Amino Acids (v.38, #3)
Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region by M. Carbonaro; A. Nucara (679-690).
Fourier transform spectroscopy in the mid-infrared (400–5,000 cm−1) (FT-IR) is being recognized as a powerful tool for analyzing chemical composition of food, with special concern to molecular architecture of food proteins. Unlike other spectroscopic techniques, it provides high-quality spectra with very small amount of protein, in various environments irrespective of the molecular mass. The fraction of peptide bonds in α-helical, β-pleated sheet, turns and aperiodic conformations can be accurately estimated by analysis of the amide I band (1,600–1,700 cm−1) in the mid-IR region. In addition, FT-IR measurement of secondary structure highlights the mechanism of protein aggregation and stability, making this technique of strategic importance in the food proteomic field. Examples of applications of FT-IR spectroscopy in the study of structural features of food proteins critical of nutritional and technological performance are discussed.
Keywords: FT-IR; Infrared spectroscopy; Food proteins; Secondary structure
A new non-natural arginine-like amino acid derivative with a sulfamoyl group in the side-chain by Rosaria De Marco; Maria L. Di Gioia; Antonella Leggio; Angelo Liguori; Francesca Perri; Carlo Siciliano; Maria C. Viscomi (691-700).
Sulfamoylation of the l-ornithine methyl ester side-chain generates a non-natural arginine isostere which can be coupled with N-Fmoc-l-proline to synthesize analogues which maintain the structural characteristics of the biologically important Pro-Arg dipeptide sequence. As a probe of its biological importance, the sulfamoylated amino acid derivative was also incorporated as P1 residue in tripeptide structures matching the C-terminal subsequence of fibrinogen. The reported results demonstrate that the functionalization of l-ornithine side-chain with a neutral sulfamoyl group can generate an arginine bioisostere which can be used for the synthesis of prototypes of a new class of human thrombin inhibitors.
Keywords: Ornithine; Sulfamoyl group; Arginine bioisosteres; Tripeptides; Thrombin inhibitors; Anticoagulants
Pseudo-peptides derived from isomannide: inhibitors of serine proteases by Thalita G. Barros; Sergio Pinheiro; J. S. Williamson; Amílcar Tanuri; M. Gomes Jr; Helena S. Pereira; R. M. Brindeiro; José B. A. Neto; O. A. C. Antunes; Estela M. F. Muri (701-709).
In this paper, we describe the synthesis of a novel class of pseudo-peptides derived from isomannide and several oxazolones as potential inhibitors of serine proteases as well as preliminary pharmacological assays for hepatitis C. Hepatitis C, dengue and West Nile fever are among the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes and are a primary target in the drug development field. Several pseudo-peptides were obtained in good yields from the reaction of isomannide and oxazolones, and their anti-HCV potential using the HCV replicon-based assay was shown.
Keywords: Hepatitis C; Dengue; Serine protease; Isomannide; Oxazolones
Voltammetric investigation on interaction of protein with chromotrope 2R and its analytical application by Ni Hui; Xue-Liang Niu; Jun-Ying Han; Wei Sun; Kui Jiao (711-719).
The electrochemical behaviors of the interaction of chromotrope 2R (CH2R) with human serum albumin (HSA) are investigated on the hanging mercury drop electrode with linear sweep voltammetry. In the acidic buffer solution (pH 2.5) CH2R has a well-defined voltammetric reductive wave at −0.34 V (SCE). On the addition of HSA into the CH2R solution, the reductive peak current of CH2R decreases with little movement of the peak potential. The voltammetric study shows that the electrochemical parameters of interaction solution do not change and a new electrochemically non-active complex is formed via interaction of CH2R with HSA, which cannot be reduced on the Hg electrode and results in the decrease of the free concentration of CH2R. The decrease of reductive peak current is proportional to HSA concentration and further used for protein detection. The binding ratio and the binding constant are further calculated with the experimental voltammetric data.
Keywords: Chromotrope 2R; Human serum albumin; Linear sweep voltammetry; Interaction; Protein
Prediction of protein structural class using a complexity-based distance measure by Taigang Liu; Xiaoqi Zheng; Jun Wang (721-728).
Knowledge of structural class plays an important role in understanding protein folding patterns. So it is necessary to develop effective and reliable computational methods for prediction of protein structural class. To this end, we present a new method called NN-CDM, a nearest neighbor classifier with a complexity-based distance measure. Instead of extracting features from protein sequences as done previously, distance between each pair of protein sequences is directly evaluated by a complexity measure of symbol sequences. Then the nearest neighbor classifier is adopted as the predictive engine. To verify the performance of this method, jackknife cross-validation tests are performed on several benchmark datasets. Results show that our approach achieves a high prediction accuracy over some classical methods.
Keywords: Symbol sequence complexity; Nearest neighbor algorithm; Jackknife cross-validation test; Performance measure
Identification of flooding stress responsible cascades in root and hypocotyl of soybean using proteome analysis by Setsuko Komatsu; Tetsuya Sugimoto; Tomoki Hoshino; Yohei Nanjo; Kiyoshi Furukawa (729-738).
Flooding inducible proteins were analyzed using a proteomic technique to understand the mechanism of soybean response to immersion in water. Soybeans were germinated for 2 days, and then subjected to flooding for 2 days. Proteins were extracted from root and hypocotyl, separated by two-dimensional polyacrylamide gel electrophoresis, stained by Coomassie brilliant blue, and analyzed by protein sequencing and mass spectrometry. Out of 803 proteins, 21 proteins were significantly up-regulated, and seven proteins were down-regulated by flooding stress. Of the total, 11 up-regulated proteins were classified as related to protein destination/storage and three proteins to energy, while four down-regulated proteins were related to protein destination/storage and three proteins to disease/defense. The expression of 22 proteins significantly changed within 1 day after flooding stress. The effects of flooding, nitrogen substitution without flooding, or flooding with aeration were analyzed for 1–4 days. The expression of alcohol dehydrogenase increased remarkably by nitrogen substitution compared to flooding. The expression of many proteins that changed due to flooding showed the same tendencies observed for nitrogen substitution; however, the expression of proteins classified into protein destination/storage did not.
Keywords: Flooding; Proteome; Soybean; Hypoxic response
Modulation of taurine release in ischemia by glutamate receptors in mouse brain stem slices by Pirjo Saransaari; Simo S. Oja (739-746).
Glutamate is the main excitatory transmitter in the brain stem, regulating many vital sensory and visceral processes. Taurine is inhibitory and functions as a neuromodulator and regulator of cell volumes in the brain, being especially important in the developing brain. Taurine release is markedly enhanced under ischemic conditions in many brain areas, providing protection against excitotoxicity. The involvement of glutamate receptors in the release of preloaded [3H]taurine was now characterized under ischemic conditions in slices prepared from the mouse brain stem from developing (7-day-old) and young adult (3-month-old) mice. The ionotropic glutamate receptor agonists N-methyl-d-aspartate, kainate, and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate had no effect on ischemic taurine release in the immature brain stem, whereas in adults the release was enhanced in a receptor-mediated manner. The metabotropic receptor agonists of group I, (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate and (S)-3,5-dihydroxyphenylglycine, potentiated both basal and K+-stimulated release in both age groups. The group III agonist l(+)-2-amino-4-phosphonobutyrate also enhanced the release. In both cases the effects were receptor-mediated, being reduced by the respective antagonists. The results show that activation of glutamate receptors in the ischemic brain stem generally enhances the release of taurine. This is beneficial to neurons in ischemia, offering protection against excitotoxicity and preventing neuronal damage.
Keywords: Glutamate receptors; Taurine release; Ischemia; Tissue slices; Brain stem; Development-mouse
Preparation of N-acetyl, tert-butyl amide derivatives of the 20 natural amino acids by A. R. Ekkati; A. A. Campanali; A. I. Abouelatta; M. Shamoun; S. Kalapugama; M. Kelley; Jeremy J. Kodanko (747-751).
N-Acetyl-AA(amino acid)-NHtBu derivatives of all 20 naturally occurring amino acids have been synthesized. Syntheses were performed via solution-phase methodology with yields that allow for access to gram quantities of substrates, in most cases. Syntheses include the coupling of a hindered amine, tert-butylamine, with each amino acid, either directly or in two steps using an activated ester isolated as an intermediate. The introduction of protecting groups was necessary in some cases. The development of synthetic sequences to access challenging substrates, such as the one derived from asparagine, are discussed.
Keywords: Amino acid substrate synthesis; N-Boc-amino acid; N-Fmoc-amino acid; Amino acid protection; Amino acid deprotection
l-Proline transport into renal OK epithelial cells: a second renal proline transport system is induced by amino acid deprivation by A. Nickel; U. Klein; D. Weitz; H. Daniel (753-761).
Influx of [3H]-l-proline into renal OK cells revealed that basal transport was mediated by the transporter SIT1. When cells were submitted for 8 h to amino acid deprivation, uptake of l-proline was now dominated by a low-affinity system with an apparent K m of 4.4 ± 0.6 mM and a V max of 10.2 ± 0.6 nmol/mg of protein/min operating in addition to the high-affinity SIT1 system with a K m of 0.12 ± 0.01 mM and a V max of 0.28 ± 0.04 nmol/mg of protein/min. The low- and high-affinity proline transporting systems were sensitive to inhibitors of JNK and PI-3 kinases, whereas a GSK-3 inhibitor affected only the upregulated transport system. Ion-replacement studies and experiments assessing substrate specificities for both systems provided strong evidence that SNAT2, that showed two- to threefold increased mRNA levels, is the responsible transporter mediating the increased proline influx under conditions of amino acid deprivation.
Keywords: Transport; Proline; Amino acid deprivation; Regulation; SIT1; SNAT2
Connective tissue growth factor is a downstream mediator for preptin-induced proliferation and differentiation in human osteoblasts by You-Shuo Liu; Ying Lu; Wei Liu; Hui Xie; Xiang-Hang Luo; Xian-Ping Wu; Ling-Qing Yuan; Er-Yuan Liao (763-769).
Preptin, a newly isolated 34-amino-acid peptide hormone that is cosecreted with insulin and amylin from pancreatic beta-cells, has emerged as a regulatory element in bone metabolism, but its mechanism remains unclear. We assessed the effects of preptin on proliferation and differentiation of human osteoblasts and investigated the mechanism involved. Our results demonstrated that preptin promoted human osteoblasts proliferation and alkaline phosphatase activity. Suppression of connective tissue growth factor (CTGF), which was upregulated by preptin in a dose- and time-dependent manner, with small interfering RNA (siRNA) abolished the preptin-induced human osteoblasts proliferation and differentiation. Preptin induced activation of ERK mitogen-activated protein kinase (MAPK), but not p38 or JNK in human osteoblasts. Furthermore, pretreatment of human osteoblasts with the ERK inhibitor PD98059 abolished the preptin-induced CTGF secretion and blocked the promoting effect of preptin on osteoblasts proliferation and differentiation. These data demonstrated that preptin is involved in bone anabolism mediated by ERK/CTGF in human osteoblasts and may contribute to the preservation of bone mass observed in hyperinsulinemic states, such as obesity.
Keywords: Preptin; Connective tissue growth factor; Osteoblasts; Extracellular signal regulated kinase
Effect of a proprietary protein supplement on recovery indices following resistance exercise in strength/power athletes by Jay R. Hoffman; Nicholas A. Ratamess; Christopher P. Tranchina; Stefanie L. Rashti; Jie Kang; Avery D. Faigenbaum (771-778).
The effect of 42 g of protein ingested pre- and post-exercise on recovery from an acute resistance exercise session was examined in 15 male strength/power athletes who were randomly divided into a supplement (SUP) or placebo (PL) group. Subjects reported to the Human Performance Laboratory (HPL) on four separate occasions (T1–T4). Maximal strength [one repetition-maximum (1-RM)] testing was performed during T1. During T2 subjects performed four sets of ten repetitions at 80% of their 1-RM in the squat, dead lift and barbell lunge exercises with 90 s of rest between each set. Blood draws occurred at baseline (BL), immediate and 15 min post-exercise to determine testosterone, cortisol and creatine kinase (CK) concentrations. Subjects reported back to the HPL 24 (T3) and 48 h (T4) post-exercise for a BL blood draw and to perform four sets of ten repetitions with 80% of 1-RM for the squat exercise only. No differences in the number of repetitions performed in the squat exercise were seen between the groups at T2. Relative to T2, PL performed significantly (P < 0.05) fewer repetitions than SUP at T3 and T4 (−9.5 ± 5.5 repetitions vs. −3.3 ± 3.6 during T3, respectively, and −10.5 ± 8.2 repetitions vs. −2.3 ± 2.9 repetitions during T4, respectively). No differences in hormonal measures were seen between the groups. CK concentrations were significantly (P < 0.05) elevated at T3 for both groups, but continued to elevate (P < 0.05) at T4 for PL only. No significant group differences were noted for CK at any time point. Results indicate that a proprietary protein SUP consumed before and after a resistance training session significantly contributes to improvements in exercise recovery 24 and 48 h post-exercise.
Keywords: Supplements; Ergogenic aids; Protein timing; Hormones
Hemolymph amino acid variations following behavioral and genetic changes in individual Drosophila larvae by Sujeewa C. Piyankarage; Hrvoje Augustin; David E. Featherstone; Scott A. Shippy (779-788).
This study investigated the effect of different sampling environments on hemolymph amino acid content of individual Drosophila melanogaster larvae. Hemolymph was collected from individual third instar larvae under cold-anesthetized, awake, and stress conditions. Qualitative and quantitative hemolymph amino acid analyses were performed via capillary electrophoresis with laser-induced fluorescence detection. The hemolymph amino acid concentrations, particularly arginine, glutamate, and taurine, changed significantly depending on the prior-to-sample-collection environments. Hemolymph amino acid analyses of six different Drosophila genotypes including two control genotypes and four mutant alleles were also carried out. Two mutant genotypes with over and under expression of a putative cystine-glutamate exchanger subunit were significantly different from each other with respect to their hemolymph glutamate, glycine, lysine, and taurine levels. Hemolymph amino acid analyses of stressed larvae of two control and two mutant genotypes indicated that behavior-related hemolymph chemical changes are also genotype dependent.
Keywords: Capillary electrophoresis; Chemical composition; Stress; Anesthetize; Cystine-glutamate exchanger; Third instar larva
Kinetic and mechanistic study of oxidation of l-methionine by Waugh-type enneamolybdomanganate(IV) in perchloric acid by V. M. Gurame; A. R. Supale; Gavisiddappa S. Gokavi (789-795).
The reaction between methionine and enneamolybdomanganate(IV) in perchloric acid was carried out under pseudo-first-order conditions keeping large excess of methionine. The orders in oxidant and substrate were found to be unity and 0.91, respectively. The reaction proceeds with rapid formation of complex between the reactants followed by its decomposition in a rate determining step. The accelerating effect of hydrogen ions on the reaction is due to the formation of active hexaprotonated oxidant species. The product of the reaction was found to be methionine sulfoxide. The reaction involves direct two-electron transfer step without any free radical intervention. The effect of ionic strength, solvent polarity and the activation parameters were also in support of the mechanism proposed.
Keywords: Kinetics; Mechanism; Oxidation; Methionine; Waugh-type enneamolybdomanganate(IV)
Testing biological activity of model Maillard reaction products: studies on gastric smooth muscle tissues by Mariana D. Argirova; Iliyana D. Stefanova; Athanas D. Krustev; Valentin I. Turiiski (797-803).
Water-soluble Maillard reaction products obtained from five different model systems were investigated for their effects upon the mechanical activity of rat gastric smooth muscle. Most of the total Maillard reaction products applied at concentration of 1.5 mg/ml evoked contractions; among them the product obtained from arginine and glucose (Arg-Glc) produced the most powerful contractions. The product obtained from glycine and ascorbic acid (Gly-AsA) was the only one that brought about relaxation response. The high molecular weight fractions (>3,500 Da) isolated from the reaction systems Arg-Glc and Gly-AsA demonstrated effects similar in type and amplitude to those evoked by non-fractioned reaction products. The results obtained suggest that moieties of molecules acting upon the muscle tonus originate mainly from lysine and arginine residues; that these structures are available in both low and high molecular pools in similar concentrations, and most likely these fragments act upon membrane-located cellular structures involved in calcium transport.
Keywords: Maillard reaction; Melanoidins; Ascorbic acid; Arginine; Gastric smooth muscle
ST-scale as a novel amino acid descriptor and its application in QSAM of peptides and analogues by Li Yang; Mao Shu; Kaiwang Ma; Hu Mei; Yongjun Jiang; Zhiliang Li (805-816).
In this study, structural topology scale (ST-scale) was recruited as a novel structural topological descriptor derived from principal component analysis on 827 structural variables of 167 amino acids. By using partial least squares (PLS), we applied ST-scale for the study of quantitative sequence-activity models (QSAMs) on three peptide datasets (58 angiotensin-converting enzyme (ACE) inhibitors, 34 antimicrobial peptides (AMPs) and 89 elastase substrates (ES)). The results of QSAMs were superior to that of the earlier studies, with determination coefficient (r 2) and cross-validated (q 2) equal to 0.855, 0.774; 0.79, 0.371 (OSC-PLS: 0.995, 0.848) and 0.846, 0.747, respectively. Therefore, ST-scale descriptors were considered to be competent to extract information from 827 structural variables and relate with their bioactivities.
Keywords: Peptides; Structural topological scale (ST-scale); Principal component analysis (PCA); Partial least squares regression (PLS); Quantitative sequence-activity models (QSAM)
d-Aspartic acid induced oxidative stress and mitochondrial dysfunctions in testis of prepubertal rats by K. N. Chandrashekar; Muralidhara (817-827).
Previously we demonstrated the potential of d-aspartic acid (d-Asp), an acidic amino acid to induce oxidative response in prepubertal rat testis in vitro. In the present study, we determined the extent of oxidative stress in the testis of prepubertal rats that were administered d-Asp (100 and 500 mg/kg bw/d, i.p. 7 days). d-Asp treatment significantly elevated the levels of reactive oxygen species, malondialdehyde and hydroperoxide in cytosol and mitochondria of testis, which were accompanied by enhanced glutathione levels, elevated activities of glutathione-dependent enzymes and catalase suggesting a state of oxidative stress. Further, the activities of d-aspartate oxidase and 3β-hydroxy steroid dehydrogenase were elevated in the testis. The testis mitochondria of d-Asp-treated rats showed altered citric acid and complex enzyme activities, reduction in membrane potential, increased permeability and intracellular Ca2+ levels. Collectively, these findings suggest the potential of d-Asp to induce oxidative perturbations in the testis of prepubertal rats and this mechanism may in part be responsible for the observed physiological effects.
Keywords: d-Aspartic acid; Oxidative stress; Testis; Mitochondrial dysfunctions; Steroidogenesis; Prepubertal rats
(S)-α-methyl,α-amino acids: a new stereocontrolled synthesis by Daniele Balducci; Ilaria Lazzari; Magda Monari; Fabio Piccinelli; Gianni Porzi (829-837).
A new and convenient stereocontrolled synthesis of the optically pure (S)-α-methyl,α-amino acids 6(a–d) that exploits the chiral synthon 1,4-N,N-[(S)-1-phenylethyl]-piperazine-2,5-dione (1) is described. The (S)-1-phenylethyl group, bonded to each of the N-atoms of the 2,5-diketopiperazine, acts as a chiral inductor in the first alkylation, while the steric hindrance appears to be the determining factor of stereocontrol in third and forth alkylation.
Keywords: Diketopiperazine derivatives; α-Methyl-α-amino acids; Asymmetric synthesis
Synthesis of proline-derived dipeptides and their catalytic enantioselective direct aldol reactions: catalyst, solvent, additive and temperature effects by Y.-H. Chen; P.-H. Sung; Kuangsen Sung (839-845).
A series of dipeptides of l-proline-l-amino acid and l-proline-d-amino acid were synthesized to evaluate the catalytic effect for asymmetric direct aldol reactions. In the direct aldol reaction, a catalyst of l-proline-l-amino acid achieves better enantioselectivity than the corresponding l-proline-d-amino acid catalyst. Solubility of the dipeptide catalysts in the solvents is a key point for achieving a better yield of the direct aldol reaction, while hydrogen bonding of solvent does not play an important role in attaining better enantioselectivity and yield. Yield and enantioselectivity of the direct aldol reaction in water were improved by NMM and SDS additives, but the results that were done in plain DMSO were even better.
Keywords: Dipeptide; Direct aldol reaction; Enantioselective reaction
Profiling histidine dipeptides in plasma and urine after ingesting beef, chicken or chicken broth in humans by Kyung-Jin Yeum; Marica Orioli; Luca Regazzoni; Marina Carini; Helen Rasmussen; Robert M. Russell; Giancarlo Aldini (847-858).
The in vitro metabolic stability of histidine-dipeptides (HD), carnosine (CAR) and anserine (ANS), in human serum, and their absorption kinetics after ingesting pure carnosine or HD rich foods in humans have been investigated. Healthy women (n = 4) went through four phases of taking one dose of either 450 mg of pure carnosine, 150 g beef (B), 150 g chicken (C), or chicken broth (CB) from 150 g chicken with a >2-week washout period between each phase. Blood samples were collected at 0, 30, 60, 100, 180, 240, and 300 min, and urine samples before and after (up to 7 h) ingesting pure carnosine or food. Both plasma and urine samples were analyzed for HD concentrations using a sensitive and selective LC–ESI-MS/MS method. CAR was undetectable in plasma after ingesting pure carnosine, B, C or CB. By contrast, plasma ANS concentration was significantly increased (P < 0.05) after ingesting C or CB, respectively. Urinary concentrations of both CAR and ANS were 13- to 14-fold increased after ingesting B, and 14.8- and 243-fold after CB ingestion, respectively. Thus, dietary HD, which are rapidly hydrolyzed by carnosinase in plasma, and excreted in urine, may act as reactive carbonyl species sequestering agents.
Keywords: Carnosine; Anserine; Histidine-dipeptides; Absorption kinetic; Chicken
Using K-minimum increment of diversity to predict secretory proteins of malaria parasite based on groupings of amino acids by Yong-Chun Zuo; Qian-Zhong Li (859-867).
Due to the complexity of Plasmodium falciparumis genome, predicting secretory proteins of P. falciparum is more difficult than other species. In this study, based on the measure of diversity definition, a new K-nearest neighbor method, K-minimum increment of diversity (K-MID), is introduced to predict secretory proteins. The prediction performance of the K-MID by using amino acids composition as the only input vector achieves 88.89% accuracy with 0.78 Mathew’s correlation coefficient (MCC). Further, the several reduced amino acids alphabets are applied to predict secretory proteins and the results show that the prediction results are improved to 90.67% accuracy with 0.83 MCC by using the 169 dipeptide compositions of the reduced amino acids alphabets obtained from Protein Blocks method.
Keywords: Secretory proteins; Increment of diversity; Reduced amino acids alphabets; Amino acid and dipeptide composition; Prediction performance
Mitochondrial damage, cytotoxicity and apoptosis in iron-potentiated alcoholic liver fibrosis: amelioration by taurine by S. Lakshmi Devi; C. V. Anuradha (869-879).
Taurine effectively prevents ischemia-induced apoptosis in the cardiomyocytes and hypothalamic nuclei. The present study explores the influence of taurine on mitochondrial damage, oxidative stress and apoptosis in experimental liver fibrosis. Male albino Wistar rats were divided into six groups and maintained for a period of 60 days as follows: Group I, control; Group II, ethanol treatment [6 g/(kg/day)]; Group III, fibrosis induced by ethanol and iron (0.5% w/w); Group IV, ethanol + iron + taurine (2% w/v); Group V, ethanol + taurine treatment and Group VI, control + taurine treatment. Hepatocytes isolated from ethanol plus iron-treated rats showed decreased cell viability and redox ratio, increased reactive oxygen species formation, lipid peroxidation, DNA fragmentation, and formation of apoptotic bodies. Liver mitochondria showed increased susceptibility to swell, diminished activities of mitochondrial respiratory chain complexes and antioxidants. Taurine administration to fibrotic rats restored mitochondrial function, reduced reactive oxygen species formation, prevented DNA damage, and apoptosis. Thus taurine might contribute to the amelioration of the disease process.
Keywords: Ethanol; Carbonyl iron; Taurine; Cytotoxicity; DNA damage; Mitochondria; Fibrosis; Apoptosis
Methods of the site-selective solid phase synthesis of peptide-derived Amadori products by Piotr Stefanowicz; Monika Kijewska; Katarzyna Kapczyńska; Zbigniew Szewczuk (881-889).
Two procedures of glycated peptides’ synthesis have been developed. The first method involves reductive alkylation of the ε-amino groups of lysine with 2,3:4,5-di-O-isopropylidene-β-d-arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium cyanoborohydride on solid support. The second one uses a new fully protected lysine derivative, which is a building block designed for direct introduction of the glycated lysine moiety into a peptide, according to the standard solid phase synthesis protocol. The applicability of the proposed methods for the synthesis of peptide-derived Amadori products is discussed. The structure of the synthesized glycated peptides was confirmed by high-resolution mass spectrometry and enzymatic hydrolysis. Circular dichroism studies, performed in water solution, revealed that the formation of the Amadori rearrangement product in the lysine side chain does not influence significantly the conformational preferences of the peptides studied. However, when the solvent was changed to trifluoroethanol, the glycated peptides preferred β-turn conformation.
Keywords: Solid phase peptide synthesis; Amadori rearrangement; Glycation; Enzymatic stability
Predicting protein–protein interactions from sequence using correlation coefficient and high-quality interaction dataset by Ming-Guang Shi; Jun-Feng Xia; Xue-Ling Li; De-Shuang Huang (891-899).
Identifying protein–protein interactions (PPIs) is critical for understanding the cellular function of the proteins and the machinery of a proteome. Data of PPIs derived from high-throughput technologies are often incomplete and noisy. Therefore, it is important to develop computational methods and high-quality interaction dataset for predicting PPIs. A sequence-based method is proposed by combining correlation coefficient (CC) transformation and support vector machine (SVM). CC transformation not only adequately considers the neighboring effect of protein sequence but describes the level of CC between two protein sequences. A gold standard positives (interacting) dataset MIPS Core and a gold standard negatives (non-interacting) dataset GO-NEG of yeast Saccharomyces cerevisiae were mined to objectively evaluate the above method and attenuate the bias. The SVM model combined with CC transformation yielded the best performance with a high accuracy of 87.94% using gold standard positives and gold standard negatives datasets. The source code of MATLAB and the datasets are available on request under firstname.lastname@example.org.
Keywords: Protein–protein interactions; Correlation coefficient; Support vector machine; Protein sequence; Gold standard positives dataset; Gold standard negatives dataset
Preoperative oral supplementation with carbohydrate and branched-chain amino acid-enriched nutrient improves insulin resistance in patients undergoing a hepatectomy: a randomized clinical trial using an artificial pancreas by Takehiro Okabayashi; Isao Nishimori; Koichi Yamashita; Takeki Sugimoto; Tsutomu Namikawa; Hiromichi Maeda; Tomoaki Yatabe; Kazuhiro Hanazaki (901-907).
Glucose metabolism is adversely affected in patients following major surgery. Patients may develop hyperglycemia due to a combination of surgical stress and postoperative insulin resistance. A randomized trial was conducted to elucidate the effect of preoperative supplementation with carbohydrates and branched-chain amino acids on postoperative insulin resistance in patients undergoing hepatic resection. A total of 26 patients undergoing a hepatectomy for the treatment of a hepatic neoplasm were randomly assigned to receive a preoperative supplement of carbohydrate and branched-chain amino acid-enriched nutrient mixture or not. The postoperative blood glucose level and the total insulin requirement for normoglycemic control during the 16 h following hepatic resection were determined using the artificial pancreas STG-22. Postoperative insulin requirements for normoglycemic control in the group with preoperative nutritional support was significantly lower than that in the control group (P = 0.039). There was no incidence of hypoglycemia (<40 mg/dL) observed in patients, including those with diabetes mellitus, when the STG-22 was used to control blood glucose levels. STG-22 is a safe and reliable tool to control postoperative glucose metabolism and evaluate insulin resistance. The preoperative oral administration of carbohydrate and branched-chain amino acid-enriched nutrient is of clinical benefit and reduces postoperative insulin resistance in patients undergoing hepatic resection.
Keywords: Branched-chain amino acids; Carbohydrate; Artificial pancreas; Insulin resistance; Hepatectomy
Safety and adequacy of a semi-elemental formula for children with gastro-intestinal disease by Yvan Vandenplas; Kathleen Plaskie; Bruno Hauser (909-914).
A prospective, open trial was conducted to evaluate the nutritional adequacy of a semi-elemental diet in 47 children with functional gastro-intestinal disorders. Nutritional adequacy was assessed based on growth relative to Euro-growth standards for body mass index (BMI)-for-age z-scores and evaluations of blood parameters. Twenty-five patients completed the study. In total, 533 l of “New-Alfare” was consumed during 775 trial-days. The mean intake per infant was 85.8 ± 26.8 kcal/kg/day or 122.5 ± 38.3 ml/kg/day. Weight and length evolution during the 4 weeks trial were within normal range. The mean BMI-for-age z-score (P < 0.05) and albumin concentration (P < 0.01) increased significantly after 4 weeks. Plasma threonine concentration decreased significantly (P = 0.01) and the tryptophan concentration increased (P = 0.06). No adverse events related to the study formula were reported. These results show that “New Alfaré” is safe and nutritionally adequate for pediatric patients with gastro-intestinal disease.
Keywords: Infant formula; Amino acids; Threonine; Milk hypersensitivity; Safety; Growth
Recognition of β-hairpin motifs in proteins by using the composite vector by Xiu-Zhen Hu; Qian-Zhong Li; Chun-Lian Wang (915-921).
A composite vector method for predicting β-hairpin motifs in proteins is proposed by combining the score of matrix, increment of diversity, the value of distance and auto-correlation information to express the information of sequence. The prediction is based on analysis of data from 3,088 non-homologous protein chains including 6,035 β-hairpin motifs and 2,738 non-β-hairpin motifs. The overall accuracy of prediction and Matthew’s correlation coefficient are 83.1% and 0.59, respectively. In addition, by using the same methods, the accuracy of 80.7% and Matthew’s correlation coefficient of 0.61 are obtained for other dataset with 2,878 non-homologous protein chains, which contains 4,884 β-hairpin motifs and 4,310 non-β-hairpin motifs. Better results are also obtained in the prediction of the β-hairpin motifs of proteins by analysis of the CASP6 dataset.
Keywords: β-Hairpin; Scoring matrix; Increment of diversity; Auto-correlation function
Plasma levels of nitric oxide related amino acids in demented subjects with Down syndrome are related to neopterin concentrations by A. M. W. Coppus; D. Fekkes; W. M. A. Verhoeven; S. Tuinier; C. M. van Duijn (923-928).
Subjects with Down syndrome (DS) have abnormalities in virtually all aspects of the immune system and almost all will be affected with Alzheimer’s disease (AD). It is thought that nitric oxide (NO) is involved in the pathophysiology of AD. In the present study, including a total of 401 elderly DS subjects, the spectrum of plasma amino acids and neopterin was investigated and related to development of AD. Concentrations of nearly all amino acids in DS subjects differed significantly from those of healthy controls. Neopterin was increased in DS subjects, especially in dementia. The production of NO as reflected by an increased citrulline/arginine ratio (Cit/Arg ratio) was enhanced during development of clinical dementia. Neopterin concentrations correlated to the Cit/Arg ratio only in the group of prevalent demented subjects (ρ = 0.48, P = 0.006). The results of this study are suggestive for an increase in oxidative processes in DS subjects with AD.
Keywords: Amino acids; Down syndrome; Neopterin; Nitric oxide; Branched chain amino acids
NMR structural elucidation of myelin basic protein epitope 83–99 implicated in multiple sclerosis by Zinovia Spyranti; Theodore Tselios; George Deraos; John Matsoukas; Georgios A. Spyroulias (929-936).
Myelin basic protein peptide 83–99 (MBP83–99) is the most immunodominant epitope playing a significant role in the multiple sclerosis (MS), an autoimmune disease of the central nervous system. Many peptide analogues, linear or cyclic have been designed and synthesized based on this segment in order to inhibit the experimental autoimmune encephalomyelitis, the best well-known animal model of MS. In this study, the solution structural motif of MBP83–99 has been performed using 2D 1H-NMR spectroscopy in dimethyl sulfoxide. A rather extended conformation, along with the formation of a well defined α-helix spanning residues Val87–Phe90 is proposed, as no long-range NOE are presented. Moreover, the residues of MBP peptide that are important for T-cell receptor recognition are solvent exposed. The spatial arrangement of the side chain all over the sequence of our NMR based model exhibits great similarity with the solid state model, while both TCR contacts occupy the same region in space.
Keywords: MBP; Multiple sclerosis; NMR conformational analysis
Cysteine enhances activity and stability of immobilized papain by Ahmad Abolpour Homaei; Reza H. Sajedi; Reyhaneh Sariri; Sara Seyfzadeh; Roberto Stevanato (937-942).
Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. After immobilization, the optimum temperature of papain activity increased by 20°C (from 60 to 80°C) and its optimum pH activity shifted from 6.5 to 8.0. Catalytic efficiency (kcat/Km) and specific activity of the immobilized enzyme do not significantly change after immobilization. The temperature profile of this form of immobilized papain showed a broad range of activity compared with both free and immobilized form of papain in the absence of cysteine. This significant behavior in terms of activation energy is also discussed.
Keywords: Papaine; Cysteine; Enzyme immobilization; Thermal stability
Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana by Takashi Ishizaki; Chieko Ohsumi; Kazuhiko Totsuka; Daisuke Igarashi (943-950).
Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO2 concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.
Keywords: Arabidopsis ; Fd-GOGAT; Glutamate synthase; Photorespiration; GLU1
Gas chromatographic determination of amino acid enantiomers in bottled and aged wines by Hatem Salama Mohammed Ali; Ralf Pätzold; Hans Brückner (951-958).
Free l- and d-amino acids were determined by chiral GC-MS in 26 wines, comprising white wines, red wines, ice wines and sparkling wines. The aim of the work was to investigate whether quantities and pattern of d-amino acids, in particular d-proline, correlate with the storage time of bottled wines. The relative quantities with respect to the corresponding l-enantiomer ranged in white wines from 0.4 to 3.9% d-Ala, 0.9–8.3% d-Asx, and 0.5–8.9% d-Glx, in red wines from 2.9 to 10.6% d-Ala, 2.2–10.9% d-Asx, and 3.9–7.4% d-Glx, and in sparkling wines from 2.2 to 9.8% d-Ala, 2.1–4.4% d-Asx and 1.3–6.1% d-Glx. Low relative quantities of 0.3–0.7% d-Pro were detected in three white wines stored for more than 20 years and did not exceed 0.2% d-Pro in two red wines stored for 10 and 20 years, respectively. An ice wine stored for 24 years contained 0.9% d-Pro, 6.4% d-Glx, 3.0% d-Asp and 1.5% d-Ala. The data confirm the presence of d-amino acids in wines. They do not provide evidence for a correlation between the storage time of bottled wines and quantities of d-amino acids.
Keywords: Wine amino acids; d-Amino acids; d-Pro; Age dating; Gas chromatography-mass spectrometry; Chirasil ® -l-Val
Plasma and urine amino acid pattern in preterm infants on enteral nutrition: impact of gestational age by Sabine Illsinger; Karl-Heinz Schmidt; Thomas Lücke; Bernhardt Vaske; Bettina Bohnhorst; Anibh Martin Das (959-972).
Plasma and urine amino acids were determined by ion-exchange chromatography in 80 healthy preterm infants divided into three groups: (1) 23 0/7–28 0/7, (2) 28 1/7–32 0/7 and (3) 32 1/7–35 0/7 weeks of gestation. Samples were collected from days 5 to 57 of life, when infants were exclusively orally fed. Infants with evidence of underlying diseases were excluded. Concentrations of most plasma amino acids increased with gestational and maturational age; urinary excretion followed an opposite course. Few amino acids depended on postnatal age. Plasma amino acids did not correlate inversely to their counterparts in urine indicating that plasma amino acids do not simply reflect kidney function. Some amino acids in blood and urine were linked to nutrient intake and body weight. Our data clearly indicate the heterogeneity of the preterm cohort; therefore, gestational age-matched reference values have to be used for diagnostic purposes in preterm infants.
Keywords: Amino acids; Preterm infants; Gestational age; Maturation; Enteral nutrition
Metabolism of polyamines and oxidative stress in the brain of cholestatic rats by Stelios F. Assimakopoulos; Dimitris Konstantinou; Christos Georgiou; Elisabeth Chroni (973-974).
In a recently published article in “Amino Acids” it was shown that obstructive jaundice of 9 days’ duration in rats induces significant alterations of polyamines’ metabolism in the brain, which might play an important pathogenetic role in cholestatic brain injury. The authors proposed that alterations of polyamines in cholestatic brain might induce neuronal toxicity through a mechanism that implicates the production of reactive oxygen species and oxidative stress, although this parameter was not evaluated in their study. This hypothesis is supported by our recent findings on brain oxidative status in rats with obstructive jaundice of 10 days’ duration. Potential interrelations of the two studies’ findings are discussed in this commentary.