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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.33, #1)
Strategies for comprehensive analysis of amino acid biomarkers of oxidative stress by A. S. Ptolemy; R. Lee; P. Britz-McKibbin (pp. 3-18).
Despite the wide interest in using modified amino acids as putative biomarkers of oxidative stress, many issues remain as to their overall reliability for early detection and diagnosis of diseases. In contrast to conventional single biomarker studies, comprehensive analysis of biomarkers offers an unbiased strategy for global assessment of modified amino acid metabolism due to reactive oxygen and nitrogen species. This review examines recent analytical techniques amenable for analysis of modified amino acids in biological samples reported during 2003–2007. Particular attention is devoted to the need for validated methods applicable to high-throughput analysis of multiple amino acid biomarkers, as well as consideration of sample pretreatment protocols on artifact formation for improved clinical relevance.
Keywords: Keywords: Oxidative stress – Modified amino acids – Analytical techniques – Comprehensive analysis – Multiple biomarkers
The five bromotryptophans by S. Bittner; R. Scherzer; E. Harlev (pp. 19-42).
The five regioisomeric bromotryptophans (BrTrps) play an important role in the life of sponges and lower marine invertebrates. These bromo-amino acids, which are formed by post-translational modifications, are not found in nature in their free state, but rather are involved in more complex structures. Any of the BrTrps can be part of a peptide, a cyclic peptide, an indole alkaloid, an ergot alkaloid, a macrocycle and others. The present review covers the synthesis, physical and spectroscopic properties of the five BrTrps. It also describes the many exiting pharmacological and biological activities played by the BrTrps and by various secondary metabolites containing brominated tryptophan moieties. Of special interest are cyclic peptides containing the 2-BrTrp unit, which were isolated from marine sponges e.g. konbamide, orbiculamide A, the various keramamides, jaspamide eusynstyelamide and more. Important families of non-cyclic peptides containing the 6-BrTrp, include the styelins, the conotoxins, the cathelicidins and several constrained macrocyclic peptides. Many marine secondary BrTrp-containing, non-peptidic metabolites also display a remarkable spectrum of bioactivities, which can be harnessed for therapeutic and other purposes. Examples are: barettin, bromotryptanthrin, tetraacetyl clionamide, cyclocinamide A, clavicipitic acid, various brominated β-carbolines. In this review we have presented the various synthetic routes leading to the preparation of the five BrTrps and many of its derivatives. Also, we have introduced the reader to many synthetic routes leading to BrTrp-containing non-peptidic natural products. Although the functional role of the various compounds in the human body is only poorly understood, its effects were extensively studied. Almost all of these compounds exhibit important therapeutic properties e.g. antifungal, antimicrobial, antihelmintic, insecticidal ichthyotoxic and anticancer activity. In the present review attempts have been made to provide synopsis, synthesis and symbiosis of chemical and biological actions, which may provide future guidance and facilitate further research in this area.
Keywords: Keywords: Tryptophan – Bromotryptophan – Synthesis – Post-translational bromination – Cyclic peptides – Antimicrobial peptides
A unified hypothesis of coeliac disease with implications for management of patients by H. J. Cornell; T. Stelmasiak (pp. 43-49).
This mini-review presents the research carried out within the context of two of the main hypotheses of the aetiology of coeliac disease. The enzymopathic hypothesis of the disease has been placed clearly as the underlying deficiency causing increased levels of toxic peptides, while the immunological hypothesis has been implicated in the pathogenesis of the disorder as the result of the action of undigested peptides in the small intestine.As a consequence, we are proposing a unified hypothesis of coeliac disease, which takes into account the actions of these undigested peptides through their direct cytotoxicity and their immunoactivity. At the same time, work aimed at defining some of these biologically active peptides, which could be said to be involved in the aetiopathogenesis of coeliac disease, will be reported.The review also focusses on the use of enzyme therapy for management of the disease, which when used in conjunction with the gluten-free diet, offers a safeguard against damage to the small intestine caused by small amounts of gluten.
Keywords: Keywords: Coeliac disease – Enzyme therapy – Gliadins – Gluten – Disease mechanisms – Peptidases
Detecting oxidative post-translational modifications in proteins by E. Gianazza; J. Crawford; I. Miller (pp. 51-56).
Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).
Keywords: Keywords: Oxidative post-translational modifications – Proteomics – Electrophoresis – Carbonylation – Glutathionylation
Euk-PLoc: an ensemble classifier for large-scale eukaryotic protein subcellular location prediction by H.-B. Shen; J. Yang; K.-C. Chou (pp. 57-67).
With the avalanche of newly-found protein sequences emerging in the post genomic era, it is highly desirable to develop an automated method for fast and reliably identifying their subcellular locations because knowledge thus obtained can provide key clues for revealing their functions and understanding how they interact with each other in cellular networking. However, predicting subcellular location of eukaryotic proteins is a challenging problem, particularly when unknown query proteins do not have significant homology to proteins of known subcellular locations and when more locations need to be covered. To cope with the challenge, protein samples are formulated by hybridizing the information derived from the gene ontology database and amphiphilic pseudo amino acid composition. Based on such a representation, a novel ensemble hybridization classifier was developed by fusing many basic individual classifiers through a voting system. Each of these basic classifiers was engineered by the KNN (K-Nearest Neighbor) principle. As a demonstration, a new benchmark dataset was constructed that covers the following 18 localizations: (1) cell wall, (2) centriole, (3) chloroplast, (4) cyanelle, (5) cytoplasm, (6) cytoskeleton, (7) endoplasmic reticulum, (8) extracell, (9) Golgi apparatus, (10) hydrogenosome, (11) lysosome, (12) mitochondria, (13) nucleus, (14) peroxisome, (15) plasma membrane, (16) plastid, (17) spindle pole body, and (18) vacuole. To avoid the homology bias, none of the proteins included has ≥25% sequence identity to any other in a same subcellular location. The overall success rates thus obtained via the 5-fold and jackknife cross-validation tests were 81.6 and 80.3%, respectively, which were 40–50% higher than those performed by the other existing methods on the same strict dataset. The powerful predictor, named “Euk-PLoc”, is available as a web-server at http://202.120.37.186/bioinf/euk . Furthermore, to support the need of people working in the relevant areas, a downloadable file will be provided at the same website to list the results predicted by Euk-PLoc for all eukaryotic protein entries (excluding fragments) in Swiss-Prot database that do not have subcellular location annotations or are annotated as being uncertain. The large-scale results will be updated twice a year to include the new entries of eukaryotic proteins and reflect the continuous development of Euk-PLoc.
Keywords: Keywords: Cellular networking – Subcellular compartment – KNN classifier – Fusion – Voting – Gene ontology – Amphiphilic pseudo amino acid composition
Prediction of protein subcellular localization by support vector machines using multi-scale energy and pseudo amino acid composition by J.-Y. Shi; S.-W. Zhang; Q. Pan; Y.-M. Cheng; J. Xie (pp. 69-74).
As more and more genomes have been discovered in recent years, there is an urgent need to develop a reliable method to predict the subcellular localization for the explosion of newly found proteins. However, many well-known prediction methods based on amino acid composition have problems utilizing the sequence-order information. Here, based on the concept of Chou’s pseudo amino acid composition (PseAA), a new feature extraction method, the multi-scale energy (MSE) approach, is introduced to incorporate the sequence-order information. First, a protein sequence was mapped to a digital signal using the amino acid index. Then, by wavelet transform, the mapped signal was broken down into several scales in which the energy factors were calculated and further formed into an MSE feature vector. Following this, combining this MSE feature vector with amino acid composition (AA), we constructed a series of MSEPseAA feature vectors to represent the protein subcellular localization sequences. Finally, according to a new kind of normalization approach, the MSEPseAA feature vectors were normalized to form the improved MSEPseAA vectors, named as IEPseAA. Using the technique of IEPseAA, C-support vector machine (C-SVM) and three multi-class SVMs strategies, quite promising results were obtained, indicating that MSE is quite effective in reflecting the sequence-order effects and might become a useful tool for predicting the other attributes of proteins as well.
Keywords: Keywords: Multi-scale energy – Wavelet transform – Support vector machines – Chou’s pseudo amino acid composition – Protein subcellular localizations
A dynamic model for the p53 stress response networks under ion radiation by J.-P. Qi; S.-H. Shao; D.-D. Li; G.-P. Zhou (pp. 75-83).
P53 controls the cell cycle arrest and cell apoptosis through interaction with the downstream genes and their signal pathways. To stimulate the investigation into the complicated responses of p53 under the circumstance of ion radiation (IR) in the cellular level, a dynamic model for the p53 stress response networks is proposed. The model can be successfully used to simulate the dynamic processes of generating the double-strand breaks (DSBs) and their repairing, ataxia telangiectasia mutated (ATM) activation, as well as the oscillations occurring in the p53-MDM2 feedback loop.
Keywords: Keywords: Cellular networks – p53 – MDM2 – IR – DNA damage – Oscillations
Concise preparation of N α-Fmoc-N ɛ-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide by Z.-P. Huang; J.-T. Du; X.-Y. Su; Y.-X. Chen; Y.-F. Zhao; Y.-M. Li (pp. 85-89).
A concise preparation of N α-Fmoc-N ɛ-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide is described. N α-Fmoc-N ɛ-(Boc, methyl)-lysine is obtained, via consecutive reductive benzylation and reductive methylation in a one-pot reaction, followed by debenzylation through catalytic hydrogenolysis and Boc protection in another one-pot reaction. A peptide containing monomethylated lysine is successfully synthesized by incorporating N α-Fmoc-N ɛ-(Boc, methyl)-lysine as a building block via solid-phase peptide synthesis.
Keywords: Keywords: Lysine – Methylation – Benzylation – Monomethylated peptide – Synthesis
Protease-catalyzed synthesis of the tripeptide CCK26–28, a fragment of CCK-8 by L. P. Meng; R. Joshi; H. Eckstein (pp. 91-96).
Two enzymatically synthetic strategies of the tripeptide derivative PhAc-Asp(OMe)-Tyr-Met-OAl are reported. The second strategy gains the advantage of more economical starting materials, less reaction steps and a higher overall isolated yield of this tripeptide fragment over the first strategy. The effect of the acyl-donor ester concentration and structure, the C-α protecting group of the nucleophile, reaction media, enzyme and the carrier on the tripeptide derivative synthesis were studied. This tripeptide selected is a fragment of the cholecystokinin C-terminal octapeptide (CCK-8), a potential therapeutic agent in the control of gastrointestinal function and also a drug candidate for the treatment of epilepsy.
Keywords: Keywords: Enzymatic peptide synthesis – Immobilized enzymes – Organic solvent system – Solvent free system – CCK fragment
A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), for the enantioresolution of amino acids on a teicoplanin phase under the elution of the methanol-based solvent mixture by T.-J. Hsien; S. Chen (pp. 97-104).
A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), is chosen to functionalize amino acids in alkaline medium before their HPLC resolution. FCC reacts with both primary and secondary amino acids to produce stable and highly fluorescent derivatives suitable for sensitive and efficient chromatographic determination and resolution on a teicoplanin chiral stationary phase (CSP) using the methanol-based solvent mixture as the mobile phase. The detection limit is in the picomole range and approximately 0.01% of the d-enantiomer in an excess of the l-enantiomer is detectable. However, the resolution is not reproducible under the elution of either the water- or the acetonitrile-based mobile phase. The increase in solubility of analyte in the mobile phase seems to be responsible. Upon comparison under the optimal chromatographic conditions, the resolution is better than that for the 9-fluorenylmethyl chloroformate (FMOC) or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives reported previously.
Keywords: Keywords: Fluorescent tagging reagent – Column liquid chromatography – Enantioresolution – Amino acids – Teicoplanin – Trace enantiomeric impurities – Enantiomeric ratio
The apoptotic effect of intercalating agents on HPV-negative cervical cancer C-33A cells by Yun Hee Kang; Kyung-Ac Lee; Young Yang; Soo-Hyun Kim; Joo Hwan Kim; Sue Nie Park; Sang-Gi Paik; Do-Young Yoon (pp. 105-112).
Cervical cancer is one of the leading causes of female cancer death worldwide with about 500,000 deaths per year. Both mitomycin C and cisplatin are alkylating agents, which bind and intercalate DNA, and thus used as anti-cancer drugs. In these studies, we focused on investigating the apoptotic effects of intercalating agents on HPV-negative cervical cancer C-33A cells. Accordingly, C-33A cells were treated with carboplatin, mitomycin C or cisplatin. Cell cycle analysis revealed that treatment with mitomycin C and cisplatin but not with carboplatin resulted in apoptosis. Both mitomycin C and cisplatin induced apoptosis in C-33A cells via caspase-8 and -3 processing in a Fas/FasL-dependent manner and also suppressed IL-18 expression, while they down-regulated IκB expression and up-regulated p65 expression. These results suggest that both mitomycin C and cisplatin induce apoptosis, not only via the caspase-8 and -3 dependent Fas/FasL pathway, but also via the regulation of NF-κB activity and IL-18 expression in HPV-negative cervical cancer C-33A cells.
Keywords: Keywords: IL-18 – FasL – Mitomycin C – Cisplatin – Apoptosis – Cervical cancer
Multi-layered network structure of amino acid (AA) metabolism characterized by each essential AA-deficient condition by N. Shikata; Y. Maki; Y. Noguchi; M. Mori; T. Hanai; M. Takahashi; M. Okamoto (pp. 113-121).
The concentrations of free amino acids in plasma change coordinately and their profiles show distinctive features in various physiological conditions; however, their behavior can not always be explained by the conventional flow-based metabolic pathway network. In this study, we have revealed the interrelatedness of the plasma amino acids and inferred their network structure with threshold-test analysis and multilevel-digraph analysis methods using the plasma samples of rats which are fed diet deficient in single essential amino acid.In the inferred network, we could draw some interesting interrelations between plasma amino acids as follows: 1) Lysine is located at the top control level and has effects on almost all of the other plasma amino acids. 2) Threonine plays a role in a hub in the network, which has direct links to the most number of other amino acids. 3) Threonine and methionine are interrelated to each other and form a loop structure.
Keywords: Keywords: Plasma amino acids – Profile – Relation – Network – Threonine – Amino acid deficiency
A facile HPLC method for optical purity and quantitative measurements of phenylalanine from the hydrolyzed aspartame under different pH and temperature after its derivatization with a fluorescent reagent by T.-J. Hsien; S. Chen (pp. 123-128).
In this paper, the artificial sweetener aspartame is deliberately hydrolyzed under different pH and temperature in the matrix, and time period for the hydrolysis. The HPLC analysis is then performed to quantitatively measure the amount and the optical purity of phenylalanine produced as a result of hydrolysis in the matrix after its functionalization with a fluorescent reagent. The results show that the amount of phenylalanine in the matrix is affected by the pH variation during the hydrolysis and found increased in low pH conditions. High temperature or long time periods for the decomposition also increases the amount, which indicates that beverages and foods containing aspartame as a sweetener may not be safe for phenylketonuria patients to consume if they are stored under these conditions. Conversely, the optical purity of phenylalanine, expressed as the percentage of d-enantiomer, is not affected by pH variations. However, it decreases as the length of time elapsed is increased or surrounding temperature is elevated during the decomposition.
Keywords: Keywords: Aspartame – Optical purity – Phenylalanine – Teicoplanin – Fluorescent reagent
Virtual screening for finding natural inhibitor against cathepsin-L for SARS therapy by S.-Q. Wang; Q.-S. Du; K. Zhao; A.-X. Li; D.-Q. Wei; K.-C. Chou (pp. 129-135).
Recently Simmons et al. reported a new mechanism for SARS virus entry into target cells, where MDL28170 was identified as an efficient inhibitor of CTSL-meditated substrate cleavage with IC50 of 2.5 nmol/l. Based on the molecule fingerprint searching method, 11 natural molecules were found in the Traditional Chinese Medicines Database (TCMD). Molecular simulation indicates that the MOL376 (a compound derived from a Chinese medicine herb with the therapeutic efficacy on the human body such as relieving cough, removing the phlegm, and relieving asthma) has not only the highest binding energy with the receptor but also the good match in geometric conformation. It was observed through docking studies that the van der Waals interactions made substantial contributions to the affinity, and that the receptor active pocket was too large for MDL21870 but more suitable for MOL736. Accordingly, MOL736 might possibly become a promising lead compound for CTSL inhibition for SARS therapy.
Keywords: Keywords: Severe acute respiratory syndrome (SARS) – MDL28170 – KZ7088 – Molecular simulation – Docking – Structural bioinformatics
The time-profile of the PBMC HSP70 response to in vitro heat shock appears temperature-dependent by R. Lovell; L. Madden; S. Carroll; L. McNaughton (pp. 137-144).
Heat shock proteins (HSPs) are synthesised by cells subsequent to a stress exposure and are known to confer protection to the cell in response to a second challenge. HSP induction and decay are correlated to thermotolerance and may therefore be used as a biomarker of thermal history. The current study tested the temperature-dependent nature of the heat shock response and characterised its time profile of induction. Whole blood from 6 healthy males (Age: 26 ± (SD) 2 yrs; Body mass 74.2 ± 3.8 kgs; VO2max: 49.1 ± 4.0 ml·kg−1·min−1) were isolated and exposed to in vitro heat shock (HS) at 37, 38, 39, 40, and 41 °C for a period of 90 min. After HS the temperature was returned to 37 °C and intracellular HSP70 was quantified from the leukocytes at 0, 2, 4, and 6 h after heat treatment. The concentration of HSP70 was not different between temperatures (P > 0.05), but the time-profile of HSP70 synthesis appeared temperature-dependent. At control (37 °C) and lower temperatures (38–39 °C) the mean HSP70 concentration increased up to 4 h post HS (P < 0.05) and then returned towards baseline values by 6 h post HS. With in vitro hyperthermic conditions (40–41 °C), the time-profile was characterised by a sharp rise in HSP70 levels immediately after treatment (P < 0.05 for 40 °C at 0 h), followed by a progressive decline over time. The results suggest a temperature-dependent time-profile of HSP70 synthesis. In addition, the temperature at which HSP70 is inducted might be lower than 37 °C.
Keywords: Keywords: Heat shock protein 70 – Temperature – In vitro, PBMCs
Variation in proton affinity of the guanidino group between free and blocked arginine by Y. Liu; L. Jin; J.-B. Hou; P.-X. Xu; Y.-F. Zhao (pp. 145-150).
In this paper, the analog of arginine residues in peptides was synthesized and characterized by ESI-MS/MS (electrospray ionization with tandem mass spectrometry), 31P NMR, 1H NMR, IR and high-resolution mass spectrometry. When the Todd reaction activity of the guanidino group in free arginine and the arginine peptide analog were compared, it was found that the proton affinity of the guanidino group was decreased when both the N- and the C-terminal were blocked. As a result, the guanidino group of arginine residues in peptides could be phosphorylated under the Todd reaction condition, but not the free arginine. This result was further proved by the theoretical calculation of their proton affinity.
Keywords: Keywords: Arginine residues – Guanidino group – Proton affinity – Todd reaction
Blood amino acids concentration during insulin induced hypoglycemia in rats: the role of alanine and glutamine in glucose recovery by R. F. Garcia; V. A. F. G. Gazola; H. C. Barrena; E. M. Hartmann; J. Berti; M. H. Toyama; A. C. Boschero; E. M. Carneiro; F. C. Manso; R. B. Bazotte (pp. 151-155).
Our purpose was to determine the blood amino acid concentration during insulin induced hypoglycemia (IIH) and examine if the administration of alanine or glutamine could help glycemia recovery in fasted rats. IIH was obtained by an intraperitoneal injection of regular insulin (1.0 U/kg). The blood levels of the majority of amino acids, including alanine and glutamine were decreased (P < 0.05) during IIH and this change correlates well with the duration than the intensity of hypoglycemia. On the other hand, the oral and intraperitoneal administration of alanine (100 mg/kg) or glutamine (100 mg/kg) accelerates glucose recovery. This effect was partly at least consequence of the increased capacity of the livers from IIH group to produce glucose from alanine and glutamine. It was concluded that the blood amino acids availability during IIH, particularly alanine and glutamine, play a pivotal role in recovery from hypoglycemia.
Keywords: Keywords: Amino acids – Alanine – Glutamine – Gluconeogenesis – Hypoglycemia – Insulin
L-methionine as immune supportive supplement: a clinical evaluation by R. Van Brummelen; D. du Toit (pp. 157-163).
The objective of the study was to test L-methioinine as a possible immune supportive supplement in HIV infected patients by means of a clinical study. A double-blind, placebo-controlled study was designed. The patients (n = 253) from four different trial centres were randomly divided into two groups, active and placebo, and regularly assessed by clinical and safety parameters. After six months from commencement, clinically and statistically significant differences were observed. The females of the active treatment group presented with a decreased level of decline in their CD4 counts (p = 0.0027), so also the patients of Centre 1 (p = 0.0377). All patients were placed onto active treatment after 12 months and were followed up for 48 months after the trial started. The same tendencies could be observed in the group as a whole, with no serious side effects directly associated to treatment. The study confirmed the supportive role of L-methionine in immune-compromised or deficient patients.
Keywords: Keywords: L-methionine – Immune support – Supplement – HIV
Free glutamine as a major precursor of brown products and fluorophores in Maillard reaction systems by C. Niquet; F. J. Tessier (pp. 165-171).
Glutamine is one of the most abundant free amino acid found in raw food. In this study, the contribution of free glutamine to nonenzymatic browning and fluorescence was investigated using an aqueous model system with methylglyoxal. The results indicated that glutamine contributed to the Maillard reaction via two pathways. First, the hydrolysis of the amide bond of glutamine led to the release of ammonia which was implicated in the formation of brown color and fluorescence. Among other nitrogen donors tested (asparagine, glutamic acid and urea) our results demonstrated that free glutamine was a major source of ammonia during heating. When heated at 120 and 180 °C, 100% of ammonia was released from glutamine after 60 and 10 min, respectively. The second pathway involved a direct Maillard reaction with the α-amino group of glutamine. Both pathways led to a rapid and complete destruction of glutamine when heated in the model systems. With reference to the Maillard browning (absorbance at 420 nm) glutamine turned out to be the most reactive amine, followed by asparagine, glutamate, ammonia and urea. Maximum fluorescence (excitation and emission wavelengths at 330 and 450 nm, respectively) was also observed with glutamine followed by urea and ammonia. Overall this study suggested that free glutamine predominantly contributes to the color and fluorescence formations of foodstuffs.
Keywords: Keywords: Glutamine – Ammonia – Maillard reaction – Browning – Fluorescence