acids. At excessive concentration, H₂S is known to severely inhibit cytochrome c oxidase, the terminal oxidase of the mitochondrial electron transport chain, and thus mitochondrial oxygen (O₂) consumption. However, the concept that sulfide is simply a metabolic troublemaker toward colonic epithelial cells has been challenged by the discovery that micromolar concentration of H₂S is able to increase the cell respiration and to energize mitochondria allowing these cells to detoxify and to recover energy from luminal sulfide. The main product of H₂S metabolism by the colonic mucosa is thiosulfate. The enzymatic activities involved in sulfide oxidation by the colonic epithelial cells appear to be sulfide quinone oxidoreductase considered as the first and rate-limiting step followed presumably by the action of sulfur dioxygenase and rhodanese. From clinical studies with human volunteers and experimental works with rodents, it appears that H₂S can exert mostly pro- but also anti-inflammatory effects on the colonic mucosa. From the available data, it is tempting to propose that imbalance between the luminal concentration of free sulfide and the capacity of colonic epithelial cells to metabolize this compound will result in an impairment of the colonic epithelial cell O₂ consumption with consequences on the process of mucosal inflammation. In addition, endogenously produced sulfide is emerging as a prosecretory neuromodulator and as a relaxant agent toward the intestinal contractibility. Lastly, sulfide has been recently described as an agent involved in nociception in the large intestine although, depending on the experimental design, both pro- and anti-nociceptive effects have been reported.

Keywords: Sulfide; Large intestine; Colon; Detoxification; Energy metabolism

Beneficial effects of l-arginine on reducing obesity: potential mechanisms and important implications for human health by Jason R. McKnight; M. Carey Satterfield; Wenjuan S. Jobgen; Stephen B. Smith; Thomas E. Spencer; Cynthia J. Meininger; Catherine J. McNeal; Guoyao Wu (pp. 349-357).

Over the past 20 years, growing interest in the biochemistry, nutrition, and pharmacology of l-arginine has led to extensive studies to explore its nutritional and therapeutic roles in treating and preventing human metabolic disorders. Emerging evidence shows that dietary l-arginine supplementation reduces adiposity in genetically obese rats, diet-induced obese rats, finishing pigs, and obese human subjects with Type-2 diabetes mellitus. The mechanisms responsible for the beneficial effects of l-arginine are likely complex, but ultimately involve altering the balance of energy intake and expenditure in favor of fat loss or reduced growth of white adipose tissue. Recent studies indicate that l-arginine supplementation stimulates mitochondrial biogenesis and brown adipose tissue development possibly through the enhanced synthesis of cell-signaling molecules (e.g., nitric oxide, carbon monoxide, polyamines, cGMP, and cAMP) as well as the increased expression of genes that promote whole-body oxidation of energy substrates (e.g., glucose and fatty acids) Thus, l-arginine holds great promise as a safe and cost-effective nutrient to reduce adiposity, increase muscle mass, and improve the metabolic profile in animals and humans.

Keywords: Arginine; Fat metabolism; Brown adipose tissue; NO

Age-related changes of muscle and plasma amino acids in healthy children by Folke Hammarqvist; Gertrud Angsten; Staffan Meurling; Kerstin Andersson; Jan Wernerman (pp. 359-366).

The aim of the study was to explore if changes in muscle and plasma amino acid concentrations developed during growth and differed from levels seen in adults. The gradient and concentrations of free amino acids in muscle and plasma were investigated in relation to age in metabolic healthy children. Plasma and specimens from the abdominal muscle were obtained during elective surgery. The children were grouped into three groups (group 1: < 1 year, n = 8; group 2: 1–4 years, n = 13 and group 3: 5–15 years, n = 15). A reference group of healthy adults (21–38 years, n = 22) was included in their comparisons and reflected specific differences between children and adults. In muscle the concentrations of 8 out of 19 amino acids analysed increased with age, namely taurine, aspartate, threonine, alanine, valine, isoleucine, leucine, histidine, as well as the total sums of branched chain amino acids (BCAA), basic amino acids (BAA) and total sum of amino acids (P < 0.05). In plasma the concentrations of threonine, glutamine, valine, cysteine, methionine, leucine, lysine, tryptophane, arginine, BCAA, BAA and the essential amino acids correlated with age (P < 0.05). These results indicate that there is an age dependency of the amino acid pattern in skeletal muscle and plasma during growth.

Keywords: Amino acids; Growth; Skeletal muscle; Plasma

Optimized synthesis of phosphatidylserine by Giuseppe Guanti; L. Banfi; A. Basso; L. Bondanza; G. Guglieri; K. Powles; R. Riva (pp. 367-373).

The synthesis of phosphatidyl serine containing saturated fatty acids was thoroughly studied and optimized in order to establish a protocol amenable to large-scale synthesis. The key step was a one-pot multicomponent reaction involving an O-benzyl phosphorodiamidite, protected serine and diacylglycerol, followed by in situ oxidation of the resulting phosphite. In order to replace expensive and poorly stable tetrazole, a screening of substitutes was carried out and imidazolium chloride was selected as the best suited one.

Keywords: Phospholipids; Multicomponent reactions; Phosphorodiamidites

Taurine restores Axl/Gas6 expression in vascular smooth muscle cell calcification model by Xiao-Bo Liao; Yi-Qun Peng; Xin-Min Zhou; Bing Yang; Zhe Zheng; Li-Ming Liu; Feng-Lin Song; Jian-Ming Li; Kang Zhou; Ji-Cai Meng; Ling-Qing Yuan; Hui Xie (pp. 375-383).

Our previous studies demonstrated that taurine inhibits osteoblastic differentiation of vascular smooth muscular cells (VSMCs) via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, but the underlying mechanism is not elucidated. The tyrosine kinase receptor Axl and its ligand growth arrest-specific protein 6 (Gas6) are expressed in VSMCs. Axl/Gas6 signaling system is known to inhibit VSMCs calcification. We herein showed that taurine partially restored Axl and Gas6 expression in β-glycerophosphate (β-GP)-induced VSMC calcification model. Taurine also induced activation of ERK, but not other two MAPKs including c-jun N-terminal Kinase (JNK) and p38 in VSMCs. Either knockdown of the taurine transporter (TAUT) or treatment with the ERK-specific inhibitor PD98059 blocked the activation of ERK by taurine and abolished taurine-induced Axl/Gas6 expression and calcium deposition reduction in β-GP-induced VSMC calcification model. These results demonstrate for the first time that taurine stimulates expression of Axl and Gas6 via TAUT/ERK signaling pathway in β-GP-induced VSMC calcification model.

Keywords: Taurine; Vascular smooth muscular cells; Extracellular signal-regulated kinase; Axl; Gas6

A novel high molecular weight glutenin subunit from Australopyrum retrofractum by Shuwei Liu; Feng Zhao; Xin Gao; Fanguo Chen; Guangmin Xia (pp. 385-392).

We describe the sequence of a gene encoding a high molecular weight glutenin subunit (HMW-GS) expressed in the endosperm of the wheat relative Australopyrum retrofractum. Although the subunit has a similar primary structure to that HMW-GS genes present in other Triticeae species, its N-terminal domain is shorter, its central repetitive domain includes a unique dodecameric motif, and its C-terminal domain contain an extra cysteine residue. A phylogenetic analysis showed that the Glu-W1 gene is neither a true x- nor a true y-type subunit, although it is more closely related to the y-type genes present in the K and E genomes than to any other published HMW-GS gene. All these results indicated that this novel subunit may undergo a special evolutionary process different from other Triticeae species. A flour supplementation experiment showed that the Glu-W1 subunit has a negative effect on dough quality, which might be the result of interaction between the two closely placed cysteine residues in the C-terminal region.

Keywords: Australopyrum retrofractum ; Evolution; Glu-1 ; HMW-GS; W genome

Molecular prediction of oseltamivir efficiency against probable influenza A (H1N1-2009) mutants: molecular modeling approach by Thanyada Rungrotmongkol; Maturos Malaisree; Nadtanet Nunthaboot; Pornthep Sompornpisut; Supot Hannongbua (pp. 393-398).

To predict the susceptibility of the probable 2009 influenza A (H1N1-2009) mutant strains to oseltamivir, MD/LIE approach was applied to oseltamivir complexed with the most frequent drug-resistant strains of neuraminidase subtypes N1 and N2: two mutations on the framework residues (N294S and H274Y) and the two others on the direct-binding residues (E119V and R292K) of oseltamivir. Relative to those of the wild type (WT), loss of drug–target interaction energies, especially in terms of electrostatic contributions and hydrogen bonds were dominantly established in the E119V and R292K mutated systems. The inhibitory potencies of oseltamivir towards the WT and mutants were predicted according to the ordering of binding-free energies: WT (−12.3 kcal mol⁻¹) > N294S (−10.4 kcal mol⁻¹) > H274Y (−9.8 kcal mol⁻¹) > E119 V (−9.3 kcal mol⁻¹) > R292K (−7.7 kcal mol⁻¹), suggesting that the H1N1-2009 influenza with R292K substitution, perhaps, conferred a high level of oseltamivir resistance, while the other mutants revealed moderate resistance levels. This result calls for an urgent need to develop new potent anti-influenza agents against the next pandemic of potentially higher oseltamivir-resistant H1N1-2009 influenza.

Keywords: 2009-H1N1 influenza A neuraminidase; Oseltamivir resistance; Mutations; Molecular dynamics simulations

Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide by Fangfang Zhong; Hongyan Wang; Tianlei Ying; Zhong-Xian Huang; Xiangshi Tan (pp. 399-408).

Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a α-subunit (690 AA) and a heme-binding β-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation, platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling was made in this study. The recombinant human sGC β1 subunit (HsGCβ619) and its truncated N-terminal fragments (HsGCβ195 and HsGCβ384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were characterized by UV–vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC.

Keywords: Human soluble guanylate cyclase; NO sensor; NO signal transduction; E. coli expression system

The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist by Mohammed Akhter Hossain; Chrishan S. Samuel; Claudia Binder; Tim D. Hewitson; Geoffrey W. Tregear; John D. Wade; Ross A. D. Bathgate (pp. 409-416).

Relaxin is a pleiotropic hormone which exerts its biological functions through its G-protein coupled receptor, RXFP1. While relaxin is well known for its reproductive and antifibrotic roles, recent studies suggest that it is produced by cancer cells and acts on RXFP1 to induce growth and metastasis. Furthermore, more recently Silvertown et al. demonstrated that lentiviral production of a human gene-2 (H2) relaxin analog reduced the growth of prostate xenograft tumors. The authors proposed that the lentivirally produced peptide was an RXFP1 antagonist; however, the processed form of the peptide produced was not demonstrated. In this study, we have chemically synthesized the H2 relaxin analog, B-R13/17K H2 relaxin, and subjected it to detailed chemical characterization by HPLC, MALDI-TOF mass spectrometry, and amino acid analysis. The biological activity of the synthetic peptide was then tested in three different cell lines. It was found to bind with 500-fold lower affinity than H2 relaxin to RXFP1 receptors over-expressed in HEK-293T cells where it acted as a partial agonist. However, in cells which natively express the RXFP1 receptor, rat renal myofibroblasts and MCF-7 cancer cells, it acted as a full antagonist. Importantly, it was able to significantly inhibit cell invasion induced by H2 relaxin in MCF-7 cells consistent with the results of the lentiviral-driven expression in prostate cancer cells. The relaxin analog, B-R13/17K H2, can now be used as a tool to further understand RXFP1 function, and serve as a template for drug design for a therapeutic to treat prostate and other cancers.

Keywords: Relaxin; H2 relaxin; RXFP1 antagonist; B-R13/17K H2 relaxin; GPCR

SANA: an algorithm for sequential and non-sequential protein structure alignment by Lin Wang; Ling-Yun Wu; Yong Wang; Xiang-Sun Zhang; Luonan Chen (pp. 417-425).

Protein structure alignment algorithms play an important role in the studies of protein structure and function. In this paper, a novel approach for structure alignment is presented. Specifically, core regions in two protein structures are first aligned by identifying connected components in a network of neighboring geometrically compatible aligned fragment pairs. The initial alignments then are refined through a multi-objective optimization method. The algorithm can produce both sequential and non-sequential alignments. We show the superior performance of the proposed algorithm by the computational experiments on several benchmark datasets and the comparisons with the well-known structure alignment algorithms such as DALI, CE and MATT. The proposed method can obtain accurate and biologically significant alignment results for the case with occurrence of internal repeats or indels, identify the circular permutations, and reveal conserved functional sites. A ranking criterion of our algorithm for fold similarity is presented and found to be comparable or superior to the Z-score of CE in most cases from the numerical experiments. The software and supplementary data of computational results are available at http://zhangroup.aporc.org/bioinfo/SANA .

Keywords: Protein structure alignment; Fold comparison; Circular permutation; Functional sites

The impact of taurine- and beta-alanine-supplemented diets on behavioral and neurochemical parameters in mice: antidepressant versus anxiolytic-like effects by Tatsuro Murakami; Mitsuhiro Furuse (pp. 427-434).

Taurine, a substrate of taurine transporter, has functions as a neuromodulator and antioxidant and beta-alanine, a taurine transporter inhibitor, has a role as a neurotransmitter in the brain, and they were expected to be involved in depression-like behavior and antidepressant treatment. These facts aroused our interest in new capabilities of taurine and beta-alanine. Thus, to investigate the effects of chronic ingestion of taurine- (22.5 mmol/kg diet) supplemented diet and beta-alanine- (22.5 mmol/kg diet) supplemented diet under acute stressful conditions, behavioral changes and brain metabolites were compared with mice fed a control diet. In the open field test, no significant difference was observed in locomotor activity among groups. In the elevated plus-maze test, however, significant increases in the percentage of time spent and entries in the open arms were observed in the beta-alanine-supplemented diet fed group compared to both controls and animals fed with taurine-supplemented diet. Moreover, a significant decrease in the duration of immobility was observed in the taurine-supplemented diet group in the forced swimming test compared to both controls and animals fed with beta-alanine-supplemented diet. Taurine-supplemented diet increased taurine and l-arginine concentrations in the hypothalamus. In contrast, beta-alanine-supplemented diet decreased the concentration of 5-hydroxyindoleacetic acid, a major metabolite of serotonin, in the hypothalamus. Beta-alanine-supplemented diet also increased carnosine (beta-alanyl-l-histidine) concentration in the cerebral cortex and hypothalamus, and brain-derived neurotrophic factor concentration in the hippocampus. These results suggested that taurine-supplemented diet had an antidepressant-like effect and beta-alanine-supplemented diet had an anxiolytic-like effect.

Keywords: Taurine; Beta-alanine; Elevated plus-maze test; Forced swimming test; Amino acids; Brain-derived neurotrophic factor

Effects of glutamine on the nuclear factor-kappaB signaling pathway of murine peritoneal macrophages by Marcelo Macedo Rogero; Primavera Borelli; Ricardo Ambrósio Fock; Maria Carolina Borges; Marco Aurélio Ramirez Vinolo; Rui Curi; Karina Nakajima; Amanda Rabello Crisma; Aline Domingas Ramos; Julio Tirapegui (pp. 435-441).

The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappaB (NF-κB) signaling pathway of murine peritoneal macrophages. Since glutamine is essential for the normal functioning of macrophages, it was hypothesized that in vitro glutamine supplementation would increase NF-κB activation. Peritoneal macrophages were pretreated with glutamine (0, 0.6, 2 and 10 mM) before incubation with lipopolysaccharide (LPS), and the effects of glutamine on the production of tumor necrosis factor-alpha and on the expression and activity of proteins involved in the NF-κB signaling pathway were studied by an enzyme linked immuno-sorbent assay, Western blotting, and an electrophoretic mobility shift assay. Glutamine treatment (2 and 10 mM) increased the activation of NF-κB in LPS-stimulated peritoneal macrophages (P < 0.05). In non-stimulated cells, glutamine treatment (2 and 10 mM) significantly reduced IκB-α protein expression (P < 0.05). Glutamine modulates NF-κB signaling pathway by reducing the level of IκB-α, leading to an increase in NF-κB within the nucleus in peritoneal macrophages.

Keywords: Macrophage; Glutamine; NF-κB; Inflammation; Tumor necrosis factor-α

An improved protocol for the preparation of (S)-vinylglycine from (S)-methionine by Christian-H. Küchenthal; Julia Migenda; Magdalena Polednia; Wolfgang Maison (pp. 443-448).

We present an optimized procedure for the synthesis of (S)-vinylglycine from (S)-methionine. The key step is a solvent free pyrolysis of an intermediate sulfoxide at high temperature. Using our optimized reaction conditions, Cbz-protected vinylglycine was obtained in high yield and with almost no side products. The protocol is scalable, fast and avoids the use of poisonous reagents.

Keywords: Amino acids; Vinylglycine; Natural products; Pyrolysis

Methionine limitation results in increased hepatic FAS activity, higher liver 18:1 to 18:0 fatty acid ratio and hepatic TAG accumulation in Atlantic salmon, Salmo salar by Marit Espe; Raja Mansingh Rathore; Zhen-Yu Du; Bjørn Liaset; Adel El-Mowafi (pp. 449-460).

The current experiment aimed to study whether interactions with lipid metabolism possibly might explain the relative increased liver weight obtained in fish fed sub-optimal methionine levels. A basal diet based on a blend of plant proteins which is low in methionine (1.6 g Met/16 g N) was compared to a methionine adequate diet (2.2 g Met/16 g N) prepared by adding dl-methionine (2.4 g/kg) to the basal diet in the expense of wheat grain. Fish oil was used as the lipid source. The diets were balanced in all nutrients except methionine. The diets were fed to Atlantic salmon (500 g BW) for a period of 3 months. Feed intake did not differ, rendering the intake of all nutrients except methionine equal. Fish fed the low methionine diet had an increased liver size relative to body weight, indicating fat deposition in the liver. Fish given the sub-optimal methionine diet showed about six times higher fatty acid synthase (FAS) activity as compared to the fish fed the adequate methionine diet, indicating a higher de novo lipogenesis. A significant rise in the liver 18:1 to 18:0 fatty acid ratios also supported storage of lipids over fatty acid oxidation. Indeed, methionine limitation resulted in significantly higher TAG concentrations in the liver. Sub-optimal dietary methionine also resulted in lower hepatic taurine concentrations and the total bile acids concentrations were reduced in faeces and tended to be reduced in plasma. Taken together, our data show that salmon fed sub-optimal methionine levels had increased relative liver weight and developed signs commonly described in the early stage of non-alcoholic fatty liver disease in rodent models (increased FAS activity, changed fatty acid ratios and TAG accumulation).

Keywords: Methionine deficiency; Fatty liver development; TAG accumulation; Taurine depletion; Bile acids; NAFLD; Atlantic salmon

Short malonyl dehydro peptides as potential scaffolds for peptidomimetics by an efficient Knoevenagel reaction by Stefania Fioravanti; Simona Gasbarri; Alberto Morreale; Lucio Pellacani; Federico Ramadori; Paolo A. Tardella (pp. 461-470).

Non-symmetric disubstituted malonamides rAA-mGly-AA′, obtained from Meldrum’s acid, were considered as methylene active compounds and a Knoevenagel condensation methodology was developed involving piperidine and activated 4 Å molecular sieves as catalysts. The reaction is efficient, broad in scope, and easy to perform and allows access to E/Z mixtures of short malonyl dehydro peptides (MDHPs) rAA-mΔ²AA″-AA′, partially modified retro peptides characterized by an interesting combination of retro and dehydro modifications in the same structure.

Keywords: Peptidomimetics; Amino acids; Malonamides; Knoevenagel reaction

Structural requirement for the agonist activity of the TLR2 ligand Pam2Cys by Weiguang Zeng; Emily Eriksson; Brendon Chua; Lara Grollo; David C. Jackson (pp. 471-480).

Synthetic lipopeptides have demonstrated great potential as a vaccine strategy for eliciting cellular and humoral immunity. One of the most potent lipid moieties used is S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys). Pam2Cys binds to and activates dendritic cells by engagement of Toll like receptor 2 (TLR 2). In this study, we have investigated the structural requirement of the agonist activity of Pam2Cys by varying the three structural elements of the core structure S-(2,3-dihydroxypropyl)-cysteine namely (1) the α-amino group of the cysteine residue (2) the sulphur atom of the cysteine residue and (3) the 2,3-dihydroxypropyl moiety. Four novel analogues of Pam2Cys were made and each of these analogues were incorporated into vaccine constructs and examined for immunogenicity. Our results demonstrate that (1) the potency of the peptide vaccine is least affected by removal of the amino group (2) substitution of the sulphur atom with an amide bond leads to significant reduction of biological activity (3) removal of the amino group and at the same time substitution of the sulphur with an amide bond significantly decreases the biological activity (4) in the two analogues in which the sulphur atom is replaced with an amide bond the analogue containing the 1,3-dihydroxypropyl moiety demonstrates higher activity than the one which contains 2,3-dihydroxypropyl. In conclusion, the results demonstrate strict structural requirements for agonist activity of the TLR2 ligand Pam2Cys.

Keywords: Vaccine; Peptide; TLR2; Structure–activity relationship; Lipopeptide

Peptide analogues of 1811–1818 loop of the A3 subunit of the light chain A3-C1–C2 of FVIII of blood coagulation: biological evaluation by K. Patsialas; C. Koutsas; P. Makris; Maria Liakopoulou-Kyriakides (pp. 481-488).

Factor VIII, the plasma protein deficient or defective in individuals with hemophilia A, is a critical member of the blood coagulation cascade. Recent studies have identified the FVIII light chain region Glu1811-Lys1818 as being involved in FIXa binding and in the assembly of the FX-activating FIXaz–FVIIIa complex. Based on this, a series of 12 peptides, analogues of the 1811–1818 loop of the A3 subunit of the light chain A3-C1–C2 of FVIIIa, were synthesized and evaluated for their anticoagulant activity. Only peptide Ac-ETKTYFWK-NH₂ showed significant anticoagulant activity by inhibiting about 40% factor VIII at a concentration of 0.43 mM. It also showed a prolongation of activated partial thromboplastin time of 6.1 s, whereas its effect on prothrombin time measurements was meaningless. All the other peptides did not show any measurable effect at the concentration of 0.43 mM. These findings are encouraging though further investigation of the effect of this active peptide in different biological settings is needed in order to evaluate its possible clinical applications.

Keywords: Factor VIII; Synthetic peptides; Anticoagulant activity; aPTT; PT

Diastereoselective functionalisation of Baylis–Hillman adducts: a convenient approach to α-methyl-α-amino acids by Gianluca Martelli; Mario Orena; Samuele Rinaldi; Piera Sabatino (pp. 489-497).

The N-tosyl carbamates 4a–e, easily prepared starting from the Baylis–Hillman adducts 3a–e, underwent cyclization carried out with I₂/NIS in the presence of NaH, to give the corresponding 2-oxo-1,3-oxazolidines 5a–e in good yield and total stereoselection when the substituent at C-5 is Ar. After the removal of tosyl group, followed by the cleavage of the heterocyclic ring, the α-methyl-α-amino acids 8a,b and 10 were obtained in good yield as hydrochlorides.

Keywords: α-Methyl-α-amino acid; Functionalisation; Diastereoselection; Baylis–Hillman adducts

Synthesis and electrochemical behaviour of β-halodehydroamino acid derivatives by Paula M. T. Ferreira; L. S. Monteiro; G. Pereira (pp. 499-513).

Several new β,β-dihalo and β-halo-β-substituted dehydroalanines and dehydrodipeptides were synthesized by reacting the corresponding dehydroamino acid derivative with a N-halosuccinimide or in the case of β,β-di-iododehydroalanines with iodine. The results obtained confirmed that the stereochemical outcome of the halogenation reaction with β-substituted dehydroamino acids depends on the substrate. Thus, an increase Z-stereoselectivity was found when the β-phenyldehydroalanines were used as substrates and when these compounds were N-protected with 4-tolylsulfonyl or with carbamates. From this study, it is also possible to conclude that when N-iodosuccinimide was used as reagent a much higher Z-stereoselectivity is found. The electrochemical behaviour of the halogenated dehydroamino acids was studied by cyclic voltammetry. The results show a shift in the reduction peak to higher potentials of the β-halogenated dehydroamino acids when compared with the corresponding non-halogenated derivatives. As expected, the β,β-dihalodehydroalanines exhibit higher peak potentials than β-halo-β-substituted dehydroalanines and the bromo derivatives have lower peak potentials when compared with the corresponding iododehydroamino acids. Controlled potential electrolysis of several β-halo-β-substituted dehydroamino acids afforded the corresponding dehalogenated dehydroamino acids as mixtures of their E and Z-isomers. In all cases, the major isomer isolated results from dehalogenation without isomerization. These new results show that electrochemical reduction constitutes a valuable method for the synthesis of the E-isomer of β-substituted dehydroalanines.

Keywords: Dehydroamino acid; N-halosuccinimides; Iodine; Cyclic voltammetry; Electrolysis

Synthesis of 2-azaspiro[3.3]heptane-derived amino acids: ornitine and GABA analogues by Dmytro S. Radchenko; Oleksandr O. Grygorenko; Igor V. Komarov (pp. 515-521).

Synthesis of 6-amino-2-azaspiro[3.3]heptane-6-carboxylic acid and 2-azaspiro[3.3]heptane-6-carboxylic acid was performed. Both four-membered rings in the spirocyclic scaffold were constructed by subsequent ring closure of corresponding 1,3-bis-electrophiles at 1,1-C- or 1,1-N-bis-nucleophiles. The two novel amino acids were added to the family of the sterically constrained amino acids for the use in chemistry, biochemistry, and drug design.

Keywords: Tailor-made amino acids; Spiro azetidines; Ornitine analogues; GABA analogues; Molecular rigidity

TG2 protects neuroblastoma cells against DNA-damage-induced stress, suppresses p53 activation by Janusz Tucholski (pp. 523-532).

Tissue transglutaminase (TG2) is a multifunctional member of the transglutaminase (TGase) family (E.C.2.3.2.13), which catalyzes in a calcium-dependent reaction the formation of covalent bonds between the γ-carboxamide groups of peptide-bound glutamine residues and various primary amines. Here, we investigated the role of TG2 in a response of the neuroblastoma SH-SY5Y cells to topoisomerase II inhibitor etoposide, known to trigger DNA-damage cell response. We found an early and transient (~2 h) increase of the TG2 protein in SH-SY5Y cells treated with etoposide, along with the increase of phosphorylated and total levels of the p53 protein. Next, we showed that SH-SY5Y cells, which overexpress wild-type TG2 were significantly protected against etoposide-induced cell death. The TG2 protective effect was associated only with the transamidation active form of TG2, because overexpression the wild-type TG2, but not its transamidation inactive C277S form, resulted in a pronounced suppression of caspase-3 activity as well as p53 phosphorylation during the etoposide-induced stress. In addition, exacerbation of cell death with a significant increase in caspase-3 and p53 activation was observed in SH/anti-TG2 cells, in which expression of the endogenous TG2 protein has been greatly reduced by the antisense cDNA construct. Though the cell signaling and molecular mechanisms of the TG2-driven suppression of the cell death machinery remain to be investigated, our findings strongly suggest that TG2 plays an active role in the response of neuroblastoma cells to DNA-damage-induced stress by exerting a strong protective effect, likely by the suppression of p53 activation and p53-driven cell signaling events.

Keywords: TG2; p53; Etoposide; Apoptosis; DNA damage; Retinoic acid

Direct synthesis of phosphinopeptides containing C-terminal α-aminoalkylphosphinic acids by Fanhua Meng; Jiaxi Xu (pp. 533-538).

A series of phosphinopeptides containing C-terminal α-aminoalkylphosphinic acids were prepared in good yields directly in one-pot reactions of 2-(N-benzoxycarbonylamino)alkanamides/peptide amides, aldehydes, and aryldichlorophosphines, followed by hydrolysis. In the current method, the peptide bond was formed in a Mannich-type reaction.

Keywords: Amino amide; Mannich reaction; Peptide; Phosphinopeptide; Synthesis

In position 7 l- and d-Tic-substituted oxytocin and deamino oxytocin: NMR study and conformational insights by Zinovia Spyranti; Maria Fragiadaki; Vassiliki Magafa; Lenka Borovickova; Georgios A. Spyroulias; Paul Cordopatis; Jirina Slaninova (pp. 539-548).

Incorporation of l- or d-Tic into position 7 of oxytocin (OT) and its deamino analogue ([Mpa¹]OT) resulted in four analogues, [l-Tic⁷]OT (1), [d-Tic⁷]OT (2), [Mpa¹,l-Tic⁷]OT (3) and [Mpa¹,d-Tic⁷]OT (4). Their biological properties were described by Fragiadaki et al. (Eur J Med Chem 42:799–806, 2007). Their NMR study (NOESY, TOCSY, ¹H–¹³C HSQC spectra) is presented here. Analogues 1, 3 and 4 showed partial agonistic activity, analogue 2 was pure antagonist, suggesting that a cis conformation between residues 6 and 7 of the molecule does not result in antagonistic activity. However, the reduction in agonistic activity of analogues 1, 3 and 4 in comparison to oxytocin is consistent with the reduction of the trans conformation form. Binding affinity for the human oxytocin receptor with IC₅₀ value of 130, 730, 103, and 380 nM for peptides 1, 2, 3, and 4, respectively, showed lower affinity in the case of d analogues. Deamination slightly increased the affinity. The existence of both cis and trans configurations of the Cys⁶-d-Tic⁷ bond is supported by observation of two sets of cross-peaks for ¹H and ¹³C nuclei for most of the residues of the peptide not only in NOESY and TOCSY but also in ¹H–¹³C HSQC spectra. The MS and HPLC indicate the presence of a single molecule/peptide, and NMR data thus suggest that this second set of peaks is due to the cis conformation.

Keywords: Oxytocin analogues; Deaminooxytocin, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); NMR; Conformation

Application of (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as a chiral derivatizing reagent for reversed-phase high-performance liquid chromatographic separation of diastereomers of amino alcohols, non-protein amino acids, and PenA by Ravi Bhushan; Charu Agarwal (pp. 549-554).

Indirect enantioresolution of 15 primary and secondary amino group containing compounds (amino alcohols, non-protein amino acids, PenA) was done using the reagent (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester [(S)-NIFE] by reversed-phase high-performance liquid chromatography. The diastereomeric derivatives were analyzed under reversed-phase conditions using linear gradient. The detection was at 205 nm and sharp peaks were obtained. The reagent used is comparatively economic than the other derivatizing reagents. Method validation was also done.

Keywords: (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester; Diastereomers; Chiral separation; HPLC; dl-PenA; Amino alcohols; Amino acids

Dietary α-ketoglutarate supplementation ameliorates intestinal injury in lipopolysaccharide-challenged piglets by Yongqing Hou; Lei Wang; Binying Ding; Yulan Liu; Huiling Zhu; Jian Liu; Yongtang Li; Xin Wu; Yulong Yin; Guoyao Wu (pp. 555-564).

Neonates are at increased risk for inflammatory bowel disease, but effective prevention and treatments are currently limited. This study was conducted with the lipopolysaccharide (LPS)-challenged piglet model to determine the effects of dietary supplementation with α-ketoglutarate (AKG) on the intestinal morphology and function. Eighteen 24-day-old pigs (weaned at 21 days of age) were assigned randomly to control, LPS, and LPS + AKG groups. The piglets in the control and LPS groups were fed a corn- and soybean meal-based diet, whereas the LPS + AKG group was fed the basal diet supplemented with 1% AKG. On days 10, 12, 14, and 16, piglets in the LPS and LPS + AKG groups received intraperitoneal administration of LPS (80 μg/kg BW), whereas piglets in the control group received the same volume of saline. On day 16, d-xylose was orally administrated to all pigs at the dose of 0.1 g/kg BW, 2 h after LPS or saline injection, and blood samples were collected 3 h thereafter. Twenty-four hours post-administration of LPS or saline, pigs were killed to obtain intestinal mucosae for analysis. Compared with the control group, LPS challenge reduced (P < 0.05) protein levels, the ratio of villus height to crypt depth, and the ratio of phosphorylated mTOR to total mTOR in duodenal, jejunal, and ileal mucosa. These adverse effects of LPS were attenuated (P < 0.05) by AKG supplementation. Moreover, AKG prevented the LPS-induced increase in intestinal HSP70 expression. Collectively, these novel results indicate that dietary supplementation with 1% AKG activates the mTOR signaling, alleviates the mucosal damage, and improves the absorptive function of the small intestine in LPS-challenged piglets. The findings not only help understand the mode of AKGs actions in the neonatal gut but also have important implications for infant nutrition under inflammatory conditions.

Keywords: α-Ketoglutarate; Intestine; mTOR signaling; Piglets; Lipopolysaccharide

Biochemical characteristics and inhibitor selectivity of mouse indoleamine 2,3-dioxygenase-2 by Christopher Jonathan Daraius Austin; B. M. Mailu; G. J. Maghzal; A. Sanchez-Perez; S. Rahlfs; K. Zocher; H. J. Yuasa; J. W. Arthur; K. Becker; R. Stocker; N. H. Hunt; H. J. Ball (pp. 565-578).

The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b ₅ as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b ₅, provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the “IDO family”. Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-d-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles.

Keywords: Cytochrome b ₅ ; Oxidation/reduction; Electron donation; IDO2

Arginine in the salt-induced peptide formation reaction: enantioselectivity facilitated by glycine, l- and d-histidine by Feng Li; Daniel Fitz; Donald G. Fraser; Bernd M. Rode (pp. 579-585).

The salt-induced peptide formation reaction has been proposed as a conceivable preliminary to the prebiotic evolution of peptides. In the present paper, the behaviour of arginine is reported for this reaction together with a discussion of the catalytic effects of glycine, and l- and d-histidine. Importantly, the behaviour of the two histidine enantiomers is different. Both histidine enantiomers perform better than glycine in enhancing the yields of arginine dipeptide with l-histidine being more effective than d-histidine. Yields in the presence of histidine are up to 70 times greater than for arginine solutions alone. This compares with 4.2 times higher in the presence of glycine. This difference is most pronounced in the most concentrated (containing 80 mM arginine) reaction solution where arginine has the lowest reactivity. A distinct preference for dimerisation of l-arginine also appears in the 80 mM cases for catalyses of other amino acids. This phenomenon is different from the behaviour of aliphatic amino acids, which display obvious inherent enantioselectivity for the l-stereomers in the SIPF reaction on their own rather than when catalysed by glycine or histidine.

Keywords: Homochirality; Arginine peptide formation; Copper complex; Chemical evolution

Amphiphilic molecular gels from ω-aminoalkylated l-glutamic acid derivatives with unique chiroptical properties by Yoshiko Kira; Yutaka Okazaki; Tsuyoshi Sawada; Makoto Takafuji; Hirotaka Ihara (pp. 587-597).

Self-assembling amphiphiles with unique chiroptical properties were derived from l-glutamic acid through ω-aminoalkylation and double long-chain alkylation. These amphiphiles can disperse in various solvents ranging from water to n-hexane. TEM and SEM observations indicate that the improvement in dispersity is induced by the formation of tubular and/or fibrillar aggregates with nanosized diameters, which makes these amphiphiles similar to aqueous lipid membrane systems. Spectroscopic observations, such as UV–visible and CD spectroscopies indicate that the aggregates are constructed on the basis of S- and R-chirally ordered structures through interamide interactions in water and organic media, respectively, and that these chiroptical properties can be controlled thermotropically and lyotropically. It is also reported that the chiral assemblies provide specific binding sites for achiral molecules and then induce chirality for the bonded molecules. Further, the applicability of the amphiphiles to template polymerization is discussed.

Keywords: Amino acid-derived amphiphilic lipid; Self-assembly; Organic nanotube; Chirally ordered structure; Induced chirality

Microwave-assisted reaction of glycosylamine with aspartic acid by Feliciana Real-Fernández; Francesca Nuti; M. Angeles Bonache; Marco Boccalini; Stefano Chimichi; Mario Chelli; Anna Maria Papini (pp. 599-604).

The synthesis of N-protected glycosyl amino acids from amines has been investigated and it was found that, under microwave conditions, glycosylamines could be hydrolyzed leading to new products containing a glycosyl ester linkage. The efficiency of the microwave-induced glycosylation of aspartic acid was studied comparing the microwave activity between amide and ester bond formation. Different sugar moieties have been employed to demonstrate the simple and reproducible coupling methodology. New glycosyl ester compounds were further characterized by NMR spectroscopy.

Keywords: Glycosylamino acids; Triazine-based coupling reagent; Microwave-assisted synthesis

Glycine oxidation and conversion into amino acids in Saccharomyces cerevisiae and Candida albicans by Nick E. Flynn; Michael E. Patyrak; Jeremiah B. Seely; Guoyao Wu (pp. 605-608).

Humans are exposed much more often to exogenous Saccharomyces cerevisiae (a baker’s yeast) than exogenous Candida albicans (a highly infectious yeast) but suffer no apparent complications from S. cerevisiae. We hypothesize that variations in characteristics between these two species may be due, in part, to differences in glycine metabolism. In this study, we examined differences in glycine oxidation between C. albicans and S. cerevisiae. Both C. albicans and S. cerevisiae were cultured in glycine enriched media, followed by determination of glycine oxidation and amino acid concentrations in cells. Glycine was degraded to a much greater extent in C. albicans than in S. cerevisiae. Threonine concentrations and glycine oxidation were also elevated in C. albicans. Almost all of the disappearance of glycine from incubation media was accounted for by the formation of serine, threonine, and CO₂ in S. cerevisiae, whereas these products represented only 50% of the metabolized glycine in C. albicans. The unidentified metabolites of glycine in C. albicans, presumably purines, could contribute to its infectious capacity and this warrants further study.

Keywords: Candida albicans ; Saccharomyces cerevisiae ; Glycine; Serine; Threonine; Metabolism

A diketoreductase exhibits unique renaturation profile from thermal-induced protein unfolding by Meiling Lu; Xu Cao; Xin Yang; Heng Zheng; Nan Liu; Yun Jiang; Donghai Lin; Yijun Chen (pp. 609-613).

Proteins can refold from thermal-induced denaturation. Apo-diketoreductase exhibited a unique refolding profile, in which the degree of refolding from higher temperature was more complete. Partial aggregation and structural change may provide possible explanation on this phenomenon.

Keywords: Diketoreductase; Refolding; Self-renaturation; Thermal induced; Unfolding

Erratum to: Glutamate is the chemotaxis-inducing factor in placental extracts by Rahul Gupta; Dhrubajyoti Chattopadhyay (pp. 615-615).

Erratum to: Involvement of individual hippocampal signaling protein levels in spatial memory formation is strain-dependent by Sudarshan S. Patil; Florentine Schlick; Harald Höger; Gert Lubec (pp. 617-618).

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.39, #2)