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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.27, #2)
Proteins interacting with the tuberous sclerosis gene products by M. Rosner; A. Freilinger; M. Hengstschläger (pp. 119-128).
Tuberous sclerosis (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 to 10000 individuals. The genes, TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for TSC. Since their identification 1997 and 1993 respectively, a variety of different functions have been described for the TSC gene products. Hamartin and tuberin form a complex, providing a tentative explanation for the similar disease phenotype in TSC patients with mutations in either of these genes. In addition, associations of hamartin or tuberin with several different proteins have been demonstrated. In this review, we summarize the current knowledge on hamartin- and tuberin-interacting proteins and discuss their role for the understanding of the functions of the TSC gene products.
Keywords: Keywords: Tuberous sclerosis – Hamartin – Tuberin – TSC1 – TSC2 – Protein interaction
Expression of proteasomal proteins in ten different tumor cell lines by L. Afjehi-Sadat; M. Gruber-Olipitz; M. Felizardo; I. Slavc; G. Lubec (pp. 129-140).
Controlled intracellular protein degradation is crucial for the maintenance of normal cell functions. An evolving concept claims that alterations in the exact timely degradation of proteins involved in growth control, apoptosis, signaling and differentiation contribute to carcinogenesis. This tightly regulated process is facilitated by the ubiquitin-26S proteasome system, a multi-enzyme complex, and inhibitors of this pathway have already been developed as potential anticancer agents.In order to generate proteasomal protein expression patterns of tumor cells and to provide an analytical tool we applied two-dimensional electrophoresis (2-DE) followed by mass spectrometry (MALDI-TOF-TOF with LIFT technology) in ten individual tumor cell lines (Saos-2; SK-N-SH; HCT-116; Caov3; A-549; HL60; A-673; A-375; MCF-7; HeLa) widely used in tumor research. A series of 39 proteasomal/proteolytic proteins was unambiguously identified by this proteomic approach, comprising proteins of the 20S core complex, the 19S regulatory complex, the 11S regulator, components of the ubiquitin pathway and proteases.Construction of individual protein maps by 2-DE and mass spectrometry provides an analytical tool and reference base for studying the pivotal importance of the proteasome and other proteolytic enzymes in tumor cells, independent of antibody availability and specificity. This preliminary database enables for designing studies in this area of research and reveals proteins that can be used as targets for new therapeutic strategies.
Keywords: Keywords: Proteasome – Tumor cell line – Cancer – Protein profiling
Proteomic basis for the possible use of lymphocytes for metabolic screenings by J. E. Oh; K. Krapfenbauer; G. Lubec (pp. 141-147).
The advent of proteomics has provided a tool for the concomitant identification and determination of a large series of proteins using two-dimensional gel electrophoresis with subsequent mass spectrometrical analysis. We tried an approach to analyse the high abundance enzyme proteome of a lymphocytic cell line.Immortalised lymphocytes were grown in RPMI 1640 in the presence of glutamine, harvested and the 100,000 × g supernatant of the homogenate was applied on two-dimensional gel electrophoresis with subsequent in-gel digestion of protein spots and MALDI-TOF (Matrix-associated laser desorption/ionization mass spectroscopy) analysis of resulting peptides using specific software.A series of 57 metabolic enzymes were identified including enzymes of carbohydrate, amino acid, purine and intermediary metabolism.We are presenting a tool for the analysis of metabolic systems including enzyme deficiencies at the protein level with the advantage of unambiguous identification of proteins and thus complementing enzyme activity determinations.
Keywords: Keywords: Two-dimensional gel electrophoresis – Lymphocyte – Metabolic enzymes – Mass spectrometry
Efficient preparation of L-cysteic acid and its esters by F. Tao; Y. Luo; Q. Wei; G. Zhang (pp. 149-151).
L-Cysteic acid and its esters were prepared in good yields from the oxidation of L-cystine by chlorine in water and in alcohols. When the reaction was carried out in alcohols the corresponding esters were produced.
Keywords: Keywords: L-Cysteic acid – L-Cysteic acid ester – L-Cystine – Oxidation – Chlorine
N-t-Boc-amino acid esters of isomannide Potential inhibitors of serine proteases by E. M. F. Muri; M. Gomes Jr.; J. S. Costa; F. L. Alencar; A. Sales Jr.; M. L. Bastos; R. Hernandez-Valdes; M. G. Albuquerque; E. F. F. da Cunha; R. B. Alencastro; J. S. Williamson; O. A. C. Antunes (pp. 153-159).
Hepatitis C, Dengue and West Nile virus are some of the most important flaviviruses, that share one important serine protease enzyme. Serine proteases are the most studied class of proteolytic enzyme and, in these cases, a primary target for drug discovery. In this paper, we describe the synthesis and preliminary molecular modeling studies of a novel class of N-t-Boc amino acid esters derived of isomannide as potential serine proteases inhibitors.
Keywords: Keywords: Isomannide derivatives – Serine protease – Dengue virus
Synthesis of Nɛ-protected-L-lysine and γ-benzyl-L-glutamate N-carboxyanhydrides (NCA) by carbamoylation and nitrosation by W. Vayaboury; O. Giani; H. Collet; A. Commeyras; F. Schué (pp. 161-167).
This paper reports on an original process to synthesize N-carboxyanhydrides, which consists of nitrosating N-carbamoylamino acids with a NO/O2 gas mixture in acetonitrile. The synthesis of several N-carbamoylamino acids of L-lysine was described using potassium cyanate in water. The latter were then nitrosated to yield the corresponding NCA with more or less efficiency. Indeed, the NCA carrying an acid-sensitive protecting group led to a partial deprotection to give the L-lysine NCA salt. The NCA of Nɛ-trifluoroacetyl-L-lysine, Nɛ-benzyloxycarbonyl-L-lysine and γ-benzyl-L-glutamate were successfully synthesized with satisfactory yields. Their polymerizability was compared to that of the Nɛ-trifluoroacetyl-L-lysine NCA initiated by n-hexylamine in N,N-dimethylformamide. It also showed that this new process of NCA synthesis could be applied to the synthesis of polypeptides and more generally to the protein chemistry.
Keywords: Keywords: N-Carboxyanhydride – Carbamoylation – Nitrosation – Poly(Nɛ-trifluoroacetyl-L-lysine) – Poly(L-lysine)
Production of amino acids by Rhizobium, Mesorhizobium and Sinorhizobium strains in chemically defined media by V. Salmeron-Lopez; M. V. Martinez-Toledo; V. Salmeron-Miron; C. Pozo; J. Gonzalez-Lopez (pp. 169-174).
Five strains of Rhizobium spp, one strain of Mesorhizobium loti and two strains of Sinorhizobium meliloti were tested for their ability to grow in chemically-defined medium lacking growth factor. Qualitative and quantitative production of aspartic acid, serine, glutamic acid, glycine, histidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine was determined by the use of mannitol as sole carbon source.Strains of Rhizobium spp. and Sinorhizobium sp. produced all the amino acids analysed with the exception of cysteine and high biological levels of serine, glycine and alanine were detected after 2 days of culture in mineral medium.Strain U226 of M. loti only produced small amounts of amino acids and glutamic acid, histidine, arginine, cysteine, methionine, lysine and phenylalanine was not liberated into the media.
Keywords: Keywords: Amino acids – Rhizobium – Mesorhizobium – Sinorhizobium
Oxidation of buried cysteines is slow and an insignificant factor in the structural destabilization of staphylococcal nuclease caused by H2O2 exposure by Y. H. Kim; W. E. Stites (pp. 175-181).
The oxidation of buried cysteine or methionine residues can destroy the enzyme activity of a protein by disrupting structure. Engineering in such an oxidatively triggered switch for enzyme activity would only be useful if the effects of substitution are relatively minor, while the effects of the oxidized side chain upon structure are significant and the oxidation relatively easy. To assess the feasibility of this strategy for controlling enzyme activity, the effects of such substitutions and their oxidation were studied in a well characterized model protein, staphylococcal nuclease. Stability and enzyme activity of the oxidized proteins was assessed and compared to the stability and enzyme activity of the unoxidized proteins. Cysteines were found to be generally well tolerated in buried positions but these mutants were not more destabilized than wild-type when oxidized. This shows that buried cysteines are difficult enough to oxidize that this is not likely to be a useful protein engineering strategy or a commonly used regulatory modification. Similar effects were observed for methionine.
Keywords: Keywords: Enzyme activity – Rate – Redox – Regulation – Solvent accessibility
Mild and effective N-phthaloylation of amino acids by Q. Zeng; Z. Liu; B. Li; F. Wang (pp. 183-186).
In the present work various free amino acids, including tryptophan and tyrosine, were effectively N-phthaloylated under reduced pressure and at lower temperature. Moreover, under these conditions, the presence of phthalic acid in phthalic anhydride did not hinder the N-phthaloylation of amino acids. This simple process is economic, environmentally friendly, and suitable for large-scale production.
Keywords: Keywords: Amino acids – Phthalic anhydride – N-Phthaloylation
Long-term taurine supplementation reduces mortality rate in streptozotocin-induced diabetic rats by M. A. S. Di Leo; S. A. Santini; N. Gentiloni Silveri; B. Giardina; F. Franconi; G. Ghirlanda (pp. 187-191).
Oxidative stress is implicated in the pathogenesis of diabetes mellitus. Taurine and vitamin E+selenium supplementation has some benefits in experimental models of diabetes mellitus. This study evaluates whether taurine and vitamin E+selenium supplementations reduce a hard end-point such as mortality due to diabetes. Streptozotocin-induced diabetic rats were fed with standard diet or taurine (5%, w/w) or vitamin E (500 UI/Kg)+selenium (8 mg/Kg) enriched diets. Taurine significantly decreased mortality rate (p < 0.04), while vitamin E failed to increase survival. In the late phase of the disease, taurine significantly decreased glycaemia, being vitamin E ineffective. No correlation between glycaemia and survival was found. None of supplementations modified body weight. Thus, only taurine decreases the mortality rate and glycaemia. These results encourage new research in the field, since classical hypoglycaemic agents are unable to decrease mortality in diabetic patients.
Keywords: Keywords: Taurine – Streptozotocin – Diabetes mellitus – Selenium – Vitamin E – Survival – Glycaemia
Kinetic study of racemization of aspartyl residues in synthetic elastin peptides by K. Kuge; N. Fujii; Y. Miura; S. Tajima; T. Saito (pp. 193-197).
We previously reported that biologically uncommon D-aspartyl residues are present in sun-damaged skin from elderly people, possibly in elastin. Here, we report the kinetics of Asp racemization in model peptides corresponding to elastin sequences from exons 6 and 26. We estimated the activation energy (E) of racemization of Asp residues, the racemization rates (RR) at 37°C and the time (t) required for the D/L ratio of Asp to approximate to 1.0 (D/L ratio of Asp=0.99) at 37°C. For an exon 6 peptide, E=29.0 kcal/mol, RR=2.59×10−2/yr and t=101.0 yr. For an exon 26A peptide E=26.2 kcal/mol, RR=4.27×10−2/yr and t=61.3 yr; and for a second exon 26A peptide E=25.7 kcal/mol, RR=5.55×10−2/yr and t=47.0 yr. These results suggest that racemization of Asp residues in elastin could occur within a human life span. We propose that D-Asp could be a useful molecular indicators of aging.
Keywords: Keywords: Aging – D-Aspartic acid – Elastin – Kinetics – Racemization – Racemization rate constant – Skin
Betaine or taurine administration prevents fibrosis and lipid peroxidation induced by rat liver by ethanol plus carbon tetrachloride intoxication by F. Erman; J. Balkan; U. Çevikbaş; N. Koçak-Toker; M. Uysal (pp. 199-205).
The aim of this study was to investigate the effect of betaine or taurine on liver fibrogenesis and lipid peroxidation in rats. Fibrosis was induced by treatment of rats with drinking water containing 5% ethanol and CCl4 (2 × weekly, 0.2 ml/kg, i.p.) for 4 weeks. Ethanol plus CCl4 treatment caused increased lipid peroxidation and disturbed antioxidant system in the liver. Histopathological findings suggested that the development of liver fibrosis was prevented in rats treated with betaine or taurine (1% v/v in drinking water) together with ethanol plus CCl4 for 4 weeks. When hepatic taurine content was depleted with β-alanine (3% v/v in drinking water), portal-central fibrosis induced by ethanol + CCl4 treatment was observed to proceed cirrhotic structure. Betaine or taurine was also found to decrease serum transaminase activities and hepatic lipid peroxidation without any change in hepatic antioxidant system in rats with hepatic fibrosis. In conclusion, the administration of betaine or taurine prevented the development of liver fibrosis probably associated with decreased oxidative stress.
Keywords: Keywords: Betaine – Taurine – Taurine depletion – Liver fibrosis – Oxidative stress – Carbon tetrachloride – Ethanol
Polyamine depletion inhibits etoposide-induced NF-κB activation in transformed mouse fibroblasts by B. Tantini; C. Pignatti; M. Fattori; E. Fiumana; A. Facchini; C. Stefanelli; C. M. Caldarera; A. E. Pegg; F. Flamigni (pp. 207-214).
In a previous research, we have shown that adequate levels of polyamines are required in transformed mouse fibroblasts for the correlated activations of MAPK subtypes (ERK and JNK) and caspases induced by etoposide and leading to apoptosis. We report now that the treatment of fibroblasts with etoposide also elicited a progressive and sustained increase of NF-κB activation. The DNA binding activity of p65 NF-κB subunit was increased up to approximately 4-fold and was accompanied by enhancement of p65 phosphorylation. A two days pre-treatment of fibroblasts with α-difluoromethylornithine (DFMO), which caused polyamine depletion, provoked a slight activating effect when given alone, but markedly inhibited the etoposide-induced increases in p65 DNA binding and phosphorylation. The NF-κB inhibiting effect of DFMO was prevented by the addition of exogenous putrescine, which restored the intracellular content of polyamines. Selective inhibitors of the etoposide-stimulated MAPK subtypes also reduced NF-κB activation. Moreover, pharmacological NF-κB inhibition reduced the increase in caspase activity and cell death elicited by etoposide, suggesting that NF-κB is involved in signaling to apoptosis. The results of the present study, together with our previous findings, suggest that polyamines play a permissive role in the pathways triggered by etoposide and leading to cell death of fibroblasts, by supporting the activation of MAPKs, NF-κB and caspases.
Keywords: Keywords: Apoptosis – Difluoromethylornithine – Etoposide – MAPK – NF-κB – Polyamines
Tryptophan administration increase contractility and change the ultrastructure of mice duodenum by Ç. Özer; B. Gönül; G. Take; D. Erdoğan; E. Tong; Z. S. Ercan (pp. 215-220).
Serotonin (5-HT) is a metabolite of tryptophan (TRP). 5-HT has been shown to induce contractions in rat duodenum and ileum. We planned to investigate the in vivo effects of TRP administration on duodenal contractility and ultrastructure together.Two equal groups of adult male Swiss-albino mice were used in the experiments.Controls (CONT) and TRP treated (100 mg/kg/24 hr in 0.2 ml. saline solution ip, 7 days).Body weights were recorded at the beginning and at the end of experiments. Duodenum tissues contractility responses to different concentration of KCl and acethycholine (ACh) were recorded on polygraph. The ultrastructural changes in duodenum observed by transmission electron microscopic (TEM) method and 5-HT levels determined by immunohistochemical method.Body weights decreased and duodenal contractile response of ACh increased significantly by TRP treatment. The duodenal ultrastructural changes in TRP group illustrated partially loss of apical surface and fusion in microvilli. Immunohistochemical detection showed that 5-HT increased by TRP treatment.There is a relation between duodenal contractility increased by TRP treatment and changes in the duodenal tissue 5-HT level and ultrastructure.
Keywords: Keywords: Tryptophan – Serotonin – Duodenal contractility – TEM – Immunohistochemistry
Library screening for D-amino-acid oxidase gene: Application of real-time PCR by R. Konno; A. Niwa (pp. 221-223).
Quantitative real-time PCR shows the quantity in addition to the presence of the target sequence. This property seemed very useful for library screening. Then, real-time PCR was employed to screen for λ phages carrying D-amino-acid oxidase gene from mouse genomic library. Using stepwise dilution screening combined with real-time PCR, positive phages were isolated in a short time.
Keywords: Keywords: Real-time PCR – D-Amino-acid oxidase – Library – Screening – Mouse
The effect of taurine administration on vitamin C levels of several tissues in mice by B. Kaplan; S. Dinçer; A. Babül; İ. Duyar (pp. 225-228).
Taurine (2-aminoethane sulphonic acid), a sulphur-containing beta amino acid, is the most prevalent free intracellular amino acid in many human and animal tissues. Vitamin C metabolism is also fluenced by sulphur-containing amino acids. The aim of this study is to investigate the effect of taurine administration on the vitamin C levels of plasma and several tissues (brain, liver, kidneys) in mice with incisional skin wounds. Animals were divided into two as control and taurine groups. Taurine was freshly dissolved in sterile saline and administered daily (60 µl, ip) for five days in the taurine group. At the end of the fifth day, the animals were killed by decapitation. The brain, liver and kidneys were immediately removed. Vitamin C levels were measured in plasma and several tissues. The administration of taurine had no effect on the plasma vitamin C levels (P>0.05) but significantly increased in liver and kidneys (P<0.001). In conclusion, taurine may affect the vitamin C metabolism in tissues by different mechanisms.
Keywords: Keywords: Taurine – Vitamin C – Mice