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Leucine
Terms modified by Leucine Selected AbstractsAmino acid substitutions in the hepatitis C virus core region are the important predictor of hepatocarcinogenesis,HEPATOLOGY, Issue 5 2007Norio Akuta We showed previously that amino acid (aa) substitutions in hepatitis C virus core region (HCV-CR) are negative predictors of virologic response to pegylated interferon (IFN) plus ribavirin therapy. HCV-CR induces hepatocellular carcinoma in transgenic mice, but the clinical impact is still unclear. To evaluate the impact of aa substitutions in HCV-CR on hepatocarcinogenesis, we performed a follow-up study on 313 noncirrhotic consecutive naïve patients infected with HCV genotype 1b who received IFN monotherapy. The median follow-up was 14.7 years. A sustained virologic response (SVR) after the first IFN was achieved by 65 patients (20.8%) (group A). Of 248 patients (79.2%) of non-SVR after first IFN, 112 (35.8%) did not receive additional IFN (group B), and the remaining 136 (43.5%) received multicourse IFN monotherapy (group C). As a whole, cumulative hepatocarcinogenesis rates in double wild-type (arginine at aa 70/leucine at aa 91) of HCV-CR were significantly lower than those in nondouble wild-type. Multivariate analyses identified 3 parameters (fibrosis stage 3, nondouble wild-type of HCV-CR, and group B) that tended to or significantly influenced hepatocarcinogenesis independently. With regard to hepatocarcinogenesis rates in group C according to HCV-CR and the mean alanine aminotransferase (ALT) during IFN-free period, significantly higher rates were noted in patients of nondouble wild-type with ALT levels of more than 1.5 times the upper limit of normal (25.7%) compared with the others (2.4%). Conclusion: Amino acid substitutions in the HCV-CR are the important predictor of hepatocarcinogenesis. In multicourse IFN therapy to nondouble wild-type, we emphasize the importance of reducing the risk of hepatocarcinogenesis by mean ALT during an IFN-free period below 1.5 times the upper limit of normal. (HEPATOLOGY 2007.) [source] Nutritional study of raw and popped seed proteins of Amaranthus caudatus L and Amaranthuscruentus LJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2004Tamer H Gamel Abstract The nutritional value of raw and popped (similar to popcorn preparation) seed proteins of two amaranth species, Amaranthus caudatus L and A cruentus L, was investigated. After popping, the true protein content in A caudatus and A cruentus decreased by 9 and 13% respectively. Among the amino acids, the loss of tyrosine due to the popping effect was the highest, followed by phenylalanine and methionine. Leucine was the first limiting amino acid in the raw samples, followed by lysine, while the reverse order was observed in the popped samples. The in vivo protein quality of raw and popped seeds was tested with male weanling rats and compared with wheat flour and casein samples. There was no difference between the in vivo digestibility of the raw and the popped seeds, although the in vitro digestibility was slightly higher for the popped samples. The protein efficiency ratio (PER) for all the amaranth seed samples was higher than that for the wheat sample, while the PER for the raw amaranth seed samples was close to that for the casein reference protein. The rat blood serum levels of total cholesterol, triglycerides and high-density lipoprotein cholesterol for all the amaranth samples were lower than those for the reference protein, while the wheat flour sample showed the lowest values. Copyright © 2004 Society of Chemical Industry [source] Leucine metabolism in regulation of insulin secretion from pancreatic beta cellsNUTRITION REVIEWS, Issue 5 2010Jichun Yang Leucine, a branched-chain amino acid that must be supplied in the daily diet, plays an important role in controlling protein synthesis and regulating cell metabolism in various cell types. In pancreatic , cells, leucine acutely stimulates insulin secretion by serving as both metabolic fuel and allosteric activator of glutamate dehydrogenase to enhance glutaminolysis. Leucine has also been shown to regulate gene transcription and protein synthesis in pancreatic islet , cells via both mTOR-dependent and -independent pathways at physiological concentrations. Long-term treatment with leucine has been shown to improve insulin secretory dysfunction of human diabetic islets via upregulation of certain key metabolic genes. In vivo, leucine administration improves glycemic control in humans and rodents with type 2 diabetes. This review summarizes and discusses the recent findings regarding the effects of leucine metabolism on pancreatic ,-cell function. [source] Widespread distribution of knockdown resistance mutations in the bed bug, Cimex lectularius (Hemiptera: Cimicidae), populations in the United StatesARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Fang Zhu Abstract We previously reported high deltamethrin resistance in bed bugs, Cimex lectularius, collected from multiple areas of the United States (Romero et al., 2007). Recently, two mutations, the Valine to Leucine mutation (V419L) and the Leucine to Isoleucine mutation (L925I) in voltage-gated sodium channel ,-subunit gene, had been identified to be responsible for knockdown resistance (kdr) to deltamethrin in bed bugs collected from New York (Yoon et al., 2008). The current study was undertaken to investigate the distribution of these two kdr mutations in 110 bed bug populations collected in the United States. Out of the 17 bed bug populations that were assayed for deltamethrin susceptibility, two resistant populations collected in the Cincinnati area and three deltamethrin-susceptible lab colonies showed neither of the two reported mutations (haplotype A). The remaining 12 populations contained L925I or both V419L and L925I mutations in voltage-gated sodium channel ,-subunit gene (haplotypes B&C). In 93 populations that were not assayed for deltamethrin susceptibility, 12 contained neither of the two mutations (haplotype A) and 81 contained L925I or V419L or both mutations (haplotypes B-D). Thus, 88% of the bed bug populations collected showed target-site mutations. These data suggest that deltamethrin resistance conferred by target-site insensitivity of sodium channel is widely spread in bed bug populations across the United States. © 2010 Wiley Periodicals, Inc. [source] Protein Synthesis Assisted by Native Chemical Ligation at LeucineCHEMBIOCHEM, Issue 9 2010Ziv Harpaz Triggering leucine: A new ligation strategy of using ,-mercaptoleucine coupled with desulfurization at leucine sites was developed, and its applicability in protein synthesis is presented. The efficiency of our Leu-NCL was examined in several model peptides and utilized for the first total synthesis of HIV-1 Tat protein. [source] Preferred Conformations of Peptides Containing tert -Leucine, a Sterically Demanding, Lipophilic ,-Amino Acid with a Quaternary Side-Chain C, AtomCHEMISTRY - A EUROPEAN JOURNAL, Issue 8 2005Fernando Formaggio Prof. Abstract Terminally protected homopeptides of tert -leucine, from the dimer to the hexamer, co-oligopeptides of tert -leucine in combination with ,-aminoisobutyric acid or glycine residues up to the hexamer level, and simple dipeptides representing known scaffolds for catalysts in asymmetric organic reactions were prepared by solution methods and fully characterized. The results of conformation analysis, performed by use of FT-IR absorption, NMR, CD, and X-ray diffraction techniques, indicate that this hydrophobic ,-amino acid with tetrasubstitution at the C, atom is structurally versatile. We show that it prefers extended or semiextended conformations, but can also be accommodated in folded structures, provided that these are biased by the presence of helicogenic residues. The current large-scale production of Tle, combined with its conformational preferences unravelled in this work, should make this bulky, hydrophobic, C, -trisubstituted ,-amino acid a regular building block of any strategy seeking to tailor peptides with improved catalytic and pharmacological properties. [source] Photolysis of rac -Leucine with Circularly Polarized Synchrotron RadiationCHEMISTRY & BIODIVERSITY, Issue 6 2010Abstract Amino acids that pass the RNA machinery in living organisms occur in L -configuration. The question on the evolutionary origin of this biomolecular asymmetry remains unanswered to this day. Amino acids were detected in artificially produced interstellar ices, and L -enantiomer-enriched amino acids were identified in CM-type meteorites. This hints at a possible interstellar/circumstellar origin of the amino acids themselves as well as their stereochemical asymmetry. Based upon the current knowledge about the occurrence of circularly-polarized electromagnetic radiation in interstellar environments, we subjected rac -leucine to far-UV circularly-polarized synchrotron radiation. Asymmetric photolysis was followed by an analysis in an enantioselective GC/MS system. Here, we report on an advanced photolysis rate of more than 99% for leucine. The results indicate that high photolysis rates can occur under the chosen conditions, favoring enantioselective photolysis. In 2014, the obtained results will be reexamined by cometary mission Rosetta. [source] Cell hydration and mTOR-dependent signallingACTA PHYSIOLOGICA, Issue 1-2 2006F. Schliess Abstract Insulin- and amino acid-induced signalling by the mammalian target of rapamycin (mTOR) involves hyperphosphorylation of the p70 ribosomal S6 protein kinase (p70S6-kinase) and the eukaryotic initiation factor 4E (eIF4E) binding protein 4E-BP1 and contributes to regulation of protein metabolism. This review considers the impact of cell hydration on mTOR-dependent signalling. Although hypoosmotic hepatocyte swelling in some instances activates p70S6-kinase, the hypoosmolarity-induced proteolysis inhibition in perfused rat liver is insensitive to mTOR inhibition by rapamycin. Likewise, swelling-dependent proteolysis inhibition by insulin and swelling-independent proteolysis inhibition by leucine, a potent activator of p70S6-kinase and 4E-BP1 hyperphosphorylation, in perfused rat liver is insensitive to rapamycin, indicating that at least rapamycin-sensitive mTOR signalling is not involved. Hyperosmotic dehydration in different cell types produces inactivation of signalling components around mTOR, thereby attenuating insulin-induced glucose uptake, glycogen synthesis, and lipogenesis in adipocytes, and MAP-kinase phosphatase MKP-1 expression in hepatoma cells. Direct inactivation of mTOR, stimulation of the AMP-activated protein kinase, and the destabilization of individual proteins may impair mTOR signalling under dehydrating conditions. Further investigation of the crosstalk between the mTOR pathway(s) and hyperosmotic signalling will improve our understanding about the contribution of cell hydration changes in health and disease and will provide further rationale for fluid therapy of insulin-resistant states. [source] Metabolism of high density lipoprotein apolipoprotein A-I and cholesteryl ester in insulin resistant dog: a stable isotope studyDIABETES OBESITY & METABOLISM, Issue 1 2007F. Briand Aims:, In reverse cholesterol transport (RCT), hepatic Scavenger Receptor class B type I (SR-BI) plays an important role by mediating the selective uptake of high-density lipoprotein cholesteryl ester (HDL-CE). However, little is known about this antiatherogenic mechanism in insulin resistance. HDL-CE selective uptake represents the main process for HDL-CE turnover in dog, a species lacking cholesteryl ester transfer protein activity. We therefore investigate the effects of diet induced insulin resistance on RCT. Methods:, Five beagle dogs, in healthy and insulin resistant states, underwent a primed constant infusion of [1,213C2]acetate and [5,5,5- 2H3]leucine, as labelled precursors of CE and apolipoprotein (apo) A-I, respectively. Data were analysed using modelling methods. Results:, HDL-apo A-I concentration did not change in insulin resistant state but apo A-I absolute production rate (APR) and fractional catabolic rate (FCR) were both higher (2.2- and 2.4-fold, respectively, p < 0.05). HDL-CE levels were lower (1.2-fold, p < 0.05). HDL-CE APR and FCR were both lower (2.3- and 2-fold, respectively, p < 0.05), as well as selective uptake (2.6-fold, p < 0.05). Conclusions:, Lower HDL-CE selective uptake suggests that RCT is impaired in obese insulin resistant dog. [source] Polymeric alkenoxy amino acid surfactants: II.,Chiral separations of ,-blockers with multiple stereogenic centersELECTROPHORESIS, Issue 6 2004Syed A. A. Rizvi Abstract Two amino acid-based (leucine and isoleucine) alkenoxy micelle polymers were employed in this study for the separation of multichiral center-bearing ,-blockers, nadolol and labetalol. These polymers include polysodium N -undecenoxy carbonyl- L -leucinate (poly- L -SUCL) and polysodium N -undecenoxy carbonyl- L -isoleucinate (poly- L -SUCIL). Detailed synthesis and characterization were reported in our previous paper [26]. It was found that poly- L -SUCIL gives better chiral separation than poly- L -SUCL for both nadolol and labetalol isomers. The use of 50,100 mM poly- L -SUCIL as a single chiral selector provided separation of four and three isomers of labetalol and nadolol, respectively. Further optimization in separation of both enantiomeric pairs of nadolol and labetalol was achieved by evaluation of type and concentration of organic solvents, capillary temperature as well type and concentration of cyclodextrins. A synergistic approach, using a combination of poly- L -SUCIL and sulfated ,-CD (S-,-CD) was evaluated and it showed dramatic separation for enantiomeric pairs of nadolol. On the other hand for labetalol enantiomers, separation was slightly decreased or remain unaffected using the dual chiral selector system. Finally, simultaneous separation of both nadolol and labetalol enantiomers was achieved in a single run using 25 mM poly- L -SUCIL and 5% w/v of S-,-CD in less then 35 min highlighting the importance of high-throughput chiral analysis. [source] Abundance and diversity of heterotrophic bacterial cells assimilating phosphate in the subtropical North Atlantic OceanENVIRONMENTAL MICROBIOLOGY, Issue 10 2010Krista Longnecker Summary Microorganisms play key roles in the cycles of carbon and nutrients in the ocean, and identifying the extent to which specific taxa contribute to these cycles will establish their ecological function. We examined the use of 33P-phosphate to identify heterotrophic bacteria actively involved in the cycling of phosphate, an essential inorganic nutrient. Seawater from the sub-tropical North Atlantic Ocean was incubated with 33P-phosphate and analysed by microautoradiography to determine the proportion and diversity of the bacterial community-assimilating phosphate. Complementary incubations using 3H-leucine and 3H-thymidine were also conducted. We found that a higher proportion of total heterotrophic bacterial cells in surface water samples assimilated phosphate compared with leucine or thymidine. Bacteria from all of the phylogenetic groups we identified by CARD-FISH were able to assimilate phosphate, although the abundances of cells within each group did not scale directly with the number found to assimilate phosphate. Furthermore, a significantly higher proportion of Alphaproteobacteria, Gammaproteobacteria and Cytophaga -like cells assimilated phosphate compared with leucine or thymidine. Our results suggest that a greater proportion of bacterial cells in surface waters are actively participating in the biogeochemical cycling of phosphorus, and possibly other elements, than is currently estimated through the use of 3H-leucine or 3H-thymidine. [source] Substrate incorporation patterns of bacterioplankton populations in stratified and mixed waters of a humic lakeENVIRONMENTAL MICROBIOLOGY, Issue 7 2009Ulrike Buck Summary Bacterial incorporation of glucose, leucine, acetate and 4-hydroxybenzoic acid (HBA) was investigated in an artificially divided humic lake (Grosse Fuchskuhle, Germany). Two basins with contrasting influx of allochthonous organic carbon were sampled during late summer stratification (oxic and anoxic layers) and after autumn mixing. High total and cell-specific incorporation rates were observed for glucose and HBA in stratified and mixed waters respectively, but only a small fraction of bacteria visibly incorporated HBA. The oxic layer of the more humic-rich basin featured a significantly lower fraction of glucose incorporating cells and substantially higher proportions of acetate assimilating bacteria. Niche differentiation was observed in two betaproteobacterial populations: cells affiliated with the Polynucleobacter C subcluster efficiently incorporated acetate but little glucose, whereas the opposite was found for members of the R-BT065 clade. By contrast, leucine incorporation was variable in both taxa. Considering the high concentrations and rapid photochemical generation of organic acids in humic waters our results may help to explain the success of the Polynucleobacter C lineage in such habitats. Specific substrate or habitat preferences were also present in three subgroups of the actinobacterial acI lineage: The numerically dominant clade in oxic waters (acI-840-1) was absent in the anoxic zone and did not incorporate acetate. A second group (acI-840-2) was found both in the epi- and hypolimnion, whereas the third one (acI-840-3) only occurred in anoxic waters. Altogether our results suggest a constitutive preference for some substrates versus an adaptive utilization of others in the studied microbial groups. [source] Universal and species-specific bacterial ,fungiphiles' in the mycospheres of different basidiomycetous fungiENVIRONMENTAL MICROBIOLOGY, Issue 2 2009J. A. Warmink Summary In previous work, several bacterial groups that show a response to fruiting bodies (the mycosphere) of the ectomycorrhizal fungus Laccaria proxima were identified. We here extend this work to a broader range of fungal fruiting bodies sampled at two occasions. PCR-DGGE analyses showed clear effects of the mycosphere of diverse fungi on the total bacterial and Pseudomonas communities in comparison with those in the corresponding bulk soil. The diversities of the Pseudomonas communities increased dramatically in most of the mycospheres tested, which contrasted with a decrease of the diversity of the total bacterial communities in these habitats. The data also indicated the existence of universal (i.e. Pseudomonas poae, P. lini, P. umsongensis, P. corrugata, P. antarctica and Rahnella aquatilis) as well as specific (i.e. P. viridiflava and candidatus Xiphinematobacter americani) fungiphiles, defined as bacteria adapted to the mycospheres of, respectively, three or more or just one fungal species. The selection of such fungiphiles was shown to be strongly related to their capacities to use particular carbonaceous compounds, as evidenced using principal components analyses of BIOLOG-based substrate utilization tests. The differentiating compounds, i.e. l -arabinose, l -leucine, m-inositol, m-arabitol, d -mannitol and d -trehalose, were tentatively linked to compounds known to occur in mycosphere exudates. [source] Microcystin-LR modulates selected immune parameters and induces necrosis/apoptosis of carp leucocytes,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2010Anna Rymuszka Abstract Microcystins (MCs) are potent hepatotoxins acting by the inhibition of protein phosphatase 1 and 2A, and may promote liver tumors. Moreover, studies also suggest they are nephrotoxic. The aim of the present study was to assess possible in vitro effects of microcystin-LR (which contains the amino acids leucine and arginine, the most widely studied and distributed variant of all microcystins) on the selected immune functions of the cells isolated from the head kidney of carp. In the experiments, pure microcystin-LR (MC-LR), was used at concentrations of 0.01, 0.1, 0.5, and 1,µg/ml RPMI-1640 medium. Leucocytes (lymphocytes and phagocytes) were isolated by centrifugation on a density gradient. Lymphocyte proliferation, intracellular production of reactive oxygen species by phagocytes, and the presence of apoptotic and/or necrotic cells were assessed. The respiratory burst activity of phagocytic cells was increased at the lowest toxin concentration used in the study, but it was decreased at higher concentrations. Using a sensitive luminescent immunoassay, MC-LR was observed to have no influence on the T-cell proliferation but decreased the proliferation of B lymphocytes. Moreover, it was noted that MC-LR induced necrosis to a higher degree than apoptosis in fish leucocytes. The results of the present study suggest the modulatory potency of microcystin-LR on fish leucocytes. Environ. Toxicol. Chem. 2010;29:569,574. © 2009 SETAC [source] Effects of dietary N -acetylcysteine on the oxidative stress induced in tilapia (Oreochromis Niloticus) exposed to a microcystin-producing cyanobacterial water bloom,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2009María Puerto Abstract Fish can be exposed to toxic cyanobacterial cells in natural waters and fish farms and suffer from oxidative damage. The present study investigates the effects of N-acetylcysteine (NAC), a glutathione (GSH) precursor, on the oxidative stress induced by Microcystis cyanobacterial cells containing microcystins (MCs) in tilapia fish (Oreochromis niloticus). Variation in lipid peroxidation (LPO) levels, carbonyl group content, reduced glutathione to oxidized glutathione ratio (GSH: GSSG), and catalase (Enzyme Commission [EC] 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1), glutathione reductase (GR; EC 1.8.1.7), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione S-transferase (EC 2.5.1.18) activities in liver and kidney of tilapia exposed to a single oral dose of 120 ,g MC-LR (with leucine [L] and arginine [R])/fish and killed in 24 h were investigated in the absence and presence of 20.0, 44.0, and 96.8 mg NAC/fish/d. Results showed a protective role of NAC, depending on the dose and the biomarker considered. The increase in LPO (1.9-and 1.4-fold in liver and kidney, respectively) and the decreased protein content and GSH:GSSG in the liver induced by MCs were recovered mainly by the lower doses of NAC employed. Antioxidant enzyme activities increased (range, 1.4-to 1.7-fold) by MCs also were ameliorated by NAC, although the highest level used induced significant alteration of some enzymatic activities, such as SOD, GPx, and GR. Thus, NAC can be considered to be a useful chemoprotectant that reduces hepatic and renal oxidative stress in the prophylaxis and treatment of MC-related intoxications in fish when careful attention is given to its application dose because of its own pro-oxidant activity, as shown in the present study at 96.8 mg NAC/ fish/d. [source] Metabolism of cholesterol ester of apolipoprotein B100-containing lipoproteins in dogs: evidence for disregarding cholesterol ester transferEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 8 2004E. Bailhache Abstract Background, It has been shown that dogs exhibit no cholesterol ester transfer protein (CETP) activity in vitro, in contrast to humans. The aim of our study was to determine modalities of in vivo plasma cholesterol ester turnover in this species, using a kinetic approach with stable isotopes. Materials and methods, Kinetics of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were studied in seven adult male Beagle dogs using a dual isotope approach through endogenous labelling of both their cholesterol moiety and their protein moiety. A primed constant infusion of both [1,213C]acetate and [5,5,5- 2H3]leucine enabled us to obtain measurable deuterium enrichments by gas chromatography-mass spectrometry for plasma leucine and apoB100, as well as measurable 13C enrichment by gas chromatography-combustion-isotopic ratio mass spectrometry for unesterified cholesterol and cholesterol ester in the VLDL and LDL. Two identical multicompartmental models (SAAM II) were used together for the analysis of tracer kinetics' data of proteins and cholesterol. Results, Characterization of the apoB100-containing lipoprotein cholesterol ester model allowed determination of kinetic parameters of VLDL and LDL cholesterol ester metabolism. We succeeded in modelling VLDL and LDL cholesterol ester metabolism and apoB100 metabolism simultaneously. Fractional catabolic rate (FCR) of apoB100 and CE had the same values. Introducing cholesterol ester transfer between lipoproteins in the model did not significantly improve the fit. Total VLDL FCR was 2·97 ± 01·47 h,1. Approximately one-quarter corresponded to the direct removal of VLDL (0·81 ± 00·34 h,1) and the remaining three-quarters corresponded to the fraction of VLDL converted to LDL, which represented a conversion of VLDL into LDL of 2·16 ± 01·16 h,1. Low-density lipoproteins were produced exclusively from VLDL conversion and were then removed (0·031 ± 0·004 h,1) from plasma. Conclusion, These kinetic data showed that VLDL cholesterol ester and LDL cholesterol ester metabolism followed VLDL and LDL apoB100 metabolism, and that consequently there is no in vivo transfer of cholesterol ester in dogs. [source] Enantioselective Recognition of Aliphatic Amino Acids by ,-Cyclodextrin Derivatives Bearing Aromatic Organoselenium Moieties on the Primary or Secondary SideEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 8 2003Yu Liu Abstract Spectrophotometric titrations have been performed in order to determine the stability constants of inclusion complexation of some aliphatic amino acids with four structurally related organoselenium-modified ,-cyclodextrins: mono(6-phenylseleno-6-deoxy)-,-cyclodextrin (1a), mono[6-(p -methoxyphenylseleno)-6-deoxy]-,-cyclodextrin (1b), mono(2-phenylseleno-2-deoxy)-,-cyclodextrin (2a), and mono[2-(p -methoxyphenylseleno)-2-deoxy]-,-cyclodextrin (2b). Conformation analysis by circular dichroism and 2D NMR spectroscopic studies revealed that the aryl-substituted ,-cyclodextrins gave self-inclusion intramolecular complexes in aqueous solution, while the extent of penetration depended both on the positions and on the structures of substituents. Quantitative investigation on the binding ability of the hosts with amino acids showed that they were able to recognize the size and the shape of guests, affording supramolecular complexes with quite small stability constants ranging from 24 to 355 M,1. The molecular recognition abilities are discussed from the viewpoints of induced-fitting mechanisms, geometric complementary, and cooperative binding processes. Furthermore, these ,-cyclodextrin derivatives displayed considerable enantioselectivity towards L/D -amino acid isomers, giving the highest L -enantioselectivity (up to 8.4) for inclusion complexation between leucine and 2a. The binding modes of L/D -leucine with 1b were elucidated from NOESY studies and the chiral recognition phenomena were interpreted accordingly. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source] Amino acid 15N in long-term bare fallow soils: influence of annual N fertilizer and manure applicationsEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 4 2008R. Bol Summary Long-term dynamics of amino acids (AAs), from a bare fallow soil experiment (established in 1928 at INRA-Versailles, France), were examined in unamended control (Con) plots and plots treated with ammonium sulphate (Amsul), ammonium nitrate (Amnit), sodium nitrate (Nanit) or with animal manure (Man). Topsoil (0,25 cm) from 1929, 1963 and 1997 was analysed for C, N and 15N content and distribution of 18 amino acids recovered after acid hydrolysis with 6 m HCl. With time, soil N, C and AA content were reduced in Con, Amsul, Amnit and Nanit, but increased in Man. However, the absolute N loss was 3,11 times larger in Man than Nanit, Amsul, Amnit and Con, due to the much higher N annual inputs applied to Man. From 1929 to 1997 in Con, Amsul, Amnit and Nanit the whole soil and non-hydrolysable-N pool ,15N increased associated with the loss of N (indicative of Rayleigh 15N/14N fractionation). No ,15N change from 1929 to 1997 was found in the hydrolysable AA-N (HAN) pool. Fertilizer N inputs aided stabilization of soil AA-N, as AA half-life in the mineral N fertilizer treatments increased from 34 years in 1963 to 50 years in 1997. The ,15N values of alanine and leucine reflected both source input and 15N/14N fractionation effects in soils. The ,15N increase of ornithine (,6,) was similar to the whole soil. The ,15N change of phenylalanine in Con (decrease of 7,) was related to its proportional loss since 1929, whereas for Amsul, Amnit, Nanit and Man it was associated with isotope effects caused by the fertilizer inputs. However, the soil ,15N value of most individual amino acids (IAAs) did not significantly change over nearly 70 years, even with mineral or organic N inputs. We conclude for these bare fallow systems that: (i) ,15N changes in the whole soil and non-hydrolysable AA pool were solely driven by microbial processes and not by the nature of fertilizer inputs, and (ii) without plant inputs, the ,15N of the HAN pool and (most) IAAs may reflect the influence of plant,soil interactions from the previous (arable cropping) rather than present (fallow) land use on these soil ,15N values. [source] Neostigmine and pilocarpine attenuated tumour necrosis factor , expression and cardiac hypertrophy in the heart with pressure overloadEXPERIMENTAL PHYSIOLOGY, Issue 1 2008Jessica Freeling The inflammatory cytokine tumour necrosis factor , (TNF,) is known to be a major factor contributing to cardiac remodelling and dysfunction. Parasympathetic nervous system cholinergic function can inhibit TNF, expression during systemic infection. In the present study, we tested the effects of a cholinesterase inhibitor, neostigmine, and a muscarinic cholinergic agonist, pilocarpine, on cardiac hypertrophy and TNF, levels during pressure overload. Rats with transverse aortic constriction exhibited elevated TNF, protein levels in the heart, increased heart weight to body weight ratios (an index of cardiac hypertrophy) and decreased left ventricular diastolic function. Two weeks of infusion with neostigmine (6 ,g kg,1 day,1) or pilocarpine (0.3 mg kg,1 day,1) significantly reduced cardiac hypertrophy, reduced TNF, levels and elevated interleukin-10 levels in heart tissues, and improved ventricular function in rats with transverse aortic constriction. Neither of these treatments significantly changed ventricular pressure load. Furthermore, in primary cultured neonatal cardiac cells, treatment with pilocarpine attenuated adrenergic agonist phenylephrine-induced increased TNF, expression and [3H]leucine (a marker of protein synthesis) incorporation in the cells. Collectively, both cholinergic agents decreased TNF, levels and attenuated cardiac hypertrophy. Since both agents potentially enhanced cholinergic function, the anti-inflammatory action may be involved in the cardioprotective effect of the treatments with these agents. [source] Nitric oxide-dependent protein synthesis in parotid and submandibular glands of anaesthetized rats upon sympathetic stimulation or isoprenaline administrationEXPERIMENTAL PHYSIOLOGY, Issue 2 2004Shariel Sayardoust In anaesthetized female rats, the ,-adrenoceptor agonist isoprenaline was intravenously infused (20 ,g kg,1 min,1) for 30 min or the ascending cervical sympathetic nerve trunk was intermittently stimulated (50 Hz, 1 s every tenth second) on one side for 30 min. The incorporation of [3H]leucine into trichloroacetic acid (TCA)-insoluble material was used as an index of protein synthesis. In response to isoprenaline, the [3H]leucine incorporation increased by 79% in the parotid glands and by 82% in the submandibular glands. The neuronal type NO-synthase inhibitor N-PLA, reduced (P < 0.001) this response to 26% and 20%, respectively. Sympathetic stimulation under ,-adrenoceptor blockade increased the [3H]leucine incorporation by 192% in the parotid glands and by 35% in the submandibular glands. N-PLA reduced the corresponding percentage figures to 86% (P < 0.01) and 8% (P < 0.05). When tested in the parotid glands, the non-selective NO-synthase inhibitor L -NAME reduced (P < 0.01) the nerve-evoked response to 91%. The increase in [3H]leucine incorporation in response to sympathetic stimulation under ,-adrenoceptor blockade was not affected by N-PLA in the parotid (139%versus 144%) and submandibular glands (39%versus 34%). In non-stimulated glands, the [3H]leucine incorporation was not influenced by the NO-synthase inhibitors. In conclusion, ,-adrenoceptor mediated salivary gland protein synthesis is largely dependent on NO generation by neuronal type NO-synthase, most likely of parenchymal origin. [source] Single amino acid repeats in signal peptidesFEBS JOURNAL, Issue 15 2010abaj There has been an increasing interest in single amino acid repeats ever since it was shown that these are the cause of a variety of diseases. Although a systematic study of single amino acid repeats is challenging, they have subsequently been implicated in a number of functional roles. In general surveys, leucine runs were among the most frequent. In the present study, we present a detailed investigation of repeats in signal peptides of secreted and type I membrane proteins in comparison with their mature parts. We focus on eukaryotic species because single amino acid repeats are generally rather rare in archaea and bacteria. Our analysis of over 100 species shows that repeats of leucine (but not of other hydrophobic amino acids) are over-represented in signal peptides. This trend is most pronounced in higher eukaryotes, particularly in mammals. In the human proteome, although less than one-fifth of all proteins have a signal peptide, approximately two-thirds of all leucine repeats are located in these transient regions. Signal peptides are cleaved early from the growing polypeptide chain and then degraded rapidly. This may explain why leucine repeats, which can be toxic, are tolerated at such high frequencies. The substantial fraction of proteins affected by the strong enrichment of repeats in these transient segments highlights the bias that they can introduce for systematic analyses of protein sequences. In contrast to a general lack of conservation of single amino acid repeats, leucine repeats were found to be more conserved than the remaining signal peptide regions, indicating that they may have an as yet unknown functional role. [source] Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406FEBS JOURNAL, Issue 11 2008Structural importance of the crown domain Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP. [source] The crystal structure of phenylpyruvate decarboxylase from Azospirillum brasilense at 1.5 Å resolutionFEBS JOURNAL, Issue 9 2007Implications for its catalytic, regulatory mechanism Phenylpyruvate decarboxylase (PPDC) of Azospirillum brasilense, involved in the biosynthesis of the plant hormone indole-3-acetic acid and the antimicrobial compound phenylacetic acid, is a thiamine diphosphate-dependent enzyme that catalyses the nonoxidative decarboxylation of indole- and phenylpyruvate. Analogous to yeast pyruvate decarboxylases, PPDC is subject to allosteric substrate activation, showing sigmoidal v versus [S] plots. The present paper reports the crystal structure of this enzyme determined at 1.5 Å resolution. The subunit architecture of PPDC is characteristic for other members of the pyruvate oxidase family, with each subunit consisting of three domains with an open ,/, topology. An active site loop, bearing the catalytic residues His112 and His113, could not be modelled due to flexibility. The biological tetramer is best described as an asymmetric dimer of dimers. A cysteine residue that has been suggested as the site for regulatory substrate binding in yeast pyruvate decarboxylase is not conserved, requiring a different mechanism for allosteric substrate activation in PPDC. Only minor changes occur in the interactions with the cofactors, thiamine diphosphate and Mg2+, compared to pyruvate decarboxylase. A greater diversity is observed in the substrate binding pocket accounting for the difference in substrate specificity. Moreover, a catalytically important glutamate residue conserved in nearly all decarboxylases is replaced by a leucine in PPDC. The consequences of these differences in terms of the catalytic and regulatory mechanism of PPDC are discussed. [source] The most C-terminal tri-glycine segment within the polyglycine stretch of the pea Toc75 transit peptide plays a critical role for targeting the protein to the chloroplast outer envelope membraneFEBS JOURNAL, Issue 7 2006Amy J. Baldwin The protein translocation channel at the outer envelope membrane of chloroplasts (Toc75) is synthesized as a larger precursor with an N-terminal transit peptide. Within the transit peptide of the pea Toc75, a major portion of the 10 amino acid long stretch that contains nine glycine residues was shown to be necessary for directing the protein to the chloroplast outer membrane in vitro[Inoue K & Keegstra K (2003) Plant J34, 661,669]. In order to get insights into the mechanism by which the polyglycine stretch mediates correct targeting, we divided it into three tri-glycine segments and examined the importance of each domain in targeting specificity in vitro. Replacement of the most C-terminal segment with alanine residues resulted in mistargeting the protein to the stroma, while exchange of either of the other two tri-glycine regions had no effect on correct targeting. Furthermore, simultaneous replacement of the N-terminal and middle tri-glycine segments with alanine repeats did not cause mistargeting of the protein as much as those of the N- and C-terminal, or the middle and C-terminal segments. These results indicate that the most C-terminal tri-glycine segment is important for correct targeting. Exchanging this portion with a repeat of leucine or glutamic acid also caused missorting of Toc75 to the stroma. By contrast, its replacement with repeats of asparagine, aspartic acid, serine, and proline did not largely affect correct targeting. These data suggest that relatively compact and nonhydrophobic side chains in this particular region play a crucial role in correct sorting of Toc75. [source] Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coliFEBS JOURNAL, Issue 3 2004Victor Croitoru Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors, IF1. Therefore, to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal infA+ gene, followed by deletion of this infA+ gene. Using this approach, the six arginine residues of IF1 were altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Differences in growth phenotypes of the mutants were observed in both minimal and rich media. Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA+ allele. Several of these recombinants showed reduced growth rate and a partial cold-sensitive phenotype. This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1. [source] Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N -acyl- d -amino acid amidohydrolase responsible for d -amino acid productionFEBS JOURNAL, Issue 19 2002Pei-Hsun Lin An N -acyl- d -amino acid amidohydrolase (N -D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N -acetyl- d -methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN -D-AAase showed significant amino acid similarity to the N -acyl- d -amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44,56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N -D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U·mg,1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N -D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 °C, respectively. After 30 min heat treatment at 45 °C, between pH 6 and pH 8, 80% activity remained. The N -D-AAase had higher hydrolysing activity against N -acetyl- d -amino acid derivates containing d -methionine, d -leucine and d -alanine and against N -chloroacetyl- d -phenylalanine. Importantly, the enzyme does not act on the N -acetyl- l -amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N -D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme. [source] Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytesFEBS JOURNAL, Issue 15 2002Ulrike Krause Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl-CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAr) or oligomycin, an inhibitor of mitochondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase-dependent mechanism but did not modify AMPK activation. Conversely, the amino-acid-induced activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK activation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino-acid-induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b) both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy-consuming biosynthetic pathways. [source] Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site-directed mutagenesisFEBS JOURNAL, Issue 22 2001Xing-Guo Wang In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards l -methionine, l -norleucine and l -norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used l -aspartate and l -leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher Km values for glutamate/2-oxoglutarate at pH 7.0, but a similar kcat/Km value and lower Km at pH 8.0, and a nearly 22-fold lower S0.5 (substrate concentration giving half-saturation under conditions where Michaelis,Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100,1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially l -norleucine and l -methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate kcat and Km separately, but for reductive amination the additional mutations have no significant effect on kcat at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in Km. At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine ,,2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants. [source] Growth response of the bacterial community to pH in soils differing in pHFEMS MICROBIOLOGY ECOLOGY, Issue 1 2010David Fernández-Calviño Abstract The effect of pH on the instantaneous growth of soil bacterial communities was studied in five soils with different pH (4.5,7.8) using leucine (Leu) and thymidine (TdR) incorporation. The pH dependency of bacterial growth was modelled using three different unimodal functions, and the pHopt for growth and the pH range in which growth was >50% of the optimal growth were compared. Leu and TdR incorporation yielded very similar results. The best fits were obtained using a third-degree polynomial function and the cardinal pH model. However, a simple second-degree function was adequate in most cases, yielding very similar pHopt values to the other two models. Bacterial growth was highly influenced by pH, showing optimum growth at a pH related to the soil pH. The lowest pHopt was found in the most acidic soil and the highest pHopt in the soil with the highest pH. The pHopt for bacterial growth was close to the soil pH measured in water, but higher (0.7,2.1 units) than the pH measured with 0.1 M KCl. The pH range in which bacterial growth was >50% of that at optimum was, on average, 1.7 units below and above the optimum pH. [source] Identification of the aceA gene encoding isocitrate lyase required for the growth of Pseudomonas aeruginosa on acetate, acyclic terpenes and leucineFEMS MICROBIOLOGY LETTERS, Issue 2 2007Alma Laura Díaz-Pérez Abstract Pseudomonas aeruginosa PAO1 mutants affected in acyclic monoterpenes, n-octanol, and acetate assimilation were isolated using transposon mutagenesis. The isocitrate lyase gene (aceA) corresponding to ORF PA2634 of the PAO1 strain genome was identified in one of these mutants. The aceA gene encodes a protein that is 72% identical to the isocitrate lyase (ICL) characterized from Colwellia maris, but is less than 30% identical to their homologues from pseudomonads. The genetic arrangement of aceA suggests that it is a monocistronic gene, and no adjacent related genes were found. The ICL protein was detected as a 60-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis from cultures grown on acetate, but not in glucose-grown PAO1 cultures. Genetic complementation further confirmed that the aceA gene encodes the ICL enzyme. The ICL enzyme activity in crude extracts from cultures of the PAO1 strain was induced by acetate, citronellol and leucine, and repressed by growth on glucose or citrate. These results suggest that ICL is involved in the assimilation of acetate, acyclic monoterpenes of the citronellol family, alkanols, and leucine, in which the final intermediary acetyl-coenzyme A may be channelled to the glyoxylate shunt. 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