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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.33, #3)
Applications and current challenges of proteomic approaches, focusing on two-dimensional electrophoresis by F. G. G. Vercauteren; L. Arckens; R. Quirion (pp. 405-414).
Since the formulation of the concept of “proteomics” in 1995, a plethora of proteomic technologies have been developed in order to study proteomes of tissues, cells and organelles. The powerful new technologies enabled by proteomic approaches have lead to the application of these methods to an exponentially increasing variety of biological questions for highly complex protein mixtures. Continuous technical optimization allows for an ever-increasing sensitivity of proteomic techniques. In this review, a brief overview of currently available proteomic techniques and their applications is given, followed by a more detailed description of advantages and technical challenges of two-dimensional electrophoresis (2-DE). Some solutions to circumvent currently encountered technical difficulties for 2-DE analyses are proposed.
Keywords: Keywords: Proteomics – Two-dimensional electrophoresis – Mass spectrometry – Applications – Shortcomings
Effects of ligands on the stability of tissue transglutaminase: studies in vitro suggest possible modulation by ligands of protein turn-over in vivo by C. M. Bergamini (pp. 415-421).
Tissue transglutaminase catalyzes irreversible post-translational modification of specific protein substrates by either crosslinkage or incorporation of primary amines into glutamine residues, through glutamyl-amide isopeptide bonds. Modulation in vivo of these reactions (collectively called “transamidation”) is brought about by both ligand dependent effects (chiefly, activation by calcium and inhibition by GTP) as well as by variation in enzyme tissue levels by transcriptional effects. Accumulating observations that the enzyme stability in vitro is greatly affected by interaction with ligands led us to postulate that also the turn-over in vivo might be modulated by ligands opening new scenarios on the regulation of the tissue transamidating activity. This proposal is consistent with data obtained in in vitro cell culture systems and has important implications for the expression of activity in vivo.
Keywords: Keywords: Transglutaminase – Protein cross-links – GTP – Polyamines
Prediction of linear B-cell epitopes using amino acid pair antigenicity scale by J. Chen; H. Liu; J. Yang; K.-C. Chou (pp. 423-428).
Identification of antigenic sites on proteins is of vital importance for developing synthetic peptide vaccines, immunodiagnostic tests and antibody production. Currently, most of the prediction algorithms rely on amino acid propensity scales using a sliding window approach. These methods are oversimplified and yield poor predicted results in practice. In this paper, a novel scale, called the amino acid pair (AAP) antigenicity scale, is proposed that is based on the finding that B-cell epitopes favor particular AAPs. It is demonstrated that, using SVM (support vector machine) classifier, the AAP antigenicity scale approach has much better performance than the existing scales based on the single amino acid propensity. The AAP antigenicity scale can reflect some special sequence-coupled feature in the B-cell epitopes, which is the essence why the new approach is superior to the existing ones. It is anticipated that with the continuous increase of the known epitope data, the power of the AAP antigenicity scale approach will be further enhanced.
Keywords: Keywords: B-cell epitope – AAP antigenicity scale – SVM classifier
Extracellular amino acid levels in the human liver during transplantation: a microdialysis study from donor to recipient by D. A. Richards; M. A. Silva; N. Murphy; S. J. Wigmore; D. F. Mirza (pp. 429-437).
Using microdialysis, we have monitored extracellular levels of amino acids and related amines in the human liver at three stages of the transplantation procedure: donor retrieval, back table preparation and during 48 h post-implantation. By comparing the ratio of mean levels at the donor and back table stages, with the ratio between early (2–6 h) and late (43–48 h) post-reperfusion, these amines were classified into one of three groups. In one group, back table levels were markedly higher than during the donor stage, with levels declining over time post-reperfusion. A second group had much lower levels in the back table than during the donor phase, and post-reperfusion levels were either stable or increased over time. Concentrations of amino acids in the final group remained relatively constant at all stages. This study illustrates the value of microdialysis in providing organ-specific metabolic data that may indicate specific mechanisms of poor graft function.
Keywords: Keywords: Liver – Transplantation – Microdialysis – Amino acids – Ethanolamine – Putrescine
Effect of the L- or D-aspartate on ecto-5′nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A2A receptors by C. R. Boeck; E. H. Kroth; M. J. Bronzatto; D. Vendite (pp. 439-444).
Glutamate increases the extracellular adenosine levels, an important endogenous neuromodulator. The neurotoxicity induced by glutamate increases the ecto-5′-nucleotidase activity in neurons, which produces adenosine from AMP. L- and D-aspartate (Asp) mimic most of the actions of glutamate in the N-methyl-D-aspartate (NMDA) receptors. In the present study, both amino acids stimulated the ecto-5′-nucleotidase activity in cerebellar granule cells. MK-801 and AP-5 prevented the L- and D-Asp-evoked activation of ecto-5′-nucleotidase. Both NMDA receptor antagonists prevented completely the damage induced by L-Asp, but partially the D-Asp-induced damage. The antagonist of adenosine A2A receptors (ZM 241385) prevented totally the L- Asp-induced cellular death, but partially the neurotoxicity induced by D-Asp and the antagonist of adenosine A1 receptors (CPT) had no effect. The results indicated a different involvement of NMDA receptors on the L- or D-Asp-evoked activation of ecto-5′-nucleotidase and on cellular damage. The adenosine formed from ecto-5′-nucleotidase stimulation preferentially acted on adenosine A2A receptor which is probably co-operating with the neurotoxicity induced by amino acids.
Keywords: Keywords: L- and D-aspartate – Neurotoxicity – Ecto-5′-nucleotidase – Adenosine receptors
Purification of branched-chain amino acid aminotransferase from Helicobacter pylori NCTC 11637 by M. Saito; K. Nishimura; S. Wakabayashi; T. Kurihara; Y. Nagata (pp. 445-449).
Branched-chain amino acid aminotransferase was purified by several column chromatographies from Helicobacter pylori NCTC 11637, and the N-terminal amino acid sequence was analyzed. The enzyme gene was sequenced based on a putative branched-chain amino acid aminotransferase gene, ilvE of H. pylori 26695, and the whole amino acid sequence was deduced from the nucleotide sequence. The enzyme existed in a homodimer with a calculated subunit molecular weight (MW) of 37,539 and an isoelectric point (pI) of 6.47. The enzyme showed high affinity to 2-oxoglutarate (K m = 0.085 mM) and L-isoleucine (K m = 0.34 mM), and V max was 27.3 µmol/min/mg. The best substrate was found to be L-isoleucine followed by L-leucine and L-valine. No activity was shown toward the D-enantiomers of these amino acids. The optimal pH and temperature were pH 8.0 and 37 °C, respectively.
Keywords: Keywords: Branched-chain amino acid aminotransferase – Purification – Helicobacter pylori – ilvE gene
Rapid analysis of taurine in energy drinks using amino acid analyzer and Fourier transform infrared (FTIR) spectroscopy as basis for toxicological evaluation by S. Triebel; C. Sproll; H. Reusch; R. Godelmann; D. W. Lachenmeier (pp. 451-457).
So-called energy drinks with very high amounts of taurine (up to 4000 mg/l are usually granted by certificates of exemption) are increasingly offered on the market. To control the currently valid maximum limits of taurine in energy drinks, a simple and rapid analytical method is required to use it routinely in food monitoring. In this article, we describe a fast and efficient analytical method (FTIR-spectroscopy) that is able to reliably characterize and quantify taurine in energy drinks. The determination of taurine in energy drinks by FTIR was compared with amino acid analyzer (ion chromatography with ninhydrin-postcolumn derivatization). During analysis of 80 energy drinks, a median concentration of 3180 mg/l was found in alcohol-free products, 314 mg/l in energy drinks with spirits, 151 mg/l in beer-containing drinks and 305 mg/l in beverages with wine. Risk analysis of these products is difficult due to the lack of valid toxicological information about taurine and its interferences with other ingredients of energy drinks (for example caffeine and alcohol). So far, the high taurine concentrations of energy drinks in comparison to the rest of the diet are scientifically doubtful, as the advertised physiological effects and the value of supplemented taurine are unproven.
Keywords: Keywords: Taurine – Amino acid analyzer – FTIR-Spectroscopy – Energy drinks
Disturbance of erythrocyte lipid bilayer by amino acid-based surfactants by V. Martínez; L. Sánchez; M. A. Busquets; M. R. Infante; M. Pilar Vinardell; M. Mitjans (pp. 459-462).
In an attempt to increase our knowledge regarding the mechanisms of surfactant membrane interaction, we studied the action of several anionic and cationic amino acid-based surfactants on membrane fluidity using fluorescence anisotropy. Anisotropy measurements demonstrated that almost all of the surfactants studied disturbed the external region of the erythrocyte membrane without affecting the core of the bilayer. How the physico-chemical properties and structure of these compounds affect dynamics of the lipid bilayer is discussed in detail.
Keywords: Keywords: Plasma membrane – Fluorescence anisotropy – Surfactants – Erythrocyte – Arginine – Lysine
Thermus thermophilus L4 ribosomal protein: purification and sensitivity alteration against erythromycin of E. coli cells harboring a single amino acid mutant of TthL4 within its extended loop by F. Leontiadou; A. Tsagkalia; T. Choli-Papadopoulou (pp. 463-468).
Protein L4 from the thermophilic bacterium Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells and purified under native conditions by using ion exchange chromatography. Although it’s known strong binding to RNA (23S rRNA as well as mRNA) the yield of the purified protein was 6 mg per 10 g of cells and it is similar to that referred for Thermotoga maritima L4 ribosomal protein.In addition, E. coli cells harboring the wild type Thermus thermophilus L4 (wtTthL4) ribosomal protein as well as its mutant having changed the highly conserved glutamic acid 56 by alanine (TthL4-Ala 56) were incorporated into E. coli ribosomes after transformation of the host cells with the recombined vector. The cells having incorporated the mutant TthL4-Ala56 are more sensitive against erythromycin related to that containing the wtTthL4 protein.The resistance to the drug indicates that the mutated amino acid Glu56 is probably critical for the local ribosomal conformation and that its mutation induces conformational disturbances that are “transferred” to the entrance of the major exit tunnel, the place where the drug does bind.
Keywords: Keywords: Ribosomal proteins – Mutations – Erythromycin sensitivity
Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27kip1 translocation and retinoblastoma protein phosphorylation by 1′-acetoxychavicol acetate by Y. Unahara; A. Kojima-Yuasa; M. Higashida; D. O. Kennedy; A. Murakami; H. Ohigashi; I. Matsui-Yuasa (pp. 469-476).
1′-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27kip1, which is an important regulator of the mammalian cell cycle, we estimated the amount of p27kip1 levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27kip1 levels, but caused a decrease in phosphorylated p27kip1 and an increase in unphosphorylated p27kip1 as well as an increase in the nuclear localization of p27kip1. These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27kip1 phosphorylation and an increase in nuclear localization of p27kip1, and these events are dependent on the cellular thiol status.
Keywords: Keywords: ACA – Tumor cell growth – Rb – p27kip1 – Glutathione – Cell cycle
Synthesis, conformation and biological activity of centrally modified pseudopeptidic analogues of For-Met-Leu-Phe-OMe by C. Giordano; G. Lucente; A. Masi; M. Paglialunga Paradisi; A. Sansone; S. Spisani (pp. 477-487).
For-Met-βAlaψ[CSNH]-Phe-OMe (3), For-Met-βAlaψ[CH2NH]-Phe-OMe (5), For-Met-NH-pC6H4-SO2-Phe-OMe (8a), For-Met-NH-mC6H4-SO2-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. 1H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated β-residue.
Keywords: Keywords: Chemotactic peptides – Conformation – Neutrophils – Pseudopeptides – β-Thiopeptides
Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly Neobellieria bullata by J. Hlaváček; B. Černý; B. Bennettová; J. Holík; R. Tykva (pp. 489-497).
A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of 3H2 to double bond of 3,4-dehydroproline residue. 3H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl β-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [3H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[3H] Pro-Ala-OH, [3H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[3H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[3H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro5-[3H] Pro-OH and H-Asp-[3H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.
Keywords: Keywords: Oostatic peptide synthesis – 3,4-Dehydroproline – 3H labeling – Incorporation in flesh fly
Suppression of myofibrillar proteolysis in chick skeletal muscles by α-ketoisocaproate by K. Nakashima; Y. Yakabe; A. Ishida; M. Yamazaki; H. Abe (pp. 499-503).
We previously reported that L-leucine suppresses myofibrillar proteolysis in chick skeletal muscles. In the current study, we compared the effects of L- and D-enantiomers of leucine on myofibrillar proteolysis in skeletal muscle of chicks. We also assessed whether leucine itself or its metabolite, α-ketoisocaproate (α-KIC), mediates the effects of leucine. Food-deprived (24 h) chicks were orally administered 225 mg/100 g body weight L-leucine, D-leucine or α-KIC and were sacrificed after 2 h. L-Leucine administration had an obvious inhibitory effect on myofibrillar proteolysis (plasma Nτ-methylhistidine concentration) in chicks while D-leucine and α-KIC were much more effective. We also examined the expression of the proteolytic-related genes (ubiquitin, proteasome, m-calpain and cathepsin B) by real-time PCR of cDNA in chick skeletal muscles. Ubiquitin mRNA expression was decreased by D-leucine and α-KIC but not L-leucine. Proteasome and m-calpain mRNA expressions as well as cathepsin B mRNA expression were likewise decreased by L-leucine, D-leucine and α-KIC. These results indicate that D-leucine and α-KIC suppress proteolytic-related genes, resulting in an decrease in myofibrillar proteolysis while L-leucine is much less effective in skeletal muscle of chicks, may be explain by conversion of D-leucine to α-KIC.
Keywords: Keywords: Myofibrillar proteolysis – Leucine – α-Ketoisocaproate – Skeletal muscle – Chick
Effects of 28 days of beta-alanine and creatine monohydrate supplementation on aerobic power, ventilatory and lactate thresholds, and time to exhaustion by R. F. Zoeller; J. R. Stout; J. A. O’Kroy; D. J. Torok; M. Mielke (pp. 505-510).
The effect of beta-alanine (β-Ala) alone or in combination with creatine monohydrate (Cr) on aerobic exercise performance is unknown. The purpose of this study was to examine the effects of 4 weeks of β-Ala and Cr supplementation on indices of endurance performance. Fifty-five men (24.5 ± 5.3 yrs) participated in a double-blind, placebo-controlled study and randomly assigned to one of 4 groups; placebo (PL, n = 13), creatine (Cr, n = 12), beta-alanine (β-Ala, n = 14), or beta-alanine plus creatine (CrBA, n = 16). Prior to and following supplementation, participants performed a graded exercise test on a cycle ergometer to determine VO2peak, time to exhaustion (TTE), and power output, VO2, and percent VO2peak associated with VT and LT. No significant group effects were found. However, within groups, a significant time effect was observed for CrBa on 5 of the 8 parameters measured. These data suggest that CrBA may potentially enhance endurance performance.
Keywords: Keywords: Beta-alanine – Creatine – Exercise – Lactate threshold – Ventilatory threshold
Pathways involved in alanyl-glutamine-induced changes in neutrophil amino- and α-keto acid homeostasis or immunocompetence by J. Mühling; D. Burchert; T. W. Langefeld; R. Matejec; H. Harbach; J. Engel; M. Wolff; I. D. Welters; M. Fuchs; T. Menges; M. Krüll; G. Hempelmann (pp. 511-524).
We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and alanyl-glutamine combined with L-NAME, SNAP, DON, β-alanine and DFMO on neutrophil amino and α-keto acid concentrations or important neutrophil immune functions in order to establish whether an inhibitor of •NO-synthase [L-NAME], an •NO donor [SNAP], an analogue of taurine and a taurine transport antagonist [β-alanine], an inhibitor of ornithine-decarboxylase [DFMO] as well as DON could influence any of the alanyl-glutamine-induced effects. In summary, irrespective of which pharmacological, metabolism-inhibiting or receptor-mediated mechanisms were involved, our results showed that impairment of granulocytic glutamine uptake, modulation of intracellular glutamine metabolisation and/or de novo synthesis as well as a blockade of important glutamine-dependent metabolic processes may led to significant modifications of physiological and immunological functions of the affected cells.
Keywords: Keywords: Alanyl-glutamine – DON – L-NAME – SNAP – β-Alanine – DFMO neutrophil – Amino acids – α-Keto acids – Immune function
Selenomethionine induces polyamine biosynthesis in regenerating rat liver tissue by G. Bjelaković; S. Beninati; D. Pavlović; D. Sokolović; I. Stojanović; T. Jevtović; G. B. Bjelaković; J. Nikolić; J. Bašić (pp. 525-529).
Our study was undertaken to elucidate the effects of selenomethionine (SeMet) on polyamine metabolism in regenerating rat liver tissue, as useful model of rapidly growing normal tissue. We have examined the levels of spermine, spermidine and putrescine in liver tissue. At the same time we have evaluated the activities of polyamine oxidase (PAO) and diamine oxidase (DAO), the catabolic enzymes of polyamine metabolism.The obtained results suggest that polyamine levels in regenerating liver tissue, at 7th day after two-thirds partial hepatectomy, were higher in comparison with control group. The administration of selenomethionine to hepatectomized animals during seven days, in a single daily dose of 2.5 µg/100 g body weight, increases the amount of spermine and spermidine; the level of putrescine does not change under the influence of SeMet in regenerating rat liver tissue.PAO activity is lower in regenerating hepatic tissue than in control group. Supplementation of hepatectomized animals with SeMet significantly decreases the activity of this enzyme. DAO activity was significantly higher in hepatectomized and in operated animals treated with SeMet compared to the sham-operated and control ones.The differential sensitivity observed in our model of highly proliferating normal tissue to SeMet, compared with the reported anticancer activity of this molecule is discussed.
Keywords: Keywords: Se-methionine – Polyamines – Polyamine oxidase – Diamine oxidase – Liver regeneration – Rats
Optimization of solid-phase synthesis of difficult peptide sequences via comparison between different improved approaches by S. Abdel Rahman; A. El-Kafrawy; A. Hattaba; M. F. Anwer (pp. 531-536).
Different approaches are applied to avoid the strong aggregation of the difficult peptide sequences, which is considered as the main reason for incomplete acylation and deprotection reactions hindering the synthesis of these sequences.Temporary protection of amide nitrogen of peptide bond using 2-hydroxy-4-methoxybenzyl (Hmb) and 2,4,6-timethoxybenzyl (Tmob) amino acid derivatives, introduction of D-Ala or Pro residues in the peptide chain sequences and utilization of microwave energy are proved to be useful methods in the enhancement of solubility and in the hindrance of the aggregation during the solid-phase synthesis of oligoalanine. Oligoalanine is chosen to demonstrate the difficult sequences and to compare the efficiencies of these methods.
Keywords: Keywords: Difficult peptide sequences – Solid-phase peptide synthesis – Oligo-alanine – N-amide protection – Microwave energy
Facile synthesis of optically pure (S)-3-p-hydroxyphenyllactic acid derivatives by Q. L. Zeng; H. Q. Wang; Z. R. Liu; B. G. Li; Y. F. Zhao (pp. 537-541).
Optically pure (S)-3-p-hydroxyphenyllactic acid derivatives are important intermediates of peroxisome proliferator-activated receptor α/γ dual agonists and heteropeptides. Many efforts have been made for synthesis of those intermediates, but there exist some flaws yet. We observed that dielectric constants of organic solvents drastically affected diazotization of O-benzyl-L-tyrosine. Optically pure (S)-3-p-benzyloxyphenyllactic acid was obtained by simple recrystallization when DMF or DMSO of higher dielectric constant was used as a co-solvent in diazotization of O-benzyl-L-tyrosine. It was easily turned into various optically pure (S)-3-p-hydroxyphenyllactic acid derivatives.
Keywords: Keywords: Solvent effects – Diazotization – O-Benzyl-L-tyrosine – (S)-3-p-Benzyloxyphenylpropionic acid – (S)-p-Hydroxyphenyllactic acid
Effect of β-alanine administration on carbon tetrachloride-induced acute hepatotoxicity by S. Y. Lee; Y. C. Kim (pp. 543-546).
Mice were supplemented with β-alanine (3%) in drinking water for one week. β-Alanine intake reduced hepatic taurine levels, but elevated cysteine levels significantly. Hepatotoxicity of CCl4 in mice fed with β-alanine was decreased as determined by changes in serum enzyme activities. Hepatic glutathione and taurine concentrations after CCl4 challenge were increased markedly by β-alanine intake. The enhanced availability of cysteine for synthesis of glutathione and/or taurine appears to account for the hepatoprotective effects of β-alanine against CCl4-induced liver injury.
Keywords: Keywords: β-Alanine – Carbon tetrachloride – Cysteine – Glutathione – Taurine