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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #3)

Antioxidative peptides: enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides by O. Power; P. Jakeman; R. J. FitzGerald (pp. 797-820).

The beneficial effects of food-derived antioxidants in health promotion and disease prevention are being increasingly recognized. Recently, there has been a particular focus on milk-derived peptides; as a source of antioxidants, these peptides are inactive within the sequence of the parent protein but can be released during enzyme hydrolysis. Once released, the peptides have been shown to possess radical scavenging, metal ion chelation properties and the ability to inhibit lipid peroxidation. A variety of methods have been used to evaluate in vitro antioxidant activity, however, there is no standardised methodology, which hinders comparison of data. This review provides an overview on the generation of antioxidative peptides from milk proteins, the proposed mechanisms of protein/peptide induced antioxidant activity, in vitro measurement of antioxidant activity, in vivo evaluation of plasma antioxidant capacity and the bioavailability of antioxidative peptides. The understanding gained from other food proteins is referred to where specific data on milk-derived peptides are limited. The potential applications and health benefits of antioxidant peptides are discussed with a particular focus on the aging population. The regulatory requirements for peptide-based antioxidant functional foods are also considered.

Keywords: Bioactive peptide; Antioxidant; Milk protein; Hydrolysate; Amino acids; Functional food

Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR by Yongchao Su; Shenhui Li; Mei Hong (pp. 821-833).

Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this review, we will summarize high-resolution structural and dynamic findings towards the understanding of the structure–activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane.

Keywords: Cationic membrane peptide; Cell-penetrating peptides; Antimicrobial peptides; Guanidinium-phosphate complex; Solid-state NMR

Short peptide constructs mimic agonist sites of AT₁R and BK receptors by Douglas D. Lopes; Renata F. F. Vieira; Luciana Malavolta; Erick F. Poletti; Suma I. Shimuta; Antonio C. M. Paiva; Shirley Schreier; Laerte Oliveira; Clovis R. Nakaie (pp. 835-846).

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT₁R and BKRB₁ or BKRB₂ G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S–S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac¹-AngII and Toac⁰-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac³-AngII and Toac³-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT₁R and BKRB cyclic constructs based on the structure of the CXCR₄, an α-chemokine GPCR-type receptor.

Keywords: Angiotensin II; Bradykinin; Receptor; Toac; Peptide–peptide interaction

Structure-based prediction of the effects of a missense variant on protein stability by Yang Yang; Biao Chen; Ge Tan; Mauno Vihinen; Bairong Shen (pp. 847-855).

Predicting the effects of amino acid substitutions on protein stability provides invaluable information for protein design, the assignment of biological function, and for understanding disease-associated variations. To understand the effects of substitutions, computational models are preferred to time-consuming and expensive experimental methods. Several methods have been proposed for this task including machine learning-based approaches. However, models trained using limited data have performance problems and many model parameters tend to be over-fitted. To decrease the number of model parameters and to improve the generalization potential, we calculated the amino acid contact energy change for point variations using a structure-based coarse-grained model. Based on the structural properties including contact energy (CE) and further physicochemical properties of the amino acids as input features, we developed two support vector machine classifiers. M47 predicted the stability of variant proteins with an accuracy of 87 % and a Matthews correlation coefficient of 0.68 for a large dataset of 1925 variants, whereas M8 performed better when a relatively small dataset of 388 variants was used for 20-fold cross-validation. The performance of the M47 classifier on all six tested contingency table evaluation parameters is better than that of existing machine learning-based models or energy function-based protein stability classifiers.

Keywords: Amino acid mutation; Physicochemical properties; Residue–residue contact energy; Support vector machine; Protein stability prediction

Acute creatine administration improves mitochondrial membrane potential and protects against pentylenetetrazol-induced seizures by Leonardo Magno Rambo; Leandro Rodrigo Ribeiro; Iuri Domingues Della-Pace; Daniel Neis Stamm; Rogério da Rosa Gerbatin; Marina Prigol; Simone Pinton; Cristina Wayne Nogueira; Ana Flávia Furian; Mauro Schneider Oliveira; Michele Rechia Fighera; Luiz Fernando Freire Royes (pp. 857-868).

A growing body of evidence indicates that creatine (Cr) exerts beneficial effects on a variety of pathologies where energy metabolism and oxidative stress play an etiological role. However, the benefits of Cr treatment for epileptics are still shrouded in controversy. In the present study, we found that acute Cr treatment (300 mg/kg, p.o.) prevented the increase in electroencephalographic wave amplitude typically elicited by PTZ (30, 45 or 60 mg/kg, i.p.). Cr treatment also increased the latency periods of first myoclonic jerks, lengthened the latency periods of the generalized tonic–clonic seizures and reduced the time spent in the generalized tonic–clonic seizures induced by PTZ (60 mg/kg). Administration of PTZ (all doses) decreased Na⁺, K⁺-ATPase activity as well as adenosine triphosphate (ATP) and adenosine diphosphate levels in the cerebral cortex, but Cr treatment prevented these effects. Cr administration also prevented increases in xanthine oxidase activity, adenosine monophosphate levels, adenosine levels, inosine levels and uric acid levels that normally occur after PTZ treatment (60 mg/kg, i.p.). We also showed that Cr treatment increased the total Cr (Cr + PCr) content, creatine kinase activity and the mitochondrial membrane potential (ΔΨ) in the cerebral cortex. In addition, Cr prevented PTZ-induced mitochondrial dysfunction characterized by decreasing ΔΨ, increasing thiobarbituric acid-reactive substance levels and increasing protein carbonylation. These experimental findings reinforce the idea that mitochondrial dysfunction plays a critical role in models of epileptic seizures and suggest that buffering brain energy levels through Cr treatment may be a promising therapeutic approach for the treatment of this neurological disease.

Keywords: Creatine; Pentylenetetrazol; Na⁺, K⁺-ATPase; Mitochondrial membrane potential; Purine catabolism

Modulation of ethanol effect on hepatocyte proliferation by polyamines by T. H. T. Do; F. Gaboriau; I. Morel; S. Lepage; I. Cannie; O. Loréal; G. Lescoat (pp. 869-877).

An occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol.

Keywords: Ethanol; Hepatocyte; Cell proliferation; Polyamine metabolism; Α-difluoromethyl ornithine

Urocortin 1 administered into the hypothalamic supraoptic nucleus inhibits food intake in freely fed and food-deprived rats by A. Fatima; S. Andrabi; G. Wolf; M. Engelmann; M. G. Spina (pp. 879-885).

Peptides of the corticotropin-releasing hormone/Urocortin (CRH/Ucn) family are known to suppress appetite primarily via CRH₂ receptors. In the rat hypothalamic supraoptic nucleus (SON), synthesis of both Ucn1 and CRH₂ receptors has been reported, yet little is known about the effects of Ucn1 in the SON on feeding behaviour. We first established the dose-related effects of Ucn1 injected into the SON on the feeding response in both freely fed and 24-h food-deprived rats. A conditioned taste avoidance paradigm was performed to investigate possible generalised effects of local Ucn1 treatment. Administration of Ucn1 into the SON at doses equal to or higher than 0.5 μg significantly decreased food intake in both freely fed and food-deprived rats. The Ucn1-mediated suppression of food intake was delayed in freely fed as compared to food-deprived animals. Conditioning for taste aversion to saccharine appeared at 0.5 and 1 μg of Ucn1. Both the early and the delayed onset of anorexia observed after intra-SON injection of Ucn1 under fasting and fed conditions, respectively, suggest the possible involvement of different CRH receptor subtypes in the two conditions, while the conditioned taste aversion seems to be responsible for the initial latency to eat the first meal in these animals.

Keywords: Urocortin 1; Corticotropin-releasing hormone 2 receptor; Feeding microstructure; Conditioned taste aversion paradigm

An empirical study on the matrix-based protein representations and their combination with sequence-based approaches by Loris Nanni; Alessandra Lumini; Sheryl Brahnam (pp. 887-901).

Many domains have a stake in the development of reliable systems for automatic protein classification. Of particular interest in recent studies of automatic protein classification is the exploration of new methods for extracting features from a protein that enhance classification for specific problems. These methods have proven very useful in one or two domains, but they have failed to generalize well across several domains (i.e. classification problems). In this paper, we evaluate several feature extraction approaches for representing proteins with the aim of sequence-based protein classification. Several protein representations are evaluated, those starting from: the position specific scoring matrix (PSSM) of the proteins; the amino-acid sequence; a matrix representation of the protein, of dimension (length of the protein) ×20, obtained using the substitution matrices for representing each amino-acid as a vector. A valuable result is that a texture descriptor can be extracted from the PSSM protein representation which improves the performance of standard descriptors based on the PSSM representation. Experimentally, we develop our systems by comparing several protein descriptors on nine different datasets. Each descriptor is used to train a support vector machine (SVM) or an ensemble of SVM. Although different stand-alone descriptors work well on some datasets (but not on others), we have discovered that fusion among classifiers trained using different descriptors obtains a good performance across all the tested datasets. Matlab code/Datasets used in the proposed paper are available at http://www.bias.csr.unibo.it anniPSSM.rar.

Keywords: Proteins classification; Machine learning; Ensemble of classifiers; Support vector machines

The influence of exogenous carbohydrate provision and pre-exercise alkalosis on the heat shock protein response to prolonged interval cycling by Daniel J. Peart; Richard J. Kirk; Leigh A. Madden; Jason C. Siegler; Rebecca V. Vince (pp. 903-910).

The aim of this study was to observe the intracellular heat shock protein 72 (HSP72) and heme oxygenase-1 (HSP32) response to prolonged interval cycling following the ingestion of carbohydrates (CHO) and sodium bicarbonate (NaHCO₃). Six recreationally active males (mean ± SD; age 23.2 ± 2.9 years, height 179.5 ± 5.5 cm, body mass 76.5 ± 6.8 kg, and peak power output 315 ± 36 W) volunteered to complete a 90 min interval cycling exercise on four occasions. The trials were completed in a random and blinded manner following ingestion of either: placebo and an artificial sweetener (P–P), NaHCO₃ and sweetener (B–P), placebo and CHO (P–CHO), and NaHCO₃ and CHO (B–CHO). Both HSP72 and HSP32 were significantly increased in monocytes and lymphocytes from 45 min post-exercise (p ≤ 0.039), with strong relationships between both cell types (HSP72, r = 0.83; HSP32, r = 0.89). Exogenous CHO had no influence on either HSP72 or HSP32, but the ingestion of NaHCO₃ significantly attenuated HSP32 in monocytes and lymphocytes (p ≤ 0.042). In conclusion, the intracellular stress protein response to 90 min interval exercise is closely related in monocytes and lymphocytes, and HSP32 appears to be attenuated with a pre-exercise alkalosis.

Keywords: Bicarbonate; Stress protein; Oxidative stress; Carbohydrates

Dietary supplementation with monosodium glutamate is safe and improves growth performance in postweaning pigs by Reza Rezaei; Darrell A. Knabe; Carmen D. Tekwe; Sudath Dahanayaka; Martin D. Ficken; Susan E. Fielder; Sarah J. Eide; Sandra L. Lovering; Guoyao Wu (pp. 911-923).

Dietary intake of glutamate by postweaning pigs is markedly reduced due to low feed consumption. This study was conducted to determine the safety and efficacy of dietary supplementation with monosodium glutamate (MSG) in postweaning pigs. Piglets were weaned at 21 days of age to a corn and soybean meal-based diet supplemented with 0, 0.5, 1, 2, and 4 % MSG (n = 25/group). MSG was added to the basal diet at the expense of cornstarch. At 42 days of age (21 days after weaning), blood samples (10 mL) were obtained from the jugular vein of 25 pigs/group at 1 and 4 h after feeding for hematological and clinical chemistry tests; thereafter, pigs (n = 6/group) were euthanized to obtain tissues for histopathological examinations. Feed intake was not affected by dietary supplementation with 0–2 % MSG and was 15 % lower in pigs supplemented with 4 % MSG compared with the 0 % MSG group. Compared with the control, dietary supplementation with 1, 2 and 4 % MSG dose-dependently increased plasma concentrations of glutamate, glutamine, and other amino acids (including lysine, methionine, phenylalanine and leucine), daily weight gain, and feed efficiency in postweaning pigs. At day 7 postweaning, dietary supplementation with 1–4 % MSG also increased jejunal villus height, DNA content, and antioxidative capacity. The MSG supplementation dose-dependently reduced the incidence of diarrhea during the first week after weaning. All variables in standard hematology and clinical chemistry tests, as well as gross and microscopic structures, did not differ among the five groups of pigs. These results indicate that dietary supplementation with up to 4 % MSG is safe and improves growth performance in postweaning pigs.

Keywords: Monosodium glutamate; Piglet; Growth; Safety; Efficacy

Glycine reduces platelet aggregation by Peter Schemmer; Zhi Zhong; Uwe Galli; Michael D. Wheeler; Li Xiangli; Blair U. Bradford; Lars O. Conzelmann; Dow Forman; José Boyer; Ronald G. Thurman (pp. 925-931).

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl⁻ influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1–10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.

Keywords: ADP; Collagen; [Ca²⁺]_i ; Glycine receptor

Synthesis, characterization, conformational analysis of a cyclic conjugated octreotate peptide and biological evaluation of ^99mTc-HYNIC-His³-Octreotate as novel tracer for the imaging of somatostatin receptor-positive tumors by Ashok Behera; Indranil Banerjee; Kakali De; Rudra Narayan Munda; Sankha Chattopadhayay; Amalesh Samanta; Bharat Sarkar; Santanu Ganguly; Mridula Misra (pp. 933-946).

Peptides are attracting increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. We therefore synthesized new cyclic octapeptides conjugated with HYNIC by Fmoc solid-phase peptide synthesis. These were purified and analyzed by RP-HPLC, MALDI mass, ¹H NMR, ¹³C NMR, HSQC, HMBC, COSY and IR spectroscopy. Conformational analysis of the peptides was performed by circular dichroism spectroscopy, in pure water and trifluoroethanol–water (1:1), revealed the presence of strong secondary structural features like β-sheet and random coils. Labeling was performed with ^99mTc using Tricine and EDDA as coligands by SnCl₂ method to get products with excellent radiochemical purity >99.5 %. Metabolic stability analysis did not show any evidence of breaking of the labeled compounds and formation of free ^99mTc. Internalization studies were done and IC₅₀ values were determined in somatostatin receptor-expressing C6 glioma cell line and rat brain cortex membrane, and the results compared with HYNIC-TOC as standard. The IC₅₀ values of ^99mTc-HYNIC-His³-Octreotate (21 ± 0.93 nM) and ^99mTc-HYNIC-TOC (2.87 ± 0.41 nM) proved to be comparable. Biodistribution and image study on normal rat under gamma camera showed very high uptake in kidney and urine, indicating kidney as primary organ for metabolism and route of excretion. Biodistribution and image study on rats bearing C6 glioma tumor found high uptake in tumor (1.27 ± 0.15) and pancreas (1.71 ± 0.03). Using these findings, new derivatives can be prepared to develop ^99mTc radiopharmaceuticals for imaging somatostatin receptor-positive tumors.

Keywords: Solid-phase peptide synthesis; SSTR; Binding affinity; C6 glioma cell; Internalization study

PEGylated dendrimer polystyrene support: synthesis, characterisation and evaluation of biologically active peptides by M. A. Siyad; G. S. Vinod Kumar (pp. 947-959).

Poly(N,N-bisethylamine) dendrimers with high content of poly(ethylene glycol) were synthesized on 3-(Acryloyloxy)-2-hydroxypropylmethacrylate-crosslinked polystyrene (PS-AHMA) resin and tested in various conditions of solid phase peptide synthesis. The dendritic templates were generated to the second generation on cross-linker active site of 3-(Acryloyloxy)-2-hydroxypropylmethacrylate (AHMA). First generation dendrimer was designed by series of four-stage reactions, such as Schiff base incorporation, acidolytic cleavage, diazotization and thionyl chloride treatment and same synthetic routes were followed for second generation also. Poly(ethylene glycol) (PEG1000) has been grafted to second-generation dendrimer and used to check various physico-chemical parameters in Fmoc/Boc peptide synthetic conditions. The utility of PEGylated dendrimer support was demonstrated by synthesizing biologically potent linear as well as disulfide-bonded peptide by Fmoc method.

Keywords: Dendrimer; Solid phase synthesis; Poly(ethylene glycol)

Vaspin attenuates the apoptosis of human osteoblasts through ERK signaling pathway by Xiao Zhu; Yi Jiang; Peng-Fei Shan; Jie Shen; Qiu-Hua Liang; Rong-Rong Cui; Yuan Liu; Guan-Ying Liu; Shan-Shan Wu; Qiong Lu; Hui Xie; You-Shuo Liu; Ling-Qing Yuan; Er-Yuan Liao (pp. 961-968).

It has been hypothesized that adipocytokines originating from adipose tissue may have an important role in bone metabolism. Vaspin is a novel adipocytokine isolated from visceral white adipose tissue, which has been reported to have anti-apoptotic effects in vascular endothelial cells. However, to the best of our knowledge there is no information regarding the effects of vaspin on osteoblast apoptosis. This study therefore examined the possible effects of vaspin on apoptosis in human osteoblasts (hOBs). Our study established that vaspin inhibits hOBs apoptosis induced by serum deprivation, as determined by ELISA and TUNEL assays. Western blot analysis revealed that vaspin upregulates the expression of Bcl-2 and downregulates that of Bax in a dose-dependent manner. Vaspin stimulated the phosphorylation of ERK, and pretreatment of hOBs with the ERK inhibitor PD98059 blocked the vaspin-induced activation of ERK, however, vaspin did not stimulate the phosphorylation of p38, JNK or Akt. Vaspin protects hOBs from serum deprivation-induced apoptosis, which may be mediated by activating the MAPK/ERK signaling pathway.

Keywords: Vaspin; Osteoblast; Apoptosis; Extracellular signal-regulated kinase

Combination of BCAAs and glutamine enhances dermal collagen protein synthesis in protein-malnourished rats by Hitoshi Murakami; Kazutaka Shimbo; Yoshinobu Takino; Hisamine Kobayashi (pp. 969-976).

Skin collagen decreases in protein-malnourished states. Amino acids regulate protein metabolism, glutamine stimulates collagen synthesis through the conversion process to proline and provides 75 % of the intracellular free proline in fibroblasts. However, the impact of these amino acids on collagen synthesis under malnutrition has not been examined. We investigated the effect of amino acids on dermal tropocollagen synthesis in protein-malnourished rats. The fractional synthesis rate (FSR, %/h) of dermal tropocollagen was evaluated by the incorporation of l-[ring-²H₅]-phenylalanine after 4 h infusion of each amino acid and the stable isotope. None of the infused 12 single amino acids (glutamine, proline, alanine, arginine, glutamate, glycine, aspartate, serine, histidine, lysine, phenylalanine and threonine) significantly increased the FSR (P = 0.343, one-way ANOVA). In contrast, amino acid mixtures of essential amino acids + glutamine + arginine (EAARQ) and branched-chain amino acids + glutamine (BCAAQ) significantly increased the FSR compared to saline, but the branched-chain amino acids (BCAAs) and amino acid mixture of collagen protein (AAC) did not alter the FSR (saline, 0.96 ± 0.24 %/h; EAARQ, 1.76 ± 0.89 %/h; BCAAQ 1.71 ± 0.36 %/h; BCAAs, 1.08 ± 0.20 %/h and AAC 1.39 ± 0.35 %/h, P < 0.05, Tukey’s test). Proline conversion from glutamine represented only 3.9 % of the free proline in skin, as evaluated by the primed-constant infusion of l-d7-proline and l-α-15N-glutamine in rats. These results suggested that the combination of BCAAQ is a key factor for the enhancement of skin collagen synthesis in protein-malnourished rats. The contribution of extracellular free glutamine on de novo proline synthesis and collagen synthesis is very low in vivo compared to the contribution in vitro.

Keywords: Amino acids; Skin collagen synthesis; Glutamine and proline metabolism; Protein-malnourished rat

Synthesis of gem-diamino acid derivatives by a Hofmann rearrangement by Emanuele Aresu; Stefania Fioravanti; Simona Gasbarri; Lucio Pellacani; Federico Ramadori (pp. 977-982).

Starting from commercially available N-protected l-α-amino acids, N,N′-protected gem-diaminic units were obtained by a two-step methodology. A Hofmann reaction performed using a primary alcohol as the solvent to trap the isocyanate intermediate represents the key step of the new synthetic procedure. Then, the methodology was applied to α-carbamoyl α′-carboxyl aziridines, also functionalized with l-α-amino esters and stable gem-diaminic units characterized by an aziridine ring and by a retro-peptide modification were obtained. The use of the latter units in the retro-peptide chemistry allows to obtain modified peptides containing an aziridine ring able to behave as an electrophilic site and as a biomimetic structural analog of proline.

Keywords: Aminals; Amino acids; Aziridines; Peptidomimetics

Acute tryptophan depletion does not improve endurance cycling capacity in a warm environment by Ruth M. Hobson; Phillip Watson; Ronald J. Maughan (pp. 983-991).

Newsholme’s theory of central fatigue suggests that acute tryptophan depletion should improve endurance exercise capacity in a warm environment by reducing serotonergic activity in the brain. Eight males cycled to volitional exhaustion at 55 % $$ dot{V}{ m O}_{2} $$ peak in 30.1 ± 0.5 °C and 30 ± 7 % relative humidity on two separate occasions, after consuming either an amino acid load to deplete their circulating tryptophan concentration (TD), or a control amino acid load (CON). Blood samples were taken before ingesting the amino acids, before the start of exercise, every 15 min during exercise and at the point of exhaustion. Heart rate (HR), core (Tc) and skin (Tsk) temperatures and ratings of perceived exertion (RPE) and thermal comfort (TC) were also monitored every 10 min during exercise. Plasma tryptophan (P = 0.003) and free tryptophan (P < 0.001) concentrations, and the free tryptophan to branched-chain amino acid ratio (P = 0.004) were all lower on the TD trial than on the CON trial. There was no difference in endurance exercise capacity (TD 99.2 ± 24.4 min as compared to CON 108.4 ± 21.6 min; P = 0.088). There was a tendency for HR (P = 0.053) and Tc (P = 0.069) to be higher on the TD trials. There were no differences for any of the other parameters. Endurance cycling capacity in a warm environment is not improved by acute tryptophan depletion, suggesting tryptophan availability is not a significant factor in the development of fatigue in such situations.

Keywords: Serotonin; Exercise capacity; Fatigue; Core temperature; Heart rate

Preference toward a polylysine enantiomer in inhibiting prions by Karen S. Jackson; Jihyun Yeom; Youngmi Han; Younsoo Bae; Chongsuk Ryou (pp. 993-1000).

Differential anti-prion activity of polylysine enantiomers was studied. Based on our recent discovery that poly-l-lysine (PLK) is a potent anti-prion agent, we investigated suppression of prions in cultured cells using poly-d-lysine (PDK). The results showed that PDK was more efficacious than PLK to inhibit prions. Protein misfolding cyclic amplification assay demonstrated improved efficacy of PDK in inhibiting plasminogen-mediated prion propagation, corresponding to the enantio-preference of PDK observed in cultured cells. Furthermore, our study demonstrated that polylysines formed a complex with plasminogen. These results propose to hypothesize a plausible mechanism that elicits prion inhibition by polylysine enantiomers.

Keywords: Polylysine; Enantiomers; Prion; Inhibition; Plasminogen

Inflammatory serum proteome pattern in mice fed a high-fat diet by Elisabetta Gianazza; Cristina Sensi; Ivano Eberini; Federica Gilardi; Marco Giudici; Maurizio Crestani (pp. 1001-1008).

To investigate the influence of diet on serum protein pattern, mice were fed for 8 weeks either control chow or a high-fat diet (containing 21 % w/w milk fat and 0.2 % w/w cholesterol); sera were collected and analyzed by 2-DE. The main positive acute-phase reactant proteins, haptoglobin and hemopexin, were significantly up-regulated in animals receiving the high-fat diet. Data on all other proteins also pointed to an inflammatory condition in these animals. The largest change in concentration was observed for carboxylesterase N, a circulating enzyme seldom connected with lipid metabolism in earlier reports. These observations agree with the notion of a link between diet-induced hyperlipidemia and the inflammatory component of its cardiovascular sequels in humans, but the effects in the experimental animals are massive and obviously affect most of the major serum proteins.

Keywords: Acute-phase reaction; Acute-phase reactant; Carboxylesterase; High-fat diet; Mouse

Triple mutated antibody scFv2F3 with high GPx activity: insights from MD, docking, MDFE, and MM-PBSA simulation by Quan Luo; Chunqiu Zhang; Lu Miao; Dongmei Zhang; Yushi Bai; Chunxi Hou; Junqiu Liu; Fei Yan; Ying Mu; Guimin Luo (pp. 1009-1019).

By combining computational design and site-directed mutagenesis, we have engineered a new catalytic ability into the antibody scFv2F3 by installing a catalytic triad (Trp²⁹–Sec⁵²–Gln⁷²). The resulting abzyme, Se-scFv2F3, exhibits a high glutathione peroxidase (GPx) activity, approaching the native enzyme activity. Activity assays and a systematic computational study were performed to investigate the effect of successive replacement of residues at positions 29, 52, and 72. The results revealed that an active site Ser⁵²/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe²⁹/Trp–Val⁷²/Gln mutations enhance the reaction rate via functional cooperation with Sec⁵². Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr¹⁰¹ occurs mainly in the loop of complementarity determining region 3. The docking studies illustrated the importance of this loop that favors the conformational shift of Tyr⁵⁴, Asn⁵⁵, and Gly⁵⁶ to stabilize substrate binding. Molecular dynamics free energy and molecular mechanics-Poisson Boltzmann surface area calculations estimated the pK _a shifts of the catalytic residue and the binding free energies of docked complexes, suggesting that dipole–dipole interactions among Trp²⁹–Sec⁵²–Gln⁷² lead to the change of free energy that promotes the residual catalytic activity and the substrate-binding capacity. The calculated results agree well with the experimental data, which should help to clarify why Se-scFv2F3 exhibits high catalytic efficiency.

Keywords: Abzyme; GPx mimic; Docking; MD simulation; MDFE simulation; MM-PBSA

Chiral bio-nanocomposites based on thermally stable poly(amide-imide) having phenylalanine linkages and reactive organoclay containing tyrosine amino acid by Shadpour Mallakpour; Mohammad Dinari (pp. 1021-1029).

Montmorillonite clay modified with the bio-active trifunctional l-tyrosine amino acid salt was used as a reactive organoclay (OC) for the preparation of poly(amide-imide) (PAI)/OC hybrid films. One of the functional groups of the l-tyrosine as the swelling agent formed an ionic bond with the negatively charged silicates, whereas the remaining functional groups were available for further reaction with polymer matrix. The soluble PAI with amine end groups including phenylalanine amino acid was synthesised under green condition using molten tetra-butylammonium bromide by direct polymerization reaction of chiral diacid and 2-(3,5-diaminophenyl)benzimidazole. PAI/OC bio-nanocomposites films containing different contents of OC were prepared via solution intercalation method through blending of OC with the PAI solution. X-ray diffraction and transmission electron microscopy revealed that the dispersion of silicate layers in the PAI created an exfoliated structure as a result of using the trifunctional groups of the swelling agent. The structure and thermal behavior of the synthesised materials were characterized by a range of methods, including X-ray diffraction, Fourier transform infrared spectroscopy, ¹H-NMR, electron microscopy, elemental and thermogravimetric analysis techniques. Thermogravimetric analysis results indicated that the addition of OC into the PAI matrix was increased in the thermal decomposition temperatures of the resulted bio-nanocomposites.

Keywords: Bio-nanocomposites; α-Amino acids; Layered silicate; Poly(amide-imide); l-Tyrosine; Green chemistry

Biocatalysts for multicomponent Biginelli reaction: bovine serum albumin triggered waste-free synthesis of 3,4-dihydropyrimidin-2-(1H)-ones by Upendra K. Sharma; Nandini Sharma; Rajesh Kumar; Arun K. Sinha (pp. 1031-1037).

Bovine serum albumin (BSA) promoted simple and efficient one-pot procedure was developed for the direct synthesis of 3,4-dihydropyrimidin-2(1H)-ones including potent mitotic kinesin Eg5 inhibitor monastrol under mild reaction conditions. The catalyst recyclability and gram scale synthesis have also been demonstrated to enhance the practical utility of process.

Keywords: Biginelli condensation; Bovine Serum Albumin; Biocatalysis; Sustainable Chemistry

Fast mass spectrometry-based enantiomeric excess determination of proteinogenic amino acids by Heidi Fleischer; Kerstin Thurow (pp. 1039-1051).

A rapid determination of the enantiomeric excess of proteinogenic amino acids is of great importance in various fields of chemical and biologic research and industries. Owing to their different biologic effects, enantiomers are interesting research subjects in drug development for the design of new and more efficient pharmaceuticals. Usually, the enantiomeric composition of amino acids is determined by conventional analytical methods such as liquid or gas chromatography or capillary electrophoresis. These analytical techniques do not fulfill the requirements of high-throughput screening due to their relative long analysis times. The method presented allows a fast analysis of chiral amino acids without previous time consuming chromatographic separation. The analytical measurements base on parallel kinetic resolution with pseudoenantiomeric mass tagged auxiliaries and were carried out by mass spectrometry with electrospray ionization. All 19 chiral proteinogenic amino acids were tested and Pro, Ser, Trp, His, and Glu were selected as model substrates for verification measurements. The enantiomeric excesses of amino acids with non-polar and aliphatic side chains as well as Trp and Phe (aromatic side chains) were determined with maximum deviations of the expected value less than or equal to 10ee%. Ser, Cys, His, Glu, and Asp were determined with deviations lower or equal to 14ee% and the enantiomeric excess of Tyr were calculated with 17ee% deviation. The total screening process is fully automated from the sample pretreatment to the data processing. The method presented enables fast measurement times about 1.38 min per sample and is applicable in the scope of high-throughput screenings.

Keywords: Amino acids; Enantiomeric excess; Laboratory automation; Parallel kinetic resolution; Mass spectrometry

Pressor response to l-cysteine injected into the cisterna magna of conscious rats involves recruitment of hypothalamic vasopressinergic neurons by Yumi Takemoto (pp. 1053-1060).

The sulfur-containing non-essential amino acid l-cysteine injected into the cisterna magna of adult conscious rats produces an increase in blood pressure. The present study examined if the pressor response to l-cysteine is stereospecific and involves recruitment of hypothalamic vasopressinergic neurons and medullary noradrenergic A1 neurons. Intracisternally injected d-cysteine produced no cardiovascular changes, while l-cysteine produced hypertension and tachycardia in freely moving rats, indicating the stereospecific hemodynamic actions of l-cysteine via the brain. The double labeling immunohistochemistry combined with c-Fos detection as a marker of neuronal activation revealed significantly higher numbers of c-Fos-positive vasopressinergic neurons both in the supraoptic and paraventricular nuclei and tyrosine hydroxylase containing medullary A1 neurons, of l-cysteine-injected rats than those injected with d-cysteine as iso-osmotic control. The results indicate that the cardiovascular responses to intracisternal injection of l-cysteine in the conscious rat are stereospecific and include recruitment of hypothalamic vasopressinergic neurons both in the supraoptic and paraventricular nuclei, as well as of medullary A1 neurons. The findings may suggest a potential function of l-cysteine as an extracellular signal such as neuromodulators in central regulation of blood pressure.

Keywords: l-Cysteine; Blood pressure; Vasopressinergic neurons; Hypothalamus; A1 neurons; c-Fos

A cysteine in the repetitive domain of a high-molecular-weight glutenin subunit interferes with the mixing properties of wheat dough by Xin Gao; Qisen Zhang; Marcus P. Newberry; Ken J. Chalmers; Diane E. Mather (pp. 1061-1071).

The quality of wheat (Triticum aestivum L.) for making bread is largely due to the strength and extensibility of wheat dough, which in turn is due to the properties of polymeric glutenin. Polymeric glutenin consists of high- and low-molecular-weight glutenin protein subunits linked by disulphide bonds between cysteine residues. Glutenin subunits differ in their effects on dough mixing properties. The research presented here investigated the effect of a specific, recently discovered, glutenin subunit on dough mixing properties. This subunit, Bx7.1, is unusual in that it has a cysteine in its repetitive domain. With site-directed mutagenesis of the gene encoding Bx7.1, a guanine in the repetitive domain was replaced by an adenine, to provide a mutant gene encoding a subunit (MutBx7.1) in which the repetitive-domain cysteine was replaced by a tyrosine residue. Bx7.1, MutBx7.1 and other Bx-type glutenin subunits were heterologously expressed in Escherichia coli and purified. This made it possible to incorporate each individual subunit into wheat flour and evaluate the effect of the cysteine residue on dough properties. The Bx7.1 subunit affected dough mixing properties differently from the other subunits. These differences are due to the extra cysteine residue, which may interfere with glutenin polymerisation through cross-linkage within the Bx7.1 subunit, causing this subunit to act as a chain terminator.

Keywords: Cysteine; Dough properties; Glutenin polymerisation; High-molecular-weight glutenin; Site-directed mutagenesis; Wheat

Catalytic mechanism of serine racemase from Dictyostelium discoideum by Tomokazu Ito; Motoki Maekawa; Shuhei Hayashi; Masaru Goto; Hisashi Hemmi; Tohru Yoshimura (pp. 1073-1084).

The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of d- and l-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to l-serine dehydrase; S81A showed no racemase activity and had significantly reduced d-serine dehydrase activity, but it completely retained its l-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove d-serine dehydration by abstracting the α-hydrogen in d-serine. Our data suggest that the abstraction and addition of α-hydrogen to l- and d-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.

Keywords: d-serine; Serine racemase; Dictyostelium discoideum ; Pyridoxal 5′-phosphate

One-pot efficient synthesis of N ^α-urethane-protected β- and γ-amino acids by Marta Cal; Mariusz Jaremko; Łukasz Jaremko; Piotr Stefanowicz (pp. 1085-1091).

1-[(4-Methylphenyl)oxy]pyrrolidine-2,5-dione and 1-[(4-methylphenyl)oxy]piperidine-2,6-dione react in a Lossen-type reaction with primary alcohols in the presence of triethylamine to furnish corresponding N ^α-urethane-protected β-alanine and γ-aminopropionic acid (GABA), respectively, with excellent yields and purities, in an essentially “one-pot” procedure.

Keywords: N-Hydroxyimides; Lossen rearrangement; β- and γ-Amino acids; N ^α-Urethane-protection; CBz; GABA; β-Alanine; Anthranilic acid

Is mTOR involved in the mechanisms of the fast-acting antidepressant effects of AMPA receptor agonists? by Yimin Hu; Xiangliu Liu; Jinchun Shen (pp. 1093-1093).

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #3)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.44, #3)