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Sample records for atp citrate lyase

  1. Compartmentation of ATP:citrate lyase in plants.

    Science.gov (United States)

    Rangasamy, D; Ratledge, C

    2000-04-01

    Extracts prepared from young leaves of Pea (Pisum sativum), tobacco (Nicotiana tabacum), rape (Brassica napus), and spinach (Spinacia oleracea) all contained ATP:citrate lyase (ACL) activity, which was most active in rape leaflets (130 nmol min(-1) g fresh weight). In rape and spinach, ACL activity was predominantly localized in the plastids (between about 78% and 90% of the total activity), whereas in pea and tobacco, distribution was mainly cytosolic (about 85% and 78%, respectively, of the total). These distributions were calculated from the relative distributions of plastid and cytosol marker enzymes. Cross-reactivity between plant and rat ACL antibody was carried out by immunoblot analysis and, in rape and spinach, showed that a 120-kD protein, presumably indicating homomeric ACL proteins, was present in both cytosolic and plastidic fractions. In pea, two cross-reacting proteins were detected, the major material being in the cytosol fraction. Therefore, ACL occurs both in the cytosol and plastids of higher plants, but the distribution of activity changes according to the species. The plastidic ACL is proposed to function for the supply of acetyl-coenzyme A for lipid biosynthesis de novo, whereas the cytosolic ACL may provide acetyl-coenzyme A for the mevalonate pathway or fatty acid elongation.

  2. Diversity of RuBisCO and ATP citrate lyase genes in soda lake sediments

    NARCIS (Netherlands)

    Kovaleva, O.L.; Tourova, T.P.; Muyzer, G.; Kolganova, T.V.; Sorokin, D.Y.

    2011-01-01

    Sediments from six soda lakes of the Kulunda Steppe (Altai, Russia) and from hypersaline alkaline lakes of Wadi Natrun (Egypt) were analyzed for the presence of cbb and aclB genes encoding key enzymes Ci assimilation (RuBisCO in Calvin-Benson and ATP citrate lyase in rTCA cycles, respectively). The

  3. Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

    2009-01-01

    factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

  4. A role for ATP-citrate lyase, malic enzyme, and pyruvate/citrate cycling in glucose-induced insulin secretion.

    Science.gov (United States)

    Guay, Claudiane; Madiraju, S R Murthy; Aumais, Alexandre; Joly, Erik; Prentki, Marc

    2007-12-07

    In pancreatic beta-cells, metabolic coupling factors generated during glucose metabolism and pyruvate cycling through anaplerosis/cataplerosis processes contribute to the regulation of insulin secretion. Pyruvate/citrate cycling across the mitochondrial membrane leads to the production of malonyl-CoA and NADPH, two candidate coupling factors. To examine the implication of pyruvate/citrate cycling in glucose-induced insulin secretion (GIIS), different steps of the cycle were inhibited in INS 832/13 cells by pharmacological inhibitors and/or RNA interference (RNAi) technology: mitochondrial citrate export, ATP-citrate lyase (ACL), and cytosolic malic enzyme (ME1). The inhibitors of the di- and tri-carboxylate carriers, n-butylmalonate and 1,2,3-benzenetricarboxylate, respectively, reduced GIIS, indicating the importance of transmitochondrial transport of tri- and dicarboxylates in the action of glucose. To directly test the role of ACL and ME1 in GIIS, small hairpin RNA (shRNA) were used to selectively decrease ACL or ME1 expression in transfected INS 832/13 cells. shRNA-ACL reduced ACL protein levels by 67%, and this was accompanied by a reduction in GIIS. The amplification/K(ATP)-independent pathway of GIIS was affected by RNAi knockdown of ACL. The ACL inhibitor radicicol also curtailed GIIS. shRNA-ME1 reduced ME1 activity by 62% and decreased GIIS. RNAi suppression of either ACL or ME1 did not affect glucose oxidation. However, because ACL is required for malonyl-CoA formation, inhibition of ACL expression by shRNA-ACL decreased glucose incorporation into palmitate and increased fatty acid oxidation in INS 832/13 cells. Taken together, the results underscore the importance of pyruvate/citrate cycling in pancreatic beta-cell metabolic signaling and the regulation of GIIS.

  5. ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch

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    Steven Zhao

    2016-10-01

    Full Text Available Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY, cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

  6. Diversity of RuBisCO and ATP citrate lyase genes in soda lake sediments.

    Science.gov (United States)

    Kovaleva, Olga L; Tourova, Tatjana P; Muyzer, Gerard; Kolganova, Tatjana V; Sorokin, Dimitry Y

    2011-01-01

    Sediments from six soda lakes of the Kulunda Steppe (Altai, Russia) and from hypersaline alkaline lakes of Wadi Natrun (Egypt) were analyzed for the presence of cbb and aclB genes encoding key enzymes Ci assimilation (RuBisCO in Calvin-Benson and ATP citrate lyase in rTCA cycles, respectively). The cbbL gene (RuBisCO form I) was found in all samples and was most diverse, while the cbbM (RuBisCO form II) and aclB were detected only in few samples and with a much lower diversity. The cbbL libraries from hypersaline lakes were dominated by members of the extremely haloalkaliphilic sulfur-oxidizing Ectothiorhodospiraceae, i.e. the chemolithotrophic Thioalkalivibrio and the phototrophic Halorhodospira. In the less saline soda lakes from the Kulunda Steppe, the cbbL gene comprised up to ten phylotypes with a domination of members of a novel phototrophic Chromatiales lineage. The cbbM clone libraries consisted of two major unidentified lineages probably belonging to chemotrophic sulfur-oxidizing Gammaproteobacteria. One of them, dominating in the haloalkaline lakes from Wadi Natrun, was related to a cbbM phylotype detected previously in a hypersaline lake with a neutral pH, and another, dominating in lakes from the Kulunda Steppe, was only distantly related to the Thiomicrospira cluster. The aclB sequences detected in two samples from the Kulunda Steppe formed a single, deep branch in the Epsilonproteobacteria, distantly related to Arcobacter sulfidicus. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  7. Genome-wide identification of citrus ATP-citrate lyase genes and their transcript analysis in fruits reveals their possible role in citrate utilization.

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    Hu, Xiao-Mei; Shi, Cai-Yun; Liu, Xiao; Jin, Long-Fei; Liu, Yong-Zhong; Peng, Shu-Ang

    2015-02-01

    ATP-citrate lyase (ACL, EC4.1.3.8) catalyzes citrate to oxaloacetate and acetyl-CoA in the cell cytosol, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. We identified three ACL genes, CitACLα1, CitACLα2, and CitACLβ1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLβ1 encodes a putative ACL β subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLβ1 had 16 exons and 15 introns. CitACLα1 and CitACLβ1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLβ1, and/or CitACLα2 in cultivars 'Niuher' and 'Owari' increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in 'Egan No. 1' and 'Iyokan', although citrate content also decreased. In 'HB pummelo', in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of CitACLα2, CitACLβ1, and/or CitACLα1 increased. Moreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only CitACLβ1 was significantly reduced by ABA treatment. Taken together, these data indicate that the effects of ACL on citrate use during fruit ripening depends on the cultivar, and the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment.

  8. Reverse genetic characterization of cytosolic acetyl-CoA generation by ATP-citrate lyase in Arabidopsis.

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    Fatland, Beth L; Nikolau, Basil J; Wurtele, Eve Syrkin

    2005-01-01

    Acetyl-CoA provides organisms with the chemical flexibility to biosynthesize a plethora of natural products that constitute much of the structural and functional diversity in nature. Recent studies have characterized a novel ATP-citrate lyase (ACL) in the cytosol of Arabidopsis thaliana. In this study, we report the use of antisense RNA technology to generate a series of Arabidopsis lines with a range of ACL activity. Plants with even moderately reduced ACL activity have a complex, bonsai phenotype, with miniaturized organs, smaller cells, aberrant plastid morphology, reduced cuticular wax deposition, and hyperaccumulation of starch, anthocyanin, and stress-related mRNAs in vegetative tissue. The degree of this phenotype correlates with the level of reduction in ACL activity. These data indicate that ACL is required for normal growth and development and that no other source of acetyl-CoA can compensate for ACL-derived acetyl-CoA. Exogenous malonate, which feeds into the carboxylation pathway of acetyl-CoA metabolism, chemically complements the morphological and chemical alterations associated with reduced ACL expression, indicating that the observed metabolic alterations are related to the carboxylation pathway of cytosolic acetyl-CoA metabolism. The observations that limiting the expression of the cytosolic enzyme ACL reduces the accumulation of cytosolic acetyl-CoA-derived metabolites and that these deficiencies can be alleviated by exogenous malonate indicate that ACL is a nonredundant source of cytosolic acetyl-CoA.

  9. Glucose-dependent de novo lipogenesis in B lymphocytes: a requirement for atp-citrate lyase in lipopolysaccharide-induced differentiation.

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    Dufort, Fay J; Gumina, Maria R; Ta, Nathan L; Tao, Yongzhen; Heyse, Shannon A; Scott, David A; Richardson, Adam D; Seyfried, Thomas N; Chiles, Thomas C

    2014-03-07

    Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation.

  10. Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen synthase by a multifunctional protein kinase (ATP-citrate lyase kinase)

    Energy Technology Data Exchange (ETDEWEB)

    Sheorain, V.S.; Ramakrishna, S.; Benjamin, W.B.; Soderling, T.R.

    1985-10-05

    A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of and)2numberSPO4/mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the TUPO4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide and protein substrate specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro.

  11. Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly.

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    Agnes Csanadi

    Full Text Available Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient's prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules '(i.e. nucleotides, aminoacids, fatty acids. In cancer, Malic enzyme (ME and ATP-citrate lyase (ACLY are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC.ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient's survival.Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite.In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.

  12. Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

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    Dulermo, Thierry; Lazar, Zbigniew; Dulermo, Rémi; Rakicka, Magdalena; Haddouche, Ramedane; Nicaud, Jean-Marc

    2015-09-01

    The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica. Copyright © 2015. Published by Elsevier B.V.

  13. Prevention of Diet-Induced Metabolic Dysregulation, Inflammation, and Atherosclerosis in Ldlr(-/-) Mice by Treatment With the ATP-Citrate Lyase Inhibitor Bempedoic Acid.

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    Samsoondar, Joshua P; Burke, Amy C; Sutherland, Brian G; Telford, Dawn E; Sawyez, Cynthia G; Edwards, Jane Y; Pinkosky, Stephen L; Newton, Roger S; Huff, Murray W

    2017-04-01

    Bempedoic acid (ETC-1002, 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel low-density lipoprotein cholesterol-lowering compound. In animals, bempedoic acid targets the liver where it inhibits cholesterol and fatty acid synthesis through inhibition of ATP-citrate lyase and through activation of AMP-activated protein kinase. In this study, we tested the hypothesis that bempedoic acid would prevent diet-induced metabolic dysregulation, inflammation, and atherosclerosis. APPROACH AND RESULTS: Ldlr(-/-) mice were fed a high-fat, high-cholesterol diet (42% kcal fat, 0.2% cholesterol) supplemented with bempedoic acid at 0, 3, 10 and 30 mg/kg body weight/day. Treatment for 12 weeks dose-dependently attenuated diet-induced hypercholesterolemia, hypertriglyceridemia, hyperglycemia, hyperinsulinemia, fatty liver and obesity. Compared to high-fat, high-cholesterol alone, the addition of bempedoic acid decreased plasma triglyceride (up to 64%) and cholesterol (up to 50%) concentrations, and improved glucose tolerance. Adiposity was significantly reduced with treatment. In liver, bempedoic acid prevented cholesterol and triglyceride accumulation, which was associated with increased fatty acid oxidation and reduced fatty acid synthesis. Hepatic gene expression analysis revealed that treatment significantly increased expression of genes involved in fatty acid oxidation while suppressing inflammatory gene expression. In full-length aorta, bempedoic acid markedly suppressed cholesteryl ester accumulation, attenuated the expression of proinflammatory M1 genes and attenuated the iNos/Arg1 ratio. Treatment robustly attenuated atherosclerotic lesion development in the aortic sinus by 44%, with beneficial changes in morphology, characteristic of earlier-stage lesions. Bempedoic acid effectively prevents plasma and tissue lipid elevations and attenuates the onset of inflammation, leading to the prevention of atherosclerotic lesion development in a mouse model of metabolic

  14. In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production.

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    Evamaria Gruchattka

    Full Text Available Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo.Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1 was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production.Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes

  15. In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production.

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    Gruchattka, Evamaria; Kayser, Oliver

    2015-01-01

    Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo. Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1) was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production. Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes are essential to

  16. mTOR complex-2 stimulates acetyl-CoA and de novo lipogenesis through ATP citrate lyase in HER2/PIK3CA-hyperactive breast cancer.

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    Chen, Yaqing; Qian, Jianchang; He, Qun; Zhao, Hui; Toral-Barza, Lourdes; Shi, Celine; Zhang, Xuesai; Wu, Jiang; Yu, Ker

    2016-05-03

    The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and is frequently dysregulated in cancer. While mTOR complex-1 (mTORC1) is a validated cancer target, the role of mTOR complex-2 (mTORC2) remains less defined. Here, we reveal mTORC2 as a critical regulator of breast cancer metabolism. We showed that hyperphosphorylation in ATP citrate lyase (ACL) occurs frequently in human breast tumors and correlates well with HER2+ and/or PIK3CA-mutant (HER2+/PIK3CAmut) status in breast tumor cell lines. In HER2+/PIK3CAmut cells, mTORC2 controls Ser-455 phosphorylation of ACL thereby promoting acetyl-CoA production, de novo lipogenesis and mitochondrial physiology, all of which were inhibited by an mTORC1/mTORC2 kinase inhibitor (mTOR-KI) or cellular depletion of mTORC2 or ACL. mTOR-KI but not rapamycin blocked the IGF-1-induced ACL phosphorylation and glucose to lipid conversion. Depletion of mTORC2 but not mTORC1 specifically inhibited the ACL-dependent acetyl-CoA production. In the HER2+/PIK3CAmut MDA361, MDA453, BT-474 and T47D cells, depletion of mTORC2 or ACL led to growth inhibition and mitochondrial hyperpolarization, which were partially rescued by an alternate source of acetyl-CoA. These same changes were not apparent in mTORC2- or ACL-depleted HER2-/PIK3CAwt MDA231 and HCC1806 cells, highlighting a differential dependence of mTORC2-ACL for survival in these two cell types. Moreover, ACL Ser-455 mutants S455E (phosphomimetic) and S455A (non-phosphorylatable) each increased or decreased, respectively, the acetyl-CoA production, mitochondrial homeostasis and survival in ACL-depleted MDA453 cells. These studies define a new and rapamycin-resistant mechanism of mTORC2-ACL in lipogenesis and acetyl-CoA biology and provide a rationale for targeting of mTORC1 and mTORC2 in HER2+/PIK3CAmut breast cancer.

  17. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism[S

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    Pinkosky, Stephen L.; Filippov, Sergey; Srivastava, Rai Ajit K.; Hanselman, Jeffrey C.; Bradshaw, Cheryl D.; Hurley, Timothy R.; Cramer, Clay T.; Spahr, Mark A.; Brant, Ashley F.; Houghton, Jacob L.; Baker, Chris; Naples, Mark; Adeli, Khosrow; Newton, Roger S.

    2013-01-01

    ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca2+/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. PMID:23118444

  18. Lactic acid bacteria involved in cocoa beans fermentation from Ivory Coast: Species diversity and citrate lyase production.

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    Ouattara, Hadja D; Ouattara, Honoré G; Droux, Michel; Reverchon, Sylvie; Nasser, William; Niamke, Sébastien L

    2017-09-01

    Microbial fermentation is an indispensable process for high quality chocolate from cocoa bean raw material. lactic acid bacteria (LAB) are among the major microorganisms responsible for cocoa fermentation but their exact role remains to be elucidated. In this study, we analyzed the diversity of LAB in six cocoa producing regions of Ivory Coast. Ribosomal 16S gene sequence analysis showed that Lactobacillus plantarum and Leuconostoc mesenteroides are the dominant LAB species in these six regions. In addition, other species were identified as the minor microbial population, namely Lactobacillus curieae, Enterococcus faecium, Fructobacillus pseudoficulneus, Lactobacillus casei, Weissella paramesenteroides and Weissella cibaria. However, in each region, the LAB microbial population was composed of a restricted number of species (maximum 5 species), which varied between the different regions. LAB implication in the breakdown of citric acid was investigated as a fundamental property for a successful cocoa fermentation process. High citrate lyase producer strains were characterized by rapid citric acid consumption, as revealed by a 4-fold decrease in citric acid concentration in the growth medium within 12h, concomitant with an increase in acetic acid and lactic acid concentration. The production of citrate lyase was strongly dependent on environmental conditions, with optimum production at acidic pH (pHfermentation. This study reveals that one of the major roles of LAB in the cocoa fermentation process involves the breakdown of citric acid during the early stage of cocoa fermentation through the activity of citrate lyase. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. The influence of ferric (III citrate on ATP-hydrolases of Desulfuromonas acetoxidans ІМV В-7384

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    O. Maslovska

    2013-02-01

    Full Text Available Desulfuromonas acetoxidans obtains energy for growth by the anaerobic oxidation of organic compounds with the carbon dioxide formation. It was found that ferrum and manganese are used as terminal electron acceptors in the processes of anaerobic respiration, such as dissimilative Fe3+- and Mn4+-reduction, carried out by these bacteria (Lovely, 1991. D. acetoxidans ІМV B-7384 can be used as anode biocatalyst in microbial fuel cell with high electron recovery through acetate oxidation to the electric current as a result of electron transfer to the anode or 3d-type transition metals, such as ferrum and manganese, in the process of their reduction. Investigation of biochemical changes of D. acetoxidans ІМV B-7384 under the influence of Fe (III compounds is important for optimization of the process of bacterial electricity generation. ATP-hydrolase is located in cytoplasmic membrane, and its subunits are exposed to both the cytoplasm and the external environment. Therefore, the changes of that enzyme activity can be used as an indicator of various stress exposure. Presence of ferric iron ions in the bacterial growth medium could catalyze generation of organic reactive oxygen species, such as peroxyl (ROO- and alkoxyl (RO- radicals. Lipid peroxidation is one of the main reasons of cell damage and it’s following death under the influence of reactive oxygen metabolites. It is known that lipid peroxidation and membrane transport processes are somehow interrelated, but mechanisms of such interaction are still unidentified. In our previous researche we have shown the influence of ferric (III citrate on the intensity of lipid peroxidation of D. аcetoxidans ІМV В-7384. Significant increase of the content of lipid peroxidation products (lipid hydroperoxides, conjugated dienes and malondialdehyde in bacterial cells has been observed under the addition of ferric (III citrate into the cultural medium. The increase of the concentration of lipid

  20. Comparative genomics and transcriptome analysis of Aspergillus niger and metabolic engineering for citrate production

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    Yin, Xian; Shin, Hyun-dong; Li, Jianghua; Du, Guocheng; Liu, Long; Chen, Jian

    2017-01-01

    Despite a long and successful history of citrate production in Aspergillus niger, the molecular mechanism of citrate accumulation is only partially understood. In this study, we used comparative genomics and transcriptome analysis of citrate-producing strains—namely, A. niger H915-1 (citrate titer: 157 g L−1), A1 (117 g L−1), and L2 (76 g L−1)—to gain a genome-wide view of the mechanism of citrate accumulation. Compared with A. niger A1 and L2, A. niger H915-1 contained 92 mutated genes, including a succinate-semialdehyde dehydrogenase in the γ-aminobutyric acid shunt pathway and an aconitase family protein involved in citrate synthesis. Furthermore, transcriptome analysis of A. niger H915-1 revealed that the transcription levels of 479 genes changed between the cell growth stage (6 h) and the citrate synthesis stage (12 h, 24 h, 36 h, and 48 h). In the glycolysis pathway, triosephosphate isomerase was up-regulated, whereas pyruvate kinase was down-regulated. Two cytosol ATP-citrate lyases, which take part in the cycle of citrate synthesis, were up-regulated, and may coordinate with the alternative oxidases in the alternative respiratory pathway for energy balance. Finally, deletion of the oxaloacetate acetylhydrolase gene in H915-1 eliminated oxalate formation but neither influence on pH decrease nor difference in citrate production were observed. PMID:28106122

  1. Ca2+-Citrate Uptake and Metabolism in Lactobacillus casei ATCC 334

    NARCIS (Netherlands)

    Mortera, Pablo; Pudlik, Agata; Magni, Christian; Alarcon, Sergio; Lolkema, Juke S.

    The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of

  2. Studies on pectin lyase

    NARCIS (Netherlands)

    Houdenhoven, van F.E.A.

    1975-01-01

    The pectin lyase activity in the commercial enzyme preparation Ultrazym originates from more then one type of enzyme; two of them, accounting for 95 % of the total activity, have been completely purified. As purity criteria specific activity, polyacrylamide disc gel electrophoresis and SDS

  3. Citrate uptake into tonoplast vesicles from acid lime (Citrus aurantifolia) juice cells.

    Science.gov (United States)

    Brune, A; Gonzalez, P; Goren, R; Zehavi, U; Echeverria, E

    1998-12-01

    Citrate transport into the vacuoles of acid lime juice cells was investigated using isolated tonoplast vesicles. ATP stimulated citrate uptake in the presence or in the absence of a Delta mu H+. Energization of the vesicles only by an artificial K+ gradient (establishing an inside-positive Delta psi) also resulted in citrate uptake as was the case of a Delta pH dominated Delta mu H+. Addition of inhibitors to endomembrane ATPases showed no direct correlation between the inhibition to the tonoplast bound H+/ATPase and citrate uptake. The data indicated that, although some citrate uptake can be accounted for by Delta psi and by a direct primary active transport mechanism involving ATP, under in vivo conditions of vacuolar pH of 2.0, citrate uptake is driven by Delta pH.

  4. Ca2+-Citrate Uptake and Metabolism in Lactobacillus casei ATCC 334

    Science.gov (United States)

    Mortera, Pablo; Pudlik, Agata; Magni, Christian; Alarcón, Sergio

    2013-01-01

    The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of Lactobacillus casei ATCC 334 metabolized citrate in complex with Ca2+ and not as free citrate or the Mg2+-citrate complex, thereby identifying Ca2+-citrate as the substrate of the transporter CitH. The pathway was induced in the presence of Ca2+ and citrate during growth and repressed by the presence of glucose and of galactose, most likely by a carbon catabolite repression mechanism. The end products of Ca2+-citrate metabolism by resting cells of Lb. casei were pyruvate, acetate, and acetoin, demonstrating the activity of the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Following pyruvate, the pathway splits into two branches. One branch is the classical citrate fermentation pathway producing acetoin by α-acetolactate synthase and α-acetolactate decarboxylase. The other branch yields acetate, for which the route is still obscure. Ca2+-citrate metabolism in a modified MRS medium lacking a carbohydrate did not significantly affect the growth characteristics, and generation of metabolic energy in the form of proton motive force (PMF) was not observed in resting cells. In contrast, carbohydrate/Ca2+-citrate cometabolism resulted in a higher biomass yield in batch culture. However, also with these cells, no generation of PMF was associated with Ca2+-citrate metabolism. It is concluded that citrate metabolism in Lb. casei is beneficial when it counteracts acidification by carbohydrate metabolism in later growth stages. PMID:23709502

  5. Citrat og nyresten

    DEFF Research Database (Denmark)

    Osther, P J

    1993-01-01

    Citrate is an important naturally occurring inhibitor of calcium stone formation in urine. Urinary citrate excretion was examined in 43 consecutive patients with recurrent idiopathic calcium nephrolithiasis and in 50 normal controls by a specific enzymatic technique. Hypocitraturia (<1.6 mmol/24h...

  6. Trisilver(I citrate

    Directory of Open Access Journals (Sweden)

    Andreas Fischer

    2011-02-01

    Full Text Available Trisilver(I citrate, 3Ag+·C6H5O73−, was obtained by evaporation of a saturated aqueous solution of the raw material that had been obtained from sodium dihydrogen citrate and silver nitrate. It features one formula unit in the asymmetric unit. There is an intramolecular O—H...O hydrogen bond between the OH group and one of the terminal carboxylate groups. Different citrate groups are linked via the three Ag+ ions, yielding a three-dimensional network with rather irregular [AgO4] polyhedra.

  7. Blood and ultrafiltrate dosage of citrate as a useful and routine tool during continuous venovenous haemodiafiltration in septic shock patients.

    Science.gov (United States)

    Mariano, Filippo; Morselli, Maurizio; Bergamo, Daniela; Hollo, Zsuzsanna; Scella, Sandro; Maio, Mariella; Tetta, Ciro; Dellavalle, Ambrogio; Stella, Maurizio; Triolo, Giorgio

    2011-12-01

    Citrate anticoagulation is gaining popularity in renal replacement therapies (RRT) for critically ill patients. In order to study whether citrate accumulates in septic shock patients, we determined citrate in plasma and dialysate during continuous venovenous haemodiafiltration (CVVHDF). An automated routine determination of citrate was set up using a commercial kit (citrate lyase method). Twelve patients with septic shock on CVVHDF and citrate anticoagulation were studied ex vivo for citrate levels in systemic and circuit blood and in the ultrafiltrate (at 0, 0.5, 1, 3, 6, 9, 12, 24, 48 and 72 h). In vitro blood studies showed a near unit correlation between the plasma measured and predicted citrate concentrations for an exclusive extracellular distribution of citrate. Median systemic arterial citratemias were 0.09 (0.06-0.12) mmol/L (Time 0) and 0.23 (0.18-0.31) mmol/L during treatment; median sieving coefficient for citrate was 0.95 (0.88-1.02) and did not change with different volumes of CVVHDF effluent (from 1350 to 5100 mL/h). Net citrate and calcium removal by filter significantly correlated with effluent volume (r = 0.85 and 0.78, respectively). Median citrate load entering in the patients' bloodstream was 13.60 (9.1-19.6, n = 68) mmol/h. Although cost analysis of the citrate test demonstrated a minimally increased daily cost (from 2.96 to 3.51€), saving costs could be potentially relevant with more extended use of citrate anticoagulation. In septic shock patients with liver dysfunction citratemia is useful in guiding clinical application of RRT, where the citrate losses in the ultrafiltrate can be efficiently modulated by increasing the effluent volume.

  8. Revue bibliographique : l'hydroperoxyde lyase

    Directory of Open Access Journals (Sweden)

    Fauconnier M.L.

    2000-01-01

    Full Text Available Hydroperoxide lyase: a review. The hydroperoxide lyase belongs to the lipoxygenase pathway, pathway in which polyunsaturated fatty acids are transformed into a large spectrum of compounds with various chemical and biological functions. The hydroperoxide lyase acts on fatty acids hydroperoxides (mainly linoleic and linolenic. It cleaves the bond between carbon carrying the hydroperoxide function and a vicinal carbon giving an omega-oxo-acid and also depending on the species, an aldehyde, a hydrocarbon or an alcohol.

  9. Citrate shows protective effects on cardiovascular and renal function in ischemia-induced acute kidney injury.

    Science.gov (United States)

    Bienholz, Anja; Reis, Jonas; Sanli, Pinar; de Groot, Herbert; Petrat, Frank; Guberina, Hana; Wilde, Benjamin; Witzke, Oliver; Saner, Fuat H; Kribben, Andreas; Weinberg, Joel M; Feldkamp, Thorsten

    2017-04-10

    Ischemia and reperfusion (I/R) is one of the major causes of acute kidney injury (AKI). Citrate reduces hypoxia-induced mitochondrial energetic deficits in isolated proximal tubules. Moreover, citrate anticoagulation is now frequently used in renal replacement therapy. In the present study a rat model of I/R-induced AKI was utilized to examine renal protection by citrate in vivo. AKI was induced by bilateral renal clamping (40 min) followed by reperfusion (3 h). Citrate was infused at three different concentrations (0.3 mmol/kg/h; 0.6 mmol/kg/h and 1.0 mmol/kg/h) continuously for 60 min before and 45 min after ischemia. Plasma calcium concentrations were kept stable by infusion of calcium gluconate. The effect of citrate was evaluated by biomonitoring, blood and plasma parameters, histopathology and tissue ATP content. In comparison to the normoxic control group bilateral renal ischemia led to an increase of creatinine and lactate dehydrogenase activity and a decrease in tissue ATP content and was accompanied by a drop in mean arterial blood pressure. Infusion of 1.0 mmol/kg/h citrate led to lower creatinine and reduced LDH activity compared to the I/R control group and a tendency for higher tissue ATP content. Pre-ischemic infusion of 1.0 mmol/kg/h citrate stabilized blood pressure during ischemia. Citrate has a protective effect during I/R-induced AKI, possibly by limiting the mitochondrial deficit as well as by beneficial cardiovascular effects. This strengthens the rationale of using citrate in continuous renal replacement therapy and encourages consideration of citrate infusion as a therapeutic treatment for AKI in humans.

  10. Citrate synthase purified from Tetrahymena mitochondria is identical with Tetrahymena 14-nm filament protein.

    Science.gov (United States)

    Kojima, H; Chiba, J; Watanabe, Y; Numata, O

    1995-07-01

    A 14-nm filament protein (designated as 49K protein) was purified from a ciliated protozoan, Tetrahymena, using the polymerization and depolymerization procedure. Previous studies in our laboratory showed that its primary structure shared a high sequence identity with citrate synthases known so far and that the 49K protein possessed citrate synthase activity. To ascertain whether or not Tetrahymena's mitochondrial citrate synthase is identical to the 49K protein, citrate synthase was purified from Tetrahymena mitochondria using ammonium sulfate fractionation, Butyl-Toyopearl and SP-Toyopearl column chromatographies, based on monitoring of the enzymatic activity. The molecular weight of the purified citrate synthase was estimated to be 49 kDa, as was that of the 49K protein and the enzyme cross-reacted with an anti-49K protein antiserum. The purified citrate synthase showed much the same optimum pH, optimum KCl concentration, effects of substrate concentrations (acetyl-CoA and oxaloacetate), and inhibitory effect by ATP as those of purified 49K protein. Furthermore, an anti-49K protein monoclonal antibody strongly suppressed the enzymatic activity of the purified citrate synthase. Thus, we suggest that mitochondrial citrate synthase and the 49K protein are identical and that the 49K protein has dual functions in the cytoskeleton in cytoplasm and as a TCA cycle enzyme, citrate synthase, in mitochondria.

  11. Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4

    DEFF Research Database (Denmark)

    McDonough, Michael A.; Kadirvelraj, Renuka; Harris, Pernille

    2004-01-01

    Rhamnogalacturonan lyase (RG-lyase) specifically recognizes and cleaves alpha-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acids in the backbone of rhamno galacturonan-I, a major component of the plant cell wall polysaccharide, pectin. The three-dimensional structure of RG-lyase from...... Aspergillus aculeatus has been determined to 1.5 Angstrom resolution representing the first known structure from polysaccharide lyase family 4 and of an enzyme with this catalytic specificity. The 508-amino acid polypeptide displays a unique arrangement of three distinct modular domains. Each domain shows...

  12. Enzymes of Krebs-Henseleit Cycle in Vitis vinifera L: II. Arginosuccinate Synthetase and Lyase.

    Science.gov (United States)

    Roubelakis, K A; Kliewer, W M

    1978-09-01

    Arginosuccinate (ASA) synthetase and lyase activities were detected in extracts from Vitis vinifera L. cv. Chenin blanc mature leaves and seedlings. Optimum reaction conditions for ASA synthetase were 10 millimolar l-citrulline, 7.5 millimolar l-aspartate, 3 to 4 millimolar ATP, 12 millimolar Mg(2+) (pH 7.5 to 8.0), enzyme extract up to equivalent of about 200 milligrams of fresh tissue, and incubation temperature of 38 to 40 C. Optimum reaction conditions for ASA lyase were 4 millimolar ASA-K salt (pH 7.3 to 7.8), amount of extract up to equivalent of about 180 milligrams of fresh tissue, and incubation temperature of 38 to 40 C.

  13. A hydrogenosome with pyruvate formate-lyase: Anaerobic chytrid fungi use an alternative route for pyruvate catabolism

    OpenAIRE

    Akhmanova, Anna; Voncken, Jan Willem; Hosea, Ken; Harhangi, Biswadjiet; Keltjens, Jan; Op Den Camp, Huub; Vogels, Godfried; Hackstein, Johannes

    1999-01-01

    textabstractThe chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytri...

  14. Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

    Science.gov (United States)

    Buch, Aditi D; Archana, G; Kumar, G Naresh

    2009-08-01

    Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525

  15. 21 CFR 582.5449 - Manganese citrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Manganese citrate. 582.5449 Section 582.5449 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5449 Manganese citrate. (a) Product. Manganese citrate. (b) Conditions of use. This...

  16. 21 CFR 184.1449 - Manganese citrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Manganese citrate. 184.1449 Section 184.1449 Food... Specific Substances Affirmed as GRAS § 184.1449 Manganese citrate. (a) Manganese citrate (Mn3(C6H5O7)2, CAS... manganese carbonate from manganese sulfate and sodium carbonate solutions. The filtered and washed...

  17. [Cloning and tissue expression pattern analysis of the human citrate synthase cDNA].

    Science.gov (United States)

    Liu, Q; Yu, L; Han, X F; Fu, Q; Zhang, J X; Tang, H; Zhao, S Y

    2000-09-01

    Tricarboxylic acid (TCA) cycle is an important way to generate ATP, which is widely distributed in the cells of animal, plant or microorganism. It catalyses the catabolism of sugar as well as protein and fat. Citrate synthase plays a key role in regulating TCA cycle and is responsible for catalysing the synthesis of citrate from oxaloacetate and acetyl CoA. Screening of genomic informatics was performed by using pig citrate synthase cDNA as a probe and a contig which is 1636 bp long and has highly homologous to the pig citrate synthase cDNA was obtained from selected ESTs with the ASSEMBLY program. According to the sequence of this contig, a pair of primers was designed and used to amplify cDNA libraries. A 1492 bp cDNA containing an open reading frame encoding 466 amino acids was cloned from human testis and skeletal muscle cDNA libraries. The deduced amino acid sequence of the cDNA showed 95%, 92% and 60.9% identity to pig, chicken and yeast citrate synthase respectively. Because the deduced amino acids sequence contains a highly conserved motif of citrate synthase from three different species, it is believed that this cDNA may be a transcript of human citrate synthase gene. Northern analysis showed that the human citrate synthase was expressed at high level in heart and muscle, at middle level in brain, kidney and pancreas tissues, not detectable in thymus and small intestine tissues, and at low level in other nine tested human tissues.

  18. Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Suurmeijer, C.N.S.P.; Pérez-Gilabert, M.; Unen, D.-J. van; Hijden, H.T.W.M. van der; Veldink, G.A.

    2000-01-01

    Fatty acid hydroperoxide lyase (HPO-lyase) was purified 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X100 and beta-mercaptoethanol to the buffer yielded an active enzyme that could be stored for several months at −80°C. The enzyme was inhibited

  19. Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.

    Science.gov (United States)

    Dangelmaier, Carol A; Holmsen, Holm

    2014-01-01

    Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.

  20. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    Science.gov (United States)

    Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

    2015-03-02

    In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza.

    Science.gov (United States)

    Gordon, W R; Koukkari, W L

    1978-10-01

    The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their "inseparability."

  2. Ranitidine bismuth citrate: A review

    Directory of Open Access Journals (Sweden)

    N Chiba

    2001-01-01

    Full Text Available Recognition of the relationship between Helicobacter pylori infection and the development of gastroduodenal disease has increased greatly in recent years. To avoid complications of H pylori infection, such as the development of recurrent duodenal and gastric ulcers, effective therapies are required for eradication of the infection. This article reviews ranitidine bismuth citrate (RBC, a novel complex of ranitidine, bismuth and citrate, which was developed specifically for the purpose of eradicating H pylori. Dual therapy with RBC in combination with clarithromycin for 14 days yields eradication rates of 76%. Triple therapy bid for one week with a proton pump inhibitor, clarithromycin and either amoxicillin or a nitroimidazole (tinidazole or metronidazole is advocated as the treatment of choice for H pylori eradication. Analogous regimens with RBC in place of proton pump inhibitors show effective eradication rates in comparative studies and with pooled data. RBC, used alone or in combination with other antibiotics, appears to be a safe and effective drug for the treatment of H pylori infection. Bismuth levels do not appear to rise to toxic levels.

  3. Arthropod venom citrate inhibits phospholipase A2.

    Science.gov (United States)

    Fenton, A W; West, P R; Odell, G V; Hudiburg, S M; Ownby, C L; Mills, J N; Scroggins, B T; Shannon, S B

    1995-06-01

    Citrate has been identified as a major component of honey bee (Apis mellifera) venom by gas liquid chromatography-mass spectrometry. A citrate concentration of 9% was found for dried bee venom by a coupled enzyme assay, aconitase-isocitric dehydrogenase. A liquid honey bee venom would contain 140 mM citrate concentration (if the solids content were 30%). Bee venom phospholipase was inhibited at a 43% level with a citrate concentration of 20 mM and calcium ion at 3 mM with the enzyme assay. Citrate was also found in the venoms of bumble bee, Bombus fervidus, 7%; yellow jacket, Vespula maculifrons, 4%; scorpion, Centruroides sculpturatus, 8%; tarantula, Grammastola cala, 8% and brown recluse spider venom gland extract, Loxoceles reclusa, 1.5% based on dried venom solids. Citrate may serve as an endogenous inhibitor of divalent metal ion-dependent enzymes in arthropod venoms as described by Francis et al. (1992, Toxicon 30, 1239-1246). Many arthropod venoms contain calcium-dependent phospholipases. A direct effect of citrate as a venom component may be possible. The presence of citrate in venoms must be considered in research on receptors, ion channels and divalent ion-dependent toxins.

  4. 21 CFR 522.800 - Droperidol and fentanyl citrate injection.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Droperidol and fentanyl citrate injection. 522.800... § 522.800 Droperidol and fentanyl citrate injection. (a) Specifications. Droperidol and fentanyl citrate injection is a sterile solution containing 20 milligrams of droperidol and 0.4 milligram of fentanyl citrate...

  5. Exogenous γ-aminobutyric acid treatment affects citrate and amino acid accumulation to improve fruit quality and storage performance of postharvest citrus fruit.

    Science.gov (United States)

    Sheng, Ling; Shen, Dandan; Luo, Yi; Sun, Xiaohua; Wang, Jinqiu; Luo, Tao; Zeng, Yunliu; Xu, Juan; Deng, Xiuxin; Cheng, Yunjiang

    2017-02-01

    The loss of organic acids during postharvest storage is one of the major factors that reduces the fruit quality and economic value of citrus. Citrate is the most important organic acid in citrus fruits. Molecular evidence has proved that γ-aminobutyric acid (GABA) shunt plays a key role in citrate metabolism. Here, we investigated the effects of exogenous GABA treatment on citrate metabolism and storage quality of postharvest citrus fruit. The content of citrate was significantly increased, which was primarily attributed to the inhibition of the expression of glutamate decarboxylase (GAD). Amino acids, including glutamate, alanine, serine, aspartate and proline, were also increased. Moreover, GABA treatment decreased the fruit rot rate. The activities of antioxidant enzymes and the content of energy source ATP were affected by the treatment. Our results indicate that GABA treatment is a very effective approach for postharvest quality maintenance and improvement of storage performance in citrus production. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. A novel, inducible, citral lyase purified from spores of Penicillium digitatum

    NARCIS (Netherlands)

    Wolken, W.A.M.; Loo, W.J.V. van; Tramper, J.; Werf, M.J. van der

    2002-01-01

    A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum. Remarkably, citral lyase activity was induced 118-fold by incubating nongerminating spores with the substrate, citral. This

  7. Anaphylaxis related to fentanyl citrate

    Directory of Open Access Journals (Sweden)

    Gaurav Singh Tomar

    2012-01-01

    Full Text Available Anaphylaxis is a fulminant, unexpected, immunoglobulin E-mediated allergic reaction that can be triggered by multiple agents. Common causative agents include neuromuscular blocking drugs, latex, antibiotics, colloids, hypnotics, and opioids. Fentanyl citrate, however, is an extremely unusual cause of anaphylaxis. Pulmonary edema, although uncommon in anaphylaxis, can be a prominent feature, as was in one of the patient. An adverse drug reaction is a noxious or unintended reaction to a drug that is administered in standard doses by the proper route for the purpose of prophylaxis, diagnosis, or treatment. Reactions are classified into two major subtypes: type A, which are dose dependent and predictable; and type B, which are not dose dependent and unpredictable. Unpredictable reactions include immune (allergic or no immune drug hypersensitivity reactions and are related to genetic susceptibilities or undefined mechanisms (formally called idiosyncratic and intolerance reactions. A drug allergy is always associated with an immune mechanism for which evidence of drug-specific antibodies or activated T lymphocytes can be shown. In the last few years, many novel drugs have entered clinical practice (i.e., biologic agents generating novel patterns of drug hypersensitivity reactions. As old drugs continue to be used, new clinical and biologic techniques enable improvement in the diagnosis of these reactions.

  8. Treatment of norovirus particles with citrate.

    Science.gov (United States)

    Koromyslova, Anna D; White, Peter A; Hansman, Grant S

    2015-11-01

    Human norovirus is a dominant cause of acute gastroenteritis around the world. Several norovirus disinfectants label citric acid as an active ingredient. In this study, we showed that norovirus virus-like particles (VLPs) treated with citrate buffer caused the particles to alter their morphology, including increased diameters associated with a new ring-like structure. We also found that epitopes on the protruding (P) domain on these particles were more readily accessible to antibodies after the citrate treatment. These results suggested that citrate had a direct effect on the norovirus particles. Using X-ray crystallography, we showed that the P domain bound citrate from lemon juice and a disinfectant containing citric acid. Importantly, citrate binds at the histo-blood group antigen binding pocket, which are attachment factors for norovirus infections. Taken together, these new findings suggested that it might be possible to treat/reduce norovirus infections with citrate, although further studies are needed. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Regional citrate anticoagulation for pediatric CRRT using integrated citrate software and physiological sodium concentration solutions.

    Science.gov (United States)

    Liet, Jean-Michel; Allain-Launay, Emma; Gaillard-LeRoux, Bénédicte; Barrière, François; Chenouard, Alexis; Dejode, Jean-Marc; Joram, Nicolas

    2014-09-01

    In continuous renal replacement therapy (CRRT), regional citrate anticoagulation offers an attractive alternative to heparinization, especially for children with a high bleeding risk. We report on a new management approach to CRRT using integrated citrate software and physiological sodium concentration solutions. Convective filtration was performed with pre-filter citrate anticoagulation using an 18 mmol/L citrate solution and a post-filter replacement fluid. The citrate flow rate was automatically adjusted to the blood flow rate by means of integrated citrate software. Similarly, calcium was automatically infused into children to maintain their blood calcium levels within normal range. Eleven CRRT sessions were performed (330 h) in seven critically ill children aged 3-15 years (extreme values 15-66 kg). Disease categories included sepsis with multiorgan dysfunction (n = 2) and hemolytic uremic syndrome (n = 5). Median effluent dose was 2.1 (extreme values 1.7-3.3) L/h/1.73 m2. No session had to be stopped because of metabolic complications. Calcium levels, both in the circuits and in the circulating blood of the children, remained stable and secure. Regional citrate anticoagulation can be used in children with a body weight of >15 kg using integrated citrate software and commercially available solutions with physiological sodium concentrations in a safe, effective and convenient procedure.

  10. Citrate Uptake in Exchange with Intermediates in the Citrate Metabolic Pathway in Lactococcus lactis IL1403

    NARCIS (Netherlands)

    Pudlik, Agata M.; Lolkema, Juke S.

    Carbohydrate/citrate cometabolism in Lactococcus lactis results in the formation of the flavor compound acetoin. Resting cells of strain IL1403(pFL3) rapidly consumed citrate while producing acetoin when substoichiometric concentrations of glucose or L-lactate were present. A proton motive force was

  11. Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination

    NARCIS (Netherlands)

    Pudlik, Agata M.; Lolkema, Juke S.

    Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate

  12. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

    Science.gov (United States)

    Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

    2003-06-01

    Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

  13. Isolation, expression and comparison of a pectate lyase produced ...

    African Journals Online (AJOL)

    Administrator

    2010-12-27

    Dec 27, 2010 ... Cultivar Cavendish is resistant to FOC1, but a newly occurring race 4 (FOC4) was found to be able to infect Cavendish. Studying the fungal pathogenicity is an important step towards the disease control. In this study, we cloned two pl1 genes (723 bp and 240 aa), encoding pectate lyase 1 (PL1) from both ...

  14. Correlation between the cystathionine-r-lyase (CES) and the ...

    African Journals Online (AJOL)

    Background: The infection of Helicobacter pylori (H. pylori) is one of the most important causes of gastric ulcer disease. The role of hydrogen sulfide (H2S) production in H. pylori-induced gastric ulcer disease. Aim: The expression of cystathionine-γ-lyase (CSE) was determined, and correlated with the severity of gastric ulcer ...

  15. Purification and characterization of pectin lyase secreted by Penicillium citrinum.

    Science.gov (United States)

    Yadav, S; Yadav, P K; Yadav, D; Yadav, K D S

    2009-07-01

    The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K(m) and k(cat) values were found to be 1 mg/ml and 76 sec(-1), respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0-12.0 range when exposed for 24 h. The optimum temperature was 50 degrees C, and the pectin lyase was found to be completely stable up to 40 degrees C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.

  16. Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases.

    Science.gov (United States)

    Devamani, Titu; Rauwerdink, Alissa M; Lunzer, Mark; Jones, Bryan J; Mooney, Joanna L; Tan, Maxilmilien Alaric O; Zhang, Zhi-Jun; Xu, Jian-He; Dean, Antony M; Kazlauskas, Romas J

    2016-01-27

    Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ∼ 100 million years ago. Both enzyme types are α/β-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.

  17. Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans

    NARCIS (Netherlands)

    Raj, Hans; Puthan Veetil, Vinod; Szymanski, Wiktor; Dekker, Frank J.; Quax, Wim J.; Feringa, Ben L.; Janssen, Dick B.; Poelarends, Gerrit J.

    Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of

  18. In Silico Characterization of Pectate Lyase Protein Sequences from Different Source Organisms

    Directory of Open Access Journals (Sweden)

    Amit Kumar Dubey

    2010-01-01

    Full Text Available A total of 121 protein sequences of pectate lyases were subjected to homology search, multiple sequence alignment, phylogenetic tree construction, and motif analysis. The phylogenetic tree constructed revealed different clusters based on different source organisms representing bacterial, fungal, plant, and nematode pectate lyases. The multiple accessions of bacterial, fungal, nematode, and plant pectate lyase protein sequences were placed closely revealing a sequence level similarity. The multiple sequence alignment of these pectate lyase protein sequences from different source organisms showed conserved regions at different stretches with maximum homology from amino acid residues 439–467, 715–816, and 829–910 which could be used for designing degenerate primers or probes specific for pectate lyases. The motif analysis revealed a conserved Pec_Lyase_C domain uniformly observed in all pectate lyases irrespective of variable sources suggesting its possible role in structural and enzymatic functions.

  19. Physicochemical action of potassium-magnesium citrate in nephrolithiasis

    Science.gov (United States)

    Pak, C. Y.; Koenig, K.; Khan, R.; Haynes, S.; Padalino, P.

    1992-01-01

    Effect of potassium-magnesium citrate on urinary biochemistry and crystallization of stone-forming salts was compared with that of potassium citrate at same dose of potassium in five normal subjects and five patients with calcium nephrolithiasis. Compared to the placebo phase, urinary pH rose significantly from 6.06 +/- 0.27 to 6.48 +/- 0.36 (mean +/- SD, p less than 0.0167) during treatment with potassium citrate (50 mEq/day for 7 days) and to 6.68 +/- 0.31 during therapy with potassium-magnesium citrate (containing 49 mEq K, 24.5 mEq Mg, and 73.5 mEq citrate per day). Urinary pH was significantly higher during potassium-magnesium citrate than during potassium citrate therapy. Thus, the amount of undissociated uric acid declined from 118 +/- 61 mg/day during the placebo phase to 68 +/- 54 mg/day during potassium citrate treatment and, more prominently, to 41 +/- 46 mg/day during potassium-magnesium citrate therapy. Urinary magnesium rose significantly from 102 +/- 25 to 146 +/- 37 mg/day during potassium-magnesium citrate therapy but not during potassium citrate therapy. Urinary citrate rose more prominently during potassium-magnesium citrate therapy (to 1027 +/- 478 mg/day from 638 +/- 252 mg/day) than during potassium citrate treatment (to 932 +/- 297 mg/day). Consequently, urinary saturation (activity product) of calcium oxalate declined significantly (from 1.49 x 10(-8) to 1.03 x 10(-8) M2) during potassium-magnesium citrate therapy and marginally (to 1.14 x 10(-8) M2) during potassium citrate therapy.(ABSTRACT TRUNCATED AT 250 WORDS).

  20. 21 CFR 582.5195 - Calcium citrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium citrate. 582.5195 Section 582.5195 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  1. Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

    National Research Council Canada - National Science Library

    Elisabeth Schmidtmann Ann-Christine Konig Anne Orwat Dario Leister Markus Hartl Iris Finkemeier

    2014-01-01

    Citrate synthase has a key role in the tricarboxylic (TCA) cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA...

  2. Fracture penis : A complication of sildenafil citrate!

    Directory of Open Access Journals (Sweden)

    Anup Kumar Kundu

    2002-01-01

    Full Text Available Our experience with 6 consecutive cases of fracture penis, a rare urological injury, in normal male without any erectile dysfunction, who consumed Sildenafil Citrate for prolonged sexual enjoyment out of curiosity. All of them were properly worked up and treated surgi-cally resulting in excellent recovery of function.

  3. 21 CFR 73.2110 - Bismuth citrate.

    Science.gov (United States)

    2010-04-01

    ..., eyebrows, or hair on parts of the body other than the scalp. (d) Labeling. (1) The label of the color... abraded scalp. Do not use to color eyelashes, eyebrows, or hair on parts of the body other than the scalp... restrictions. The color additive bismuth citrate may be safely used in cosmetics intended for coloring hair on...

  4. Cultivable Alginate Lyase-Excreting Bacteria Associated with the Arctic Brown Alga Laminaria

    Directory of Open Access Journals (Sweden)

    Yu-Zhong Zhang

    2012-11-01

    Full Text Available Although some alginate lyases have been isolated from marine bacteria, alginate lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65, Psychromonas (10/65 and Polaribacter (8/65 were the predominant groups. Moreover, 21 alginate lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate lyase-excreting strains belong to five genera. Psychromonas (8/21, Psedoalteromonas (6/21 and Polaribacter (4/21 are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate lyases. The optimal temperatures for the growth and algiante lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate lyase activity around 200 U/mL. These results suggest that these algiante lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate lyases.

  5. Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

    Energy Technology Data Exchange (ETDEWEB)

    Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Yamasaki, Masayuki; Mikami, Bunzo [Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru; Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2006-05-01

    The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2{sub 1} and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β = 91.5°.

  6. Reduction and aggregation of silver ions in aqueous citrate solutions.

    Science.gov (United States)

    Chadha, Ridhima; Maiti, Nandita; Kapoor, Sudhir

    2014-05-01

    Radiolytic reduction of Ag(+) ions and the subsequent formation of Ag clusters were studied in aqueous citrate solutions. Pulse-radiolysis studies show that the presence of citrate in the solution affects the early processes, via complexation of Ag(+) ions with the carboxyl moieties of the citrate. The ratio of citrate to Ag(+) determines the kinetic consequences of the reduction and agglomeration processes. The complexation reduces somewhat the rate of reduction by hydrated electrons. However, when all the ions are complexed to the citrate, the surface plasmon absorption band becomes broader, albeit small, but nevertheless it provides extreme stability to the formed nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Eliminylation: a post-translational modification catalyzed by phosphothreonine lyases.

    Science.gov (United States)

    Brennan, Damian F; Barford, David

    2009-03-01

    We propose the classification of a protein post-translational modification, eliminylation, based on the recently delineated mechanism of the Shigella OspF and Salmonella SpvC phosphothreonine lyases. These bacterial type-III secretion-system virulence factors are injected into eukaryotic cells and inhibit signalling by irreversibly inactivating mitogen-activated protein kinases (MAPKs). Remarkably, they employ an unusual beta-elimination reaction, removing the phosphate from phosphothreonine and converting it into dehydrobutyrine (an alkene). Eliminylated cysteine can also be produced by decarboxylation and eliminylated serine and threonine by dehydration; these residues are found in the eye lens and in bacterial lantibiotics. We postulate that eliminylation might be a widespread regulatory modification, and we propose the use of phosphothreonine lyases as in vivo MAPK inhibitors both therapeutically and to investigate MAPK signalling regulation.

  8. Quantitation of heparosan with heparin lyase III and spectrophotometry.

    Science.gov (United States)

    Huang, Haichan; Zhao, Yingying; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

    2014-02-15

    Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Enhancing RGI lyase thermostability by targeted single point mutations

    DEFF Research Database (Denmark)

    Silva, Inês R.; Larsen, Dorte Møller; Jers, Carsten

    2013-01-01

    approach involving single amino acid substitution. Nine individual amino acids were selected as targets for site-saturated mutagenesis by the use of a predictive consensus approach in combination with prediction of protein mutant stability changes and B-factor iteration testing. After extensive...... the applicability of a combinatorial predictive approach for designing a small site saturation library for improving enzyme thermostability. In addition, new thermostable RGI lyases suitable for enzymatic upgrading of pectinaceous plant biomass materials at elevated temperatures were produced....

  10. Citrate anticoagulation for CRRT in children: comparison with heparin.

    Science.gov (United States)

    Fernández, Sara Nicole; Santiago, Maria José; López-Herce, Jesús; García, Miriam; Del Castillo, Jimena; Alcaraz, Andrés José; Bellón, Jose María

    2014-01-01

    Regional anticoagulation with citrate is an alternative to heparin in continuous renal replacement therapies, which may prolong circuit lifetime and decrease hemorrhagic complications. A retrospective comparative cohort study based on a prospective observational registry was conducted including critically ill children undergoing CRRT. Efficacy, measured as circuit survival, and secondary effects of heparin and citrate were compared. 12 patients on CRRT with citrate anticoagulation and 24 patients with heparin anticoagulation were analyzed. Median citrate dose was 2.6 mmol/L. Median calcium dose was 0.16 mEq/kg/h. Median heparin dose was 15 UI/kg/h. Median circuit survival was 48 hours with citrate and 31 hours with heparin (P = 0.028). 66.6% of patients treated with citrate developed mild metabolic alkalosis, which was directly related to citrate dose. There were no cases of citrate intoxication: median total calcium/ionic calcium index (CaT/I) of 2.16 and a maximum CaT/I of 2.33, without metabolic acidosis. In the citrate group, 45.5% of patients developed hypochloremia and 27.3% hypomagnesemia. In the heparin group, 27.8% developed hypophosphatemia. Three patients were moved from heparin to citrate to control postoperatory bleeding. In conclusion citrate is a safe and effective anticoagulation method for CRRT in children and it achieves longer circuit survival than heparin.

  11. Citrate Anticoagulation for CRRT in Children: Comparison with Heparin

    Directory of Open Access Journals (Sweden)

    Sara Nicole Fernández

    2014-01-01

    Full Text Available Regional anticoagulation with citrate is an alternative to heparin in continuous renal replacement therapies, which may prolong circuit lifetime and decrease hemorrhagic complications. A retrospective comparative cohort study based on a prospective observational registry was conducted including critically ill children undergoing CRRT. Efficacy, measured as circuit survival, and secondary effects of heparin and citrate were compared. 12 patients on CRRT with citrate anticoagulation and 24 patients with heparin anticoagulation were analyzed. Median citrate dose was 2.6 mmol/L. Median calcium dose was 0.16 mEq/kg/h. Median heparin dose was 15 UI/kg/h. Median circuit survival was 48 hours with citrate and 31 hours with heparin (P=0.028. 66.6% of patients treated with citrate developed mild metabolic alkalosis, which was directly related to citrate dose. There were no cases of citrate intoxication: median total calcium/ionic calcium index (CaT/I of 2.16 and a maximum CaT/I of 2.33, without metabolic acidosis. In the citrate group, 45.5% of patients developed hypochloremia and 27.3% hypomagnesemia. In the heparin group, 27.8% developed hypophosphatemia. Three patients were moved from heparin to citrate to control postoperatory bleeding. In conclusion citrate is a safe and effective anticoagulation method for CRRT in children and it achieves longer circuit survival than heparin.

  12. Concentration-dependent Sildenafil citrate (Viagra) effects on ROS production, energy status, and human sperm function.

    Science.gov (United States)

    Sousa, Maria Inês; Amaral, Sandra; Tavares, Renata Santos; Paiva, Carla; Ramalho-Santos, João

    2014-04-01

    Literature regarding the effects of sildenafil citrate on sperm function remains controversial. In the present study, we specifically wanted to determine if mitochondrial dysfunction, namely membrane potential, reactive oxygen species production, and changes in energy content, are involved in in vitro sildenafil-induced alterations of human sperm function. Sperm samples of healthy men were incubated in the presence of 0.03, 0.3, and 3 μM sildenafil citrate in a phosphate buffered saline (PBS)-based medium for 2, 3, 12, and 24 hours. Sperm motility and viability were evaluated and mitochondrial function, i.e., mitochondrial membrane potential and mitochondrial superoxide production were assessed using flow-cytometry. Additionally, adenosine triphosphate (ATP) levels were determined by high performance liquid chromatography (HPLC) analysis. Results show a decrease in sperm motility correlated with the level of mitochondria-generated superoxide, without a visible effect on mitochondrial membrane potential or viability upon exposure to sildenafil. The effect on both motility and superoxide production was higher for the intermediate concentration of sildenafil (0.3 µM) indicating that the in vitro effects of sildenafil on human sperm do not vary linearly with drug concentration. Adenosine triphosphate levels also decreased following sildenafil exposure, but this decrease was only detected after a decrease in motility was already evident. These results suggest that along with the level of ATP and mitochondrial function other factors are involved in the early sildenafil-mediated decline in sperm motility. However, the further decrease in ATP levels and increase in mitochondria-generated reactive oxygen species after 24 hours of exposure might further contribute towards declining sperm motility.

  13. The effect of pH and free Mg2+ on ATP linked enzymes and the calculation of Gibbs free energy of ATP hydrolysis.

    Science.gov (United States)

    Bergman, Christian; Kashiwaya, Yoshihiro; Veech, Richard L

    2010-12-16

    The apparent equilibrium constants, K′, of biochemical reactions containing substrates which bind [Mg2+] unequally can be significantly altered by changes in free intracellular [Mg2+]. Intracellular free [Mg2+] can be estimated by measurements of [citrate]/[isocitrate], a ratio known to vary with tissue free [Mg2+]. The combined equilibrium constant for glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and triose phosphate isomerase for the three reactions (K(GG-TPI)′) was corrected using new binding constants for dihydroxyacetone-phosphate and 3-phosphoglycerate. The result of this calculation is demonstrated in the calculation of the free energy of ATP hydrolysis. In addition, the dependence of the equilibrium constant for the glutamine synthetase reaction on pH and free [Mg2+] was demonstrated. Furthermore, a theory linking the ΔG′ value of mitochondrial complex I−II and the cytosolic ΔG′ value of ATP hydrolysis is discussed with evidence from previous publications.

  14. Citrate Pharmacokinetics in Critically Ill Patients with Acute Kidney Injury.

    Directory of Open Access Journals (Sweden)

    Yin Zheng

    Full Text Available Regional citrate anticoagulation (RCA is gaining popularity in continous renal replacement therapy (CRRT for critically ill patients. The risk of citrate toxicity is a primary concern during the prolonged process. The aim of this study was to assess the pharmacokinetics of citrate in critically ill patients with AKI, and used the kinetic parameters to predict the risk of citrate accumulation in this population group undergoing continuous veno-venous hemofiltration (CVVH with RCA.Critically ill patients with AKI (n = 12 and healthy volunteers (n = 12 were investigated during infusing comparative dosage of citrate. Serial blood samples were taken before, during 120 min and up to 120 min after infusion. Citrate pharmacokinetics were calculated and compared between groups. Then the estimated kinetic parameters were applied to the citrate kinetic equation for validation in other ten patients' CVVH sessions with citrate anticoagulation.Total body clearance of citrate was similar in critically ill patients with AKI and healthy volunteers (648.04±347.00 L/min versus 686.64±353.60 L/min; P = 0.624. Basal and peak citrate concentrations were similar in both groups (p = 0.423 and 0.247, respectively. The predicted citrate curve showed excellent fit to the measurements.Citrate clearance is not impaired in critically ill patients with AKI in the absence of severe liver dysfunction. Citrate pharmacokinetic data can provide a basis for the clinical use of predicting the risk of citrate accumulation.ClinicalTrials.gov Identifier NCT00948558.

  15. ATP as a biological hydrotrope.

    Science.gov (United States)

    Patel, Avinash; Malinovska, Liliana; Saha, Shambaditya; Wang, Jie; Alberti, Simon; Krishnan, Yamuna; Hyman, Anthony A

    2017-05-19

    Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells. Copyright © 2017, American Association for the Advancement of Science.

  16. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  17. ATP synthase from slow and fast growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  18. Alfalfa contains substantial 9-hydroperoxide lyase activity and a 3Z:2E-enal isomerase

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Noordermeer, M.A.; Veldink, G.A.

    1999-01-01

    Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been

  19. Study on optimal production of 3-ketovalidoxylamine A C-N lyase ...

    African Journals Online (AJOL)

    3-Ketovalidoxylamine A C-N lyase and glucoside 3-dehydrogenase (G3DH), two key enzymes for valienamine synthesis, are produced by Stenotrophomonas maltrophilia. The condition of producing 3- ketovalidoxylamine A C-N lyase and G3DH was optimized. Validamycin A was showed to be suitable carbon source for ...

  20. Functional characterization and Me2+ ion specificity of a Ca2+-citrate transporter from Enterococcus faecalis

    NARCIS (Netherlands)

    Blancato, Victor S.; Magni, Christian; Lolkema, Juke S.

    2006-01-01

    Secondary transporters of the bacterial CitMHS family transport citrate in complex with a metal ion. Different members of the family are specific for the metal ion in the complex and have been shown to transport Mg2+-citrate, Ca2+-citrate or Fe3+-citrate. The Fe3+-citrate transporter of

  1. Aspergillus niger Secretes Citrate to Increase Iron Bioavailability

    Science.gov (United States)

    Odoni, Dorett I.; van Gaal, Merlijn P.; Schonewille, Tom; Tamayo-Ramos, Juan A.; Martins dos Santos, Vitor A. P.; Suarez-Diez, Maria; Schaap, Peter J.

    2017-01-01

    Aspergillus niger has an innate ability to secrete various organic acids, including citrate. The conditions required for A. niger citrate overproduction are well described, but the physiological reasons underlying extracellular citrate accumulation are not yet fully understood. One of the less understood culture conditions is the requirement of growth-limiting iron concentrations. While this has been attributed to iron-dependent citrate metabolizing enzymes, this straightforward relationship does not always hold true. Here, we show that an increase in citrate secretion under iron limited conditions is a physiological response consistent with a role of citrate as A. niger iron siderophore. We found that A. niger citrate secretion increases with decreasing amounts of iron added to the culture medium and, in contrast to previous findings, this response is independent of the nitrogen source. Differential transcriptomics analyses of the two A. niger mutants NW305 (gluconate non-producer) and NW186 (gluconate and oxalate non-producer) revealed up-regulation of the citrate biosynthesis gene citA under iron limited conditions compared to iron replete conditions. In addition, we show that A. niger can utilize Fe(III) citrate as iron source. Finally, we discuss our findings in the general context of the pH-dependency of A. niger organic acid production, offering an explanation, besides competition, for why A. niger organic acid production is a sequential process influenced by the external pH of the culture medium. PMID:28824560

  2. Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications

    Science.gov (United States)

    Zhu, Benwei; Yin, Heng

    2015-01-01

    Alginate lyases catalyze the degradation of alginate, a complex copolymer of α-L-guluronate and its C5 epimer β-D-mannuronate. The enzymes have been isolated from various kinds of organisms with different substrate specificities, including algae, marine mollusks, marine and terrestrial bacteria, and some viruses and fungi. With the progress of structural biology, many kinds of alginate lyases of different polysaccharide lyases families have been characterized by obtaining crystal structures, and the catalytic mechanism has also been elucidated. Combined with various studies, we summarized the source, classification and properties of the alginate lyases from different polysaccharide lyases families. The relationship between substrate specificity and protein sequence was also investigated. PMID:25831216

  3. HCN production and hydroxynitrile lyase: a natural activity in plants and a renewed biotechnological interest.

    Science.gov (United States)

    Kassim, Muhammad Arshad; Rumbold, Karl

    2014-02-01

    Over 3,000 plant species are cyanogenic. Cyanogenesis is defined as the hydroxynitrile lyase catalysed release of a cyanide group in the form of HCN and the corresponding aldehyde or ketone. When a plant is attacked, HCN released is a self defence mechanism. A special characteristic of enzymatic reactions is that all enzymatic reactions are reversible-hydroxynitrile lyases can also be used for the synthesis of enantiomerically pure cyanohydrins which are of great importance in industry. This article presents a comprehensive review of the role of hydroxynitrile lyases, both in nature and industry, and an insightful. Areas covered include: history, discovery and natural sources of the hydroxynitrile lyase. Molecular cloning for mass production of this enzyme, including detailed information about several successful recombinant hydroxynitrile lyases is also included.

  4. ATP Release and Effects in Pancreas

    DEFF Research Database (Denmark)

    Novak, Ivana; Amstrup, Jan; Henriksen, Katrine Lütken

    2003-01-01

    ATP and other nucleotides are released from various cells, but the pathway and physiological stimulus for ATP release are often unclear. The focus of our studies is the understanding of ATP release and signaling in rat exocrine pancreas. In acinar suspension mechanical stimulation, hypotonic shock...

  5. Extracellular ATP induces albuminuria in pregnant rats

    NARCIS (Netherlands)

    Faas, M.M.; van der Schaaf, G.; Borghuis, T.; Jongman, R.M.; van Pampus, Maria; de Vos, P.; van Goor, Harry; Bakker, W.W.

    BACKGROUND: As circulating plasma ATP concentrations are increased in pre-eclampsia, we tested whether increased plasma ATP is able to induce albuminuria during pregnancy. METHODS: Pregnant (day 14) and non-pregnant rats were infused with ATP (3000 microg/kg bw) via a permanent jugular vein cannula.

  6. Lithium citrate as treatment of Canavan disease.

    Science.gov (United States)

    Solsona, María Dolores Edo; Fernández, Laura Lorente; Boquet, Emilio Monte; Andrés, José Luís Poveda

    2012-01-01

    Canavan disease is a rare autosomal recessive leukodystrophy characterized by abnormal accumulation of N-acetyl aspartate (NAA) in brain white matter. Currently, there is no cure for this disease, and management of patients consists mainly of treating symptoms. We describe a 3-month-old girl who was hospitalized for poor head control and decreased muscle tone. A battery of laboratory and genetic (homozygous mutation p.C218X) analysis revealed the presence of Canavan disease. Lithium citrate was initiated at a dosage of 45 mg/kg per day after diagnosis. Periodic controls of thyroid and liver function, and lithium levels in blood showed that this drug was sure and well tolerated. After 1 year of treatment, NAA levels decreased by approximately 20% in the brain region, urinary NAA levels showed a reduction of 80%, and patient improved alertness and visual tracking but continued with no heat support, axial hypotonia, and spastic diplegia. In our patient, the results obtained after drug administration are important with respect to the decrease in NAA and more discreet in clinical improvement. However, given the absence of adverse effects and limited treatment options, lithium citrate may be a good alternative to stop the progression of the disease and improve the quality of life of patients.

  7. Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza 1

    Science.gov (United States)

    Gordon, William R.; Koukkari, Willard L.

    1978-01-01

    The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their “inseparability.” PMID:16660569

  8. Importance of citrate and the calcium : citrate ratio in patients with calcium renal lithiasis and severe lithogenesis.

    Science.gov (United States)

    Arrabal-Polo, Miguel Angel; Arrabal-Martin, Miguel; Arias-Santiago, Salvador; Garrido-Gomez, Juan; Poyatos-Andujar, Antonio; Zuluaga-Gomez, Armando

    2013-04-01

    Different studies have shown the importance of citrate in the formation of calcium stones. It has further been shown that the states of metabolic acidosis result in an increase in bone resorption and lower urinary citrate levels. Increasing the intake of citrate in these patients can reduce the lithogenic risk and improve bone mineral density (BMD), contributing to control of both diseases. The study shows the importance of citrate in patients with calcium stones and BMD loss. The deficit in citrate excretion is associated with a decrease in bone mineralization and increased β-crosslaps. A calcium : citrate ratio >0.25 in patients with calcium stones and loss of mineral density may predict severe lithogenic activity. To analyse the importance of urinary citrate and the urinary calcium : citrate ratio in patients with calcium renal lithiasis and severe lithogenesis compared with a control group of patients without lithiasis. A cross-sectional study of 115 patients in eastern Andalusia, Spain was conducted. The patients were divided into two groups: Group A: 56 patients aged 25-60 years without calcium renal lithiasis; Group B: 59 patients aged 25-60 years, presenting with calcium renal lithiasis and severe lithogenesis. The citrate levels and the calcium : citrate ratio in the patients' urine and the relationship of these two factors to lithiasic activity were analysed and compared. In Group B, 32.2% of the patients presented with hypocitraturia, compared with 14.3% of the patients in Group A (P = 0.02). The urinary citrate levels were lower in Group B than in Group A (P = 0.001) and the calcium : citrate ratio was higher in Group B than in Group A (P = 0.005). The results suggest that a patient urinary calcium : citrate ratio > 0.25 indicates severe lithogenesis (with a sensitivity of 89% and a specificity of 57%). After linear regression analysis, we found that the urinary citrate level is an independent factor associated with the changes in bone densitometry T

  9. Development of pH-responsive polymer and citrate aqueous two-phase system for extractive bioconversion of cefprozil.

    Science.gov (United States)

    Chen, Jialing; Ding, Zhaoyang; Pan, Hongxia; Cao, Xuejun

    2017-11-01

    A pH-responsive aqueous two-phase system (pH-ATPS) has been developed by sodium citrate and a recyclable pH-responsive polymer PADB6.8 that can response to the change of pH values. Phase separation mechanism is studied through Low field-NMR. All variables affecting the phase separation are evaluated. Phase characteristics (viscosity, density, interfacial tension) and phase separation kinetic are studied for understanding of separation process and operational parameters in applications. This pH-ATPS has the characters of low interfacial tension, high recovery leading efficient mass transfer and low cost. The proposed system can be used as a mild medium for extractive bioconversion with low cost. We applied this pH-ATPS in extractive bioconversion of cefprozil. Cefprozil is partitioned towards the polymer-rich phase while the substrates tended to be partitioned in the salt-rich phase. Extractive bioconversion of cefprozil in this pH-ATPS can improve yield of the enzymatic process and reduce the product hydrolysis in optimal conditions. The maximal conversion yield of cefprozil in the studied system is 91.0%. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. [sup 99m]Tc-Citrate versus [sup 67]Ga-citrate for the scintigraphic visualization of inflammatory lesions

    Energy Technology Data Exchange (ETDEWEB)

    Ercan, M.T.; Aras, T.; Unlenen, E.; Unsal, I.S.; Hascelik, Z. (Hacettepe Univ., Ankara (Turkey)); Unlu, M. (Gazi Univ., Ankara (Turkey). Dept. of Nuclear Medicine)

    1993-10-01

    Citric acid was labeled with [sup 99m]Tc with an efficiency of > 99%. The biodistribution of [sup 99m]Tc-citrate was studied in mice with turpentine-induced abscesses in comparison to [sup 67]Ga-citrate. The max. abscess/muscle concentration ratios were 4.61 [+-] 1.92 (3 h) for [sup 99m]Tc-citrate and 4.76 [+-] 2.04 (4 h) for [sup 67]Ga-citrate. Arthritis was induced in 10 rabbits by intra-articular injection of ovalbumin Scintigrams obtained 4 days later and at 3 h post-injection of [sup 99m]Tc-citrate showed increased activity involving the synovium. The max. arthritic/contralateral knee ratio was 3.19 [+-] 1.29 (3 h) and 6.47 [+-] 3.71 (24 h) for [sup 99m]Tc- and [sup 67]Ga-citrate, respectively. The blood clearance curve of [sup 99m]Tc-citrate in rabbits was biexponential with a fast and a slow component, compared to mono-exponential clearance of [sup 67]Ga-citrate. In 10 patients with rheumatoid arthritis whole-body scintigrams and spot images of involved joints indicated localization of the tracer in inflamed tissues. The mean target-to-soft tissue ratios were 3.04 [+-] 0.81 and 4.95 [+-] 2.56 for [sup 99m]Tc-citrate and [sup 99m]Tc-MDP, respectively. Renal clearance of radioactivity was evident from the scintigrams. Our results demonstrated that [sup 99m]Tc-citrate is effective as a radiopharmaceutical for the visualization of inflammatory lesions and may be preferred to [sup 67]Ga-citrate due to the ideal physical characteristics of the radionuclide, easy preparation, low cost, early accumulation and the preference for the renal route of excretion. (Author).

  11. 21 CFR 522.300 - Carfentanil citrate injection.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Carfentanil citrate injection. 522.300 Section 522....300 Carfentanil citrate injection. (a) Specifications. Each milliliter of sterile aqueous solution..., shoulder, back, or hindquarter. Avoid intrathoracic, intra-abdominal, or subcutaneous injection. To reverse...

  12. 21 CFR 520.622 - Diethylcarbamazine citrate oral dosage forms.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Diethylcarbamazine citrate oral dosage forms. 520... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS ORAL DOSAGE FORM NEW ANIMAL DRUGS § 520.622 Diethylcarbamazine citrate oral dosage forms. ...

  13. Pre-process desilication of wheat straw with citrate

    DEFF Research Database (Denmark)

    Le, Duy Michael; Sorensen, Hanne R.; Meyer, Anne S.

    2017-01-01

    Effects of treatment time, citrate concentration, temperature, and pH on Si extraction from wheat straw prior to hydrothermal pretreatment were investigated for maximising Si removal and biomass recovery before biomass refining. With citrate, an almost linear negative correlation between Si conte...

  14. Enhanced dissolution of sildenafil citrate as dry foam tablets.

    Science.gov (United States)

    Sawatdee, Somchai; Atipairin, Apichart; Sae Yoon, Attawadee; Srichana, Teerapol; Changsan, Narumon

    2017-01-30

    Dry foam formulation technology is alternative approach to enhance dissolution of the drug. Sildenafil citrate was suspended in sodium dodecyl sulfate solution and adding a mixture of maltodextrin and mannitol as diluent to form a paste. Sildenafil citrate paste was passed through a nozzle spray bottle to obtain smooth foam. The homogeneous foam was dried in a vacuum oven and sieved to obtain dry foam granules. The granules were mixed with croscarmellose sodium, magnesium stearate and compressed into tablet. All formulations were evaluated for their physicochemical properties and dissolution profiles. All the tested excipients were compatible with sildenafil citrate by both differential scanning calorimetry (DSC) and infrared (IR) analysis. There are no X-ray diffraction (XRD) peaks representing crystals of sildenafil citrate observed form dry foam formulations. The hardness of tablets was about 5 kg, friability test <1% with a disintegration time <5 min. The sildenafil citrate dry foam tablet had higher dissolution rate in 0.1 N HCl in comparison with commercial sildenafil citrate tablet, sildenafil citrate prepared by direct compression and wet granulation method. Sildenafil citrate dry foam tablet with the high-level composition of surfactant, water and diluent showed enhanced dissolution rate than that of the lower-level composition of these excipients. This formulation was stable under accelerated conditions for at least 6 months.

  15. Structural basis for norovirus inhibition and fucose mimicry by citrate.

    Science.gov (United States)

    Hansman, Grant S; Shahzad-Ul-Hussan, Syed; McLellan, Jason S; Chuang, Gwo-Yu; Georgiev, Ivelin; Shimoike, Takashi; Katayama, Kazuhiko; Bewley, Carole A; Kwong, Peter D

    2012-01-01

    Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 Å and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 μM). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 μM) and H type 2 trisaccharide (390 μM), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.

  16. Structural Basis for Norovirus Inhibition and Fucose Mimicry by Citrate

    Energy Technology Data Exchange (ETDEWEB)

    Hansman, Grant S.; Shahzad-ul-Hussan, Syed; McLellan, Jason S.; Chuang, Gwo-Yu; Georgiev, Ivelin; Shimoike, Takashi; Katayama, Kazuhiko; Bewley, Carole A.; Kwong, Peter D. (NIAID)

    2012-01-20

    Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 {angstrom} and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 {mu}M). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 {mu}M) and H type 2 trisaccharide (390 {mu}M), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.

  17. Citrate increases glass transition temperature of vitrified sucrose preparations

    NARCIS (Netherlands)

    Kets, E.P.W.; Lipelaar, P.J.; Hoekstra, F.A.; Vromans, H.

    2004-01-01

    The aim of this study was to investigate the effect of sodium citrate on the properties of dried amorphous sucrose glasses. Addition of sodium citrate to a sucrose solution followed by freeze-drying or convective drying resulted in a glass transition temperature (T-g) that was higher than the

  18. Association between clomiphene citrate and central retinal vein ...

    African Journals Online (AJOL)

    Central retinal vein occlusion (CRVO) is a common retinal vascular disorder associated with cardiovascular disorders and other related risk factors. A case of CRVO secondary to clomiphene citrate has been reported. We present a case that also illustrates the association between clomiphene citrate and CRVO, and hope ...

  19. Screening of alginate lyase-excreting microorganisms from the surface of brown algae.

    Science.gov (United States)

    Wang, Mingpeng; Chen, Lei; Zhang, Zhaojie; Wang, Xuejiang; Qin, Song; Yan, Peisheng

    2017-12-01

    Alginate lyase is a biocatalyst that degrades alginate to produce oligosaccharides, which have many bioactive functions and could be used as renewable biofuels. Here we report a simple and sensitive plate assay for screening alginate lyase-excreting microorganisms from brown algae. Brown algae Laminaria japonica, Sargassum horneri and Sargassum siliquatrum were cultured in sterile water. Bacteria growing on the surface of seaweeds were identified and their capacity of excreting alginate lyase was analyzed. A total of 196 strains were recovered from the three different algae samples and 12 different bacterial strains were identified capable of excreting alginate lyases. Sequence analysis of the 16S rRNA gene revealed that these alginate lyase-excreting strains belong to eight genera: Paenibacillus (4/12), Bacillus (2/12), Leclercia (1/12), Isoptericola (1/12), Planomicrobium (1/12), Pseudomonas (1/12), Lysinibacillus (1/12) and Sphingomonas (1/12). Further analysis showed that the LJ-3 strain (Bacillus halosaccharovorans) had the highest enzyme activity. To our best knowledge, this is the first report regarding alginate lyase-excreting strains in Paenibacillus, Planomicrobium and Leclercia. We believe that our method used in this study is relatively easy and reliable for large-scale screening of alginate lyase-excreting microorganisms.

  20. Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524

    Directory of Open Access Journals (Sweden)

    Xiu-Lan Chen

    2016-07-01

    Full Text Available Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7 was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30oC and remained 19% of the highest activity at 5oC. AlyPM was unstable at temperatures above 30oC and had a low Tm of 37oC. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5-1.2 M NaCl was 6-fold higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20 folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a novel PL7 endo-alginate lyase with special characteristics.

  1. Uniport of Anionic Citrate and Proton Consumption in Citrate Metabolism Generates a Proton Motive Force in Leuconostoc oenos

    NARCIS (Netherlands)

    RAMOS, A; POOLMAN, B; SANTOS, H; LOLKEMA, JS; KONINGS, WN

    The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (Delta Psi) and a pH gradient (Delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous

  2. L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus, a bifunctional enzyme involved in autotrophic CO(2) fixation.

    Science.gov (United States)

    Herter, Sylvia; Busch, Andreas; Fuchs, Georg

    2002-11-01

    The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta-methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.

  3. Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

    Energy Technology Data Exchange (ETDEWEB)

    Han, Young-Hyun; Garron, Marie-Line; Kim, Hye-Yeon; Kim, Wan-Seok; Zhang, Zhenqing; Ryu, Kyeong-Seok; Shaya, David; Xiao, Zhongping; Cheong, Chaejoon; Kim, Yeong Shik; Linhardt, Robert J.; Jeon, Young Ho; Cygler, Miroslaw; (SNU); (Korea BSI); (McGill); (UST-Korea); (Rensselaer)

    2010-01-12

    Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a {beta}-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an ({alpha}/{alpha}){sub 6} fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.

  4. Circadian urinary citrate excretion in a rat model of exercise.

    Science.gov (United States)

    Nuñez, Paula; Diaz, Irene; Perillan, Carmen; Arguelles, Juan; Diaz, Elena

    2017-01-15

    Circadian rhythms are the approximate 24h biological cycles that function to prepare an organism for daily environmental changes. Circadian rhythms unquestionably play critical roles in metabolic homeostasis and the exercise has emerged as a strong non-photic time cue or zeitgeber in animal models and humans. Numerous studies about the effects of exercise on the citrate synthase activity have been published. Citrate is used to assess energy production or expenditure because it is a substrate of the Krebs Cycle, a cycle for oxidative energy production. We tested the existence of a rhythmic urinary citrate excretion in a rat model that is made to exercise at six different points during the day. The data obtained by the enzyme assays were fitted to a mathematical model (Fourier series), showing for the first time, the existence of a distinct ultradian rhythm in the urinary citrate excretion. The aerobic exercise led to the increase in the period length of the ultradian rhythm and raised the acrophase value of the urinary citrate excretion. Therefore, the urinary citrate excretion pattern changed after exercise, showing a clear circadian rhythm fitted to the mathematical model. The citrate urine samples could provide accurate data for ranking an individual's metabolic status. Using exercise to maintain the circadian clock at an appropriate phase and amplitude might be effective to prevent cardiometabolic disease development. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism.

    Science.gov (United States)

    Akhmanova, A; Voncken, F G; Hosea, K M; Harhangi, H; Keltjens, J T; op den Camp, H J; Vogels, G D; Hackstein, J H

    1999-06-01

    The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.

  6. Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Hayoz Sébastien

    2012-05-01

    Full Text Available Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP and a G protein-coupled P2Y receptor agonist (UTP. Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP

  7. High cerebral guanidinoacetate and variable creatine concentrations in argininosuccinate synthetase and lyase deficiency : Implications for treatment?

    NARCIS (Netherlands)

    van Spronsen, F. J.; Reijngoud, D. J.; Verhoeven, N. M.; Soorani-Lunsing, R. J.; Jakobs, C.; Sijens, P. E.

    2006-01-01

    Cerebral creatine and guanidinoacetate and blood and urine metabolites were studied in four patients with argininosuccinate synthetase (ASS) or argininosuccinate lyase (ASL) deficiency receiving large doses of arginine. Urine and blood metabolites varied largely. Cerebral guanidinoacetate was

  8. Densities, cellulases, alginate and pectin lyases of luminous and other heterotrophic bacteria associated with marine algae

    Digital Repository Service at National Institute of Oceanography (India)

    Ramaiah, N.; Chandramohan, D.

    enzymes. No luminous bacteria examined produced cellulases, but both V. harveyi and V. fischeri strains produced substantial amounts of alginate and pectin lyases. In contrast, cellulase activities were pronounced in non-luminous vibrio, pseudomonad...

  9. Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme

    OpenAIRE

    Shisler, Krista A.; Hutcheson, Rachel U.; Horitani, Masaki; Duschene, Kaitlin S.; Crain, Adam V.; Byer, Amanda S.; Shepard, Eric M.; Rasmussen, Ashley; Yang, Jian; Broderick, William E.; Vey, Jessica L.; Drennan, Catherine L.; Hoffman, Brian M.; Broderick, Joan B

    2017-01-01

    Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na+ as the most likely ...

  10. Cloning of a Putative Pectate Lyase Gene Expressed in the Subventral Esophageal Glands of Heterodera glycines.

    Science.gov (United States)

    De Boer, J M; Davis, E L; Hussey, R S; Popeijus, H; Smant, G; Baum, T J

    2002-03-01

    We report the cloning of a Heterodera glycines cDNA that has 72% identity at the amino acid level to a pectate lyase from Globodera rostochiensis. In situ hybridizations showed that the corresponding gene (Hg-pel-1) is expressed in the subventral esophageal gland cells of second-stage juveniles. The deduced amino acid sequence of the H. glycines cDNA shows homology to class III pectate lyases of bacterial and fungal origin.

  11. PbSO4 Leaching in Citric Acid/Sodium Citrate Solution and Subsequent Yielding Lead Citrate via Controlled Crystallization

    Directory of Open Access Journals (Sweden)

    Dongsheng He

    2017-06-01

    Full Text Available Lead citrate is a key precursor for the green recycling of spent lead acid battery paste in a citric acid/sodium citrate (CA/SC solution. In this study, the main paste component, PbSO4, was leached and crystallized to yield lead citrate. Results showed that the leaching of PbSO4 in citric acid/sodium citrate solution was remarkably enhanced by an increase in temperature from 35 °C to 95 °C and an increase in sodium citrate (SC concentration from 50 to 650 g/L. In comparison, increasing the citric acid/sodium citrate molar ratio inhibited this leaching. Controlled crystallization through cooling the solution or adjusting the pH of the solution can effectively produce lead citrate crystals. The X-ray diffraction patterns of four products obtained in a comparison test were all consistent with Pb3(C6H5O72. However, the scanning electron microscopy analysis suggested that the morphology was distinct from rods to sheets, which were mainly affected by the temperature variation.

  12. Acute effects of calcium carbonate, calcium citrate and potassium citrate on markers of calcium and bone metabolism in young women.

    Science.gov (United States)

    Karp, Heini J; Ketola, Maarit E; Lamberg-Allardt, Christel J E

    2009-11-01

    Both K and Ca supplementation may have beneficial effects on bone through separate mechanisms. K in the form of citrate or bicarbonate affects bone by neutralising the acid load caused by a high protein intake or a low intake of alkalising foods, i.e. fruits and vegetables. Ca is known to decrease serum parathyroid hormone (S-PTH) concentration and bone resorption. We compared the effects of calcium carbonate, calcium citrate and potassium citrate on markers of Ca and bone metabolism in young women. Twelve healthy women aged 22-30 years were randomised into four controlled 24 h study sessions, each subject serving as her own control. At the beginning of each session, subjects received a single dose of calcium carbonate, calcium citrate, potassium citrate or a placebo in randomised order. The diet during each session was identical, containing 300 mg Ca. Both the calcium carbonate and calcium citrate supplement contained 1000 mg Ca; the potassium citrate supplement contained 2250 mg K. Markers of Ca and bone metabolism were followed. Potassium citrate decreased the bone resorption marker (N-terminal telopeptide of type I collagen) and increased Ca retention relative to the control session. Both Ca supplements decreased S-PTH concentration. Ca supplements also decreased bone resorption relative to the control session, but this was significant only for calcium carbonate. No differences in bone formation marker (bone-specific alkaline phosphatase) were seen among the study sessions. The results suggest that potassium citrate has a positive effect on the resorption marker despite low Ca intake. Both Ca supplements were absorbed well and decreased S-PTH efficiently.

  13. The effect of alginate lyase on the gentamicin resistance of Pseudomonas aeruginosa in mucoid biofilms.

    Science.gov (United States)

    Germoni, L A P; Bremer, P J; Lamont, I L

    2016-07-01

    Pseudomonas aeruginosa can secrete large amounts of alginate during chronic infections and this has been associated with high resistance to antibiotics. The major aim of this study was to investigate whether degradation of extracellular alginate by alginate lyase would increase the sensitivity of Ps. aeruginosa to gentamicin, an aminoglycoside antibiotic. Degradation of alginate from Ps. aeruginosa was monitored using a spectrometric assay. Alginate lyase depolymerized alginate, but calcium and zinc cations at concentrations found in the cystic fibrosis lung reduced enzyme activity. Biofilms formed on agar were partially degraded by alginate lyase, but staining with crystal violet showed that the biomass of biofilms grown in liquid was not significantly affected by the enzyme. Viability testing showed that the sensitivity to gentamicin of biofilm bacteria and of bacteria released from biofilms was unaffected by alginate lyase. Our results show that at least under the conditions used here alginate lyase does not affect gentamicin resistance of Ps. aeruginosa. Our study indicates that alginate does not contribute to resistance to gentamicin and so does not provide support for the concept of treating patients with alginate lyase in order to increase the antibiotic sensitivity of Ps. aeruginosa. © 2016 The Society for Applied Microbiology.

  14. Purification and Characterization of Hyaluronate Lyase from Arthrobacter globiformis A152.

    Science.gov (United States)

    Zhu, Changliang; Zhang, Jingliang; Li, Liyan; Zhang, Jing; Jiang, Yanhui; Shen, Zhaopeng; Guan, Huashi; Jiang, Xiaolu

    2017-05-01

    A hyaluronate lyase was obtained by cultivating Arthrobacter globiformis strain A152. The enzyme was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q Sepharose Fast Flow, and Sephadex G-100 chromatography. The purification resulted in a 32.78-fold increase in hyaluronate lyase activity with specific activity of 297.2 U/mg. The molecular weight of the enzyme determined by SDS-PAGE was approximately 73.7 kDa. Using hyaluronic acid (HA) as a substrate, the maximal reaction rate (Vmax) and the Michaelis-Menten constant (Km) of hyaluronate lyase were found to be 4.76 μmol/min/ml and 0.11 mg/ml, respectively. The optimum pH and temperature values for hyaluronate lyase activity were pH 6.0 and 42 °C, respectively. This enzyme was stable at pH 4-10, 5-7, and 5-7 at 4, 37, and 42 °C, respectively. Investigation about temperature effects on hyaluronate lyase displayed that it was stable at 30-37 °C and also showed high activity at 37 °C. The enzymatic activity was enhanced by Ca2+ and was strongly inhibited by Cu2+ and SDS. These properties suggested that the hyaluronate lyase in this study could bring promising prospects in medical and industry applications.

  15. Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A.

    Directory of Open Access Journals (Sweden)

    Steven M Swift

    Full Text Available Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.

  16. Simplified Citrate Anticoagulation for CRRT Without Calcium Replacement.

    Science.gov (United States)

    Broman, Marcus; Klarin, Bengt; Sandin, Karin; Carlsson, Ola; Wieslander, Anders; Sternby, Jan; Godaly, Gabriela

    2015-01-01

    Since 2012, citrate anticoagulation is the recommended anticoagulation strategy for continuous renal replacement therapy (CRRT). The main drawback using citrate as anticoagulant compared with heparin is the need for calcium replacement and the rigorous control of calcium levels. This study investigated the possibility to achieve anticoagulation while eliminating the need for calcium replacement. This was successfully achieved by including citrate and calcium in all CRRT solutions. Thereby the total calcium concentration was kept constant throughout the extracorporeal circuit, whereas the ionized calcium was kept at low levels enough to avoid clotting. Being a completely new concept, only five patients with acute renal failure were included in a short, prospective, intensely supervised nonrandomized pilot study. Systemic electrolyte levels and acid-base parameters were stable and remained within physiologic levels. Ionized calcium levels declined slightly initially but stabilized at 1.1 mmol/L. Plasma citrate concentrations stabilized at approximately 0.6 mmol/L. All postfilter ionized calcium levels were CRRT.

  17. Formulation, Characterization and Physicochemical Evaluation of Potassium Citrate Effervescent Tablets

    National Research Council Canada - National Science Library

    Fatemeh Fattahi; Abolfazl Aslani

    2013-01-01

    Purpose: The aim of this study was to design and formulation of potassium citrate effervescent tablet for reduction of calcium oxalate and urate kidney stones in patients suffering from kidney stones. Methods...

  18. Post Ejaculatory Effects of Sildenafil Citrate (Viagra) On Sexual ...

    African Journals Online (AJOL)

    Viagra) administration on sexual dysfunctions associated with diabetic neuropathy/ erectile impotence prevailing in the male population. Aim: To investigate whether sildenafil citrate administration maintains improved erectile functions in diabetic ...

  19. Glucose and the ATP paradox in yeast.

    NARCIS (Netherlands)

    Somsen, O.J.G.; Hoeben, M.A.; Esgalhado, M.E.L.M.; Snoep, J.L.; Visser, D.; van der Heijden, R.T.J.M.; Heijnen, J.J.; Westerhoff, H.V.

    2000-01-01

    A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be

  20. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    aliquots of standards increase quality control and ease daily operation. The medium (Lumin(PM) buffer, tap water or MilliQ water) for preparation of ATP-standard dilution significantly affected the rlu response of the ATP-standard dilutions (20% difference). The effect of dilution media and of sample...

  1. Genomic diversity of citrate fermentation in Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    Liu Yen-Ming

    2009-08-01

    Full Text Available Abstract Background It has long been recognized that Klebsiella pneumoniae can grow anaerobically on citrate. Genes responsible for citrate fermentation of K. pneumoniae were known to be located in a 13-kb gene cluster on the chromosome. By whole genome comparison of the available K. pneumoniae sequences (MGH 78578, 342, and NTUH-K2044, however, we discovered that the fermentation gene cluster was present in MGH 78578 and 342, but absent in NTUH-K2044. In the present study, the previously unknown genome diversity of citrate fermentation among K. pneumoniae clinical isolates was investigated. Results Using a genomic microarray containing probe sequences from multiple K. pneumoniae strains, we investigated genetic diversity among K. pneumoniae clinical isolates and found that a genomic region containing the citrate fermentation genes was not universally present in all strains. We confirmed by PCR analysis that the gene cluster was detectable in about half of the strains tested. To demonstrate the metabolic function of the genomic region, anaerobic growth of K. pneumoniae in artificial urine medium (AUM was examined for ten strains with different clinical histories and genomic backgrounds, and the citrate fermentation potential was found correlated with the genomic region. PCR detection of the genomic region yielded high positive rates among a variety of clinical isolates collected from urine, blood, wound infection, and pneumonia. Conserved genetic organizations in the vicinity of the citrate fermentation gene clusters among K. pneumoniae, Salmonella enterica, and Escherichia coli suggest that the13-kb genomic region were not independently acquired. Conclusion Not all, but nearly half of the K. pneumoniae clinical isolates carry the genes responsible for anaerobic growth on citrate. Genomic variation of citrate fermentation genes in K. pneumoniae may contribute to metabolic diversity and adaptation to variable nutrient conditions in different

  2. Formulation, Characterization and Physicochemical Evaluation of Potassium Citrate Effervescent Tablets

    OpenAIRE

    Fatemeh Fattahi; Abolfazl Aslani

    2013-01-01

    Purpose: The aim of this study was to design and formulation of potassium citrate effervescent tablet for reduction of calcium oxalate and urate kidney stones in patients suffering from kidney stones. Methods: In this study, 13 formulations were prepared from potassium citrate and effervescent base in different concentration. The flowability of powders and granules was studied. Then effervescent tablets were prepared by direct compression, fusion and wet granulation methods. The prepared tabl...

  3. Coconut milk - citrate as extender for West African dwarf buck ...

    African Journals Online (AJOL)

    motility of 52.6% was highly significant (p = 0.018) at 2 hours post-extension in preserving motility of extended buck semen un-refrigerated compared to both D3 (40% coconut milk and 60% citrate buffer) and D4 (50% coconut milk and 50% citrate buffer). D2 also maintained mean sperm cell motility of 45% and was highly ...

  4. Coupled Enzyme Reactions Performed in Heterogeneous Reaction Media: Experiments and Modeling for Glucose Oxidase and Horseradish Peroxidase in a PEG/Citrate Aqueous Two-Phase System

    Science.gov (United States)

    2015-01-01

    The intracellular environment in which biological reactions occur is crowded with macromolecules and subdivided into microenvironments that differ in both physical properties and chemical composition. The work described here combines experimental and computational model systems to help understand the consequences of this heterogeneous reaction media on the outcome of coupled enzyme reactions. Our experimental model system for solution heterogeneity is a biphasic polyethylene glycol (PEG)/sodium citrate aqueous mixture that provides coexisting PEG-rich and citrate-rich phases. Reaction kinetics for the coupled enzyme reaction between glucose oxidase (GOX) and horseradish peroxidase (HRP) were measured in the PEG/citrate aqueous two-phase system (ATPS). Enzyme kinetics differed between the two phases, particularly for the HRP. Both enzymes, as well as the substrates glucose and H2O2, partitioned to the citrate-rich phase; however, the Amplex Red substrate necessary to complete the sequential reaction partitioned strongly to the PEG-rich phase. Reactions in ATPS were quantitatively described by a mathematical model that incorporated measured partitioning and kinetic parameters. The model was then extended to new reaction conditions, i.e., higher enzyme concentration. Both experimental and computational results suggest mass transfer across the interface is vital to maintain the observed rate of product formation, which may be a means of metabolic regulation in vivo. Although outcomes for a specific system will depend on the particulars of the enzyme reactions and the microenvironments, this work demonstrates how coupled enzymatic reactions in complex, heterogeneous media can be understood in terms of a mathematical model. PMID:24517887

  5. Characterization of an Eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia yezoensis.

    Science.gov (United States)

    Inoue, Akira; Mashino, Chieco; Uji, Toshiki; Saga, Naotsune; Mikami, Koji; Ojima, Takao

    2015-08-01

    Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL-7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis. The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated. PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35(o)C and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage. This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.

  6. Effect of clomifene citrate plus metformin and clomifene citrate plus placebo on induction of ovulation in women with newly diagnosed polycystic ovary syndrome: randomised double blind clinical trial

    NARCIS (Netherlands)

    Moll, Etelka; Bossuyt, Patrick M. M.; Korevaar, Johanna C.; Lambalk, Cornelis B.; van der Veen, Fulco

    2006-01-01

    OBJECTIVE: To compare the effectiveness of clomifene citrate plus metformin and clomifene citrate plus placebo in women with newly diagnosed polycystic ovary syndrome. DESIGN: Randomised clinical trial. SETTING: Multicentre trial in 20 Dutch hospitals. PARTICIPANTS: 228 women with polycystic ovary

  7. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    DEFF Research Database (Denmark)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured...... the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency...... for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A...

  8. Cloning and expression in Escherichia coli of a gene coding for a chondroitin lyase from Bacteroides thetaiotaomicron

    Energy Technology Data Exchange (ETDEWEB)

    Guthrie, E.P.; Shoemaker, N.B.; Salyers, A.A.

    1985-11-01

    The authors cloned the gene for one of the two chondroitin lyases of Bacteroides thetaiotaomicron into the cosmid vector pHC79 and subcloned it into pBR328. No proteins the size of B. thetaiotamicron chondroitin lyase I or II (104 to 108 kilodaltons) were detectable in maxicell or in vitro transcription-translation preparations. However, partial purification of the chondroitin lyase activity from the Escherichia coli subclone showed that its properties were similar to those of the B. thetaiotaomicron chondroitin lyases. Antibodies to the chondroitin lyase that was produced in E. coli cross-reacted with the B. thetaiotaomicron chondroitin lyase II but not with chondroitin lyase I. The molecular weight of the enzyme produced in E. coli was slightly lower than those of the two chondroitin lyases from B. thetaiotaomicron. The chondroitin lyase gene was located on the subcloned 7.8-kilobase EcoRI fragment. The size of the gene was approximately 3.3 kilobases, as expected for a protein with a molecular weight of 104,000.

  9. Aroma compounds generation in citrate metabolism of Enterococcus faecium: Genetic characterization of type I citrate gene cluster.

    Science.gov (United States)

    Martino, Gabriela P; Quintana, Ingrid M; Espariz, Martín; Blancato, Victor S; Magni, Christian

    2016-02-02

    Enterococcus is one of the most controversial genera belonging to Lactic Acid Bacteria. Research involving this microorganism reflects its dual behavior as regards its safety. Although it has also been associated to nosocomial infections, natural occurrence of Enterococcus faecium in food contributes to the final quality of cheese. This bacterium is capable of fermenting citrate, which is metabolized to pyruvate and finally derives in the production of the aroma compounds diacetyl, acetoin and 2,3 butanediol. Citrate metabolism was studied in E. faecium but no data about genes related to these pathways have been described. A bioinformatic approach allowed us to differentiate cit(-) (no citrate metabolism genes) from cit(+) strains in E. faecium. Furthermore, we could classify them according to genes encoding for the transcriptional regulator, the oxaloacetate decarboxylase and the citrate transporter. Thus we defined type I organization having CitI regulator (DeoR family), CitM cytoplasmic soluble oxaloacetate decarboxylase (Malic Enzyme family) and CitP citrate transporter (2-hydroxy-carboxylate transporter family) and type II organization with CitO regulator (GntR family), OAD membrane oxaloacetate decarboxylase complex (Na(+)-transport decarboxylase enzyme family) and CitH citrate transporter (CitMHS family). We isolated and identified 17 E. faecium strains from regional cheeses. PCR analyses allowed us to classify them as cit(-) or cit(+). Within the latter classification we could differentiate type I but no type II organization. Remarkably, we came upon E. faecium GM75 strain which carries the insertion sequence IS256, involved in adaptative and evolution processes of bacteria related to Staphylococcus and Enterococcus genera. In this work we describe the differential behavior in citrate transport, metabolism and aroma generation of three strains and we present results that link citrate metabolism and genetic organizations in E. faecium for the first time

  10. Citrate-release-mediated aluminum resistance is coupled to the inducible expression of mitochondrial citrate synthase gene in Paraserianthes falcataria.

    Science.gov (United States)

    Osawa, Hiroki; Kojima, Katsumi

    2006-05-01

    Aluminum (Al) resistance in some leguminous plants is achieved by enhanced citrate release from roots. Enhancement requires several hours for complete activation and is postulated to involve Al-responsive genes or components. We examined the mechanism of Al-induced citrate release by studying the relationship between citrate release and expression of the mitochondrial citrate synthase (mCS) gene in three leguminous trees. Root elongation in Leucaena leucocephala (Lam.) de Wit was arrested within 24 h by 30 microM Al, whereas root elongation in Paraserianthes falcataria (L.) Neilson and Acacia mangium Willd. was inhibited mangium maintained enhanced release and accumulation of citrate for at least 28 days in response to Al treatment. Aluminum increased the accumulation of mCS transcripts in P. falcataria roots, but not in L. leucocephala roots, and thus up-regulation decreased following removal of Al. Lanthanum did not alter the expression level of mCS. Aluminum increased mCS activity concomitantly with enhanced mCS gene expression in P. falcataria, whereas it did not affect mCS activity in L. leucocephala. Aluminum content in root apices of P. falcataria was increased by cycloheximide, supporting the idea that de novo synthesis of proteins is a prerequisite for Al resistance. Our findings suggest that Al-inducible expression of mCS coupled with enhanced citrate release mediates Al resistance in P. falcataria.

  11. Regulation of phenylalanine ammonia lyase in Rhodotorula glutinis.

    Science.gov (United States)

    Kane, J F; Fiske, M J

    1985-01-01

    In the red yeast Rhodotorula glutinis, phenylalanine ammonia lyase (PAL) was induced 10-fold during carbon starvation even in the absence of exogenous phenylalanine, although maximal induction occurred when phenylalanine was the nitrogen (40-fold) or carbon (100-fold) source. Apparent regulatory mutations that affected the expression of PAL were isolated by selecting mutants resistant to the analog p-fluoro-D,L-phenylalanine (PFP). One such mutant, designated FP1, could use phenylalanine as a nitrogen source but not as a carbon source. Similarly, FP1 failed to utilize intermediates of the phenylalanine degradative pathway, namely, benzoate, p-hydroxybenzoate, or 3,4-dihydroxybenzoate, as carbon sources. Although the PFP-resistant mutant contained a low level of PAL, no increase was found when it was grown with phenylalanine as the nitrogen source. A derivative of FP1, FP1a, was isolated that simultaneously regained an inducible PAL and the ability to use phenylalanine, benzoate, p-hydroxybenzoate, and 3,4-dihydroxybenzoate as carbon sources. In addition, when p-hydroxybenzoate was the carbon source, PAL was induced in the mutant FP1a but not in the PFP-sensitive parental strain. We propose that the mutation to PFP resistance occurred in a regulatory gene that controls the entire phenylalanine degradative pathway. Secondary mutations at this locus, as found in strain FP1a, not only restored expression of this pathway, but also altered the induction of PAL by metabolites of this pathway. PMID:4038704

  12. Sexual Dimorphism in the Selenocysteine Lyase Knockout Mouse

    Directory of Open Access Journals (Sweden)

    Ashley N. Ogawa-Wong

    2018-01-01

    Full Text Available Selenium (Se is an essential micronutrient known for its antioxidant properties and health benefits, attributed to its presence in selenoproteins as the amino acid, selenocysteine. Selenocysteine lyase (Scly catalyzes hydrolysis of selenocysteine to selenide and alanine, facilitating re-utilization of Se for de novo selenoprotein synthesis. Previously, it was reported that male Scly−/− mice develop increased body weight and body fat composition, and altered lipid and carbohydrate metabolism, compared to wild type mice. Strikingly, females appeared to present with a less severe phenotype, suggesting the relationship between Scly and energy metabolism may be regulated in a sex-specific manner. Here, we report that while body weight and body fat gain occur in both male and female Scly−/− mice, strikingly, males are susceptible to developing glucose intolerance, whereas female Scly−/− mice are protected. Because Se is critical for male reproduction, we hypothesized that castration would attenuate the metabolic dysfunction observed in male Scly−/− mice by eliminating sequestration of Se in testes. We report that fasting serum insulin levels were significantly reduced in castrated males compared to controls, but islet area was unchanged between groups. Finally, both male and female Scly−/− mice exhibit reduced hypothalamic expression of selenoproteins S, M, and glutathione peroxidase 1.

  13. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

    Directory of Open Access Journals (Sweden)

    Karthikeyan Thiyagarajan

    Full Text Available Phenylalanine Ammonia Lyase (PAL gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum. The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.

  14. Phenylalanine ammonia-lyase through evolution: A bioinformatic approach

    Directory of Open Access Journals (Sweden)

    Shiva Hemmati

    2015-03-01

    Full Text Available Phenylalanine ammonia-lyase (PAL is the first entry enzyme of the phenylpropanoid pathway that converts phenylalanine to cinnamic acid which is the precursor of various secondary metabolites. PAL is recently formulated for phenylketonuric patients in pegylated forms; therefore, screening a PAL with the highest affinity to the substrate is of a great importance. PAL exists in all higher plants and some fungi and few bacteria. Ancestors of land plants have been adopted by evolving metabolic pathways. A multi-gene family encodes PAL by gene duplication events in most plants. In this study, the taxonomic distribution and phylogeny of pal gene found in land plants, fungi and bacteria have been analyzed. It seems that the ancestor of plants acquired a pal gene via horizontal gene transfer in symbioses with bacteria and fungi. Gymnosperms have kept a diverse set of pal genes that arose from gene duplication events. In angiosperms, after the divergence of dicotyledons from monocots, pal genes were duplicated many times. The close paralogues of pal genes in some species indicate expansion of gene families after the divergence in plant pal gene evolution. Interestingly, some of the plant pals clustered by species in a way that pals within one species are more closely related to each other than to homologs in the other species which indicates this duplication event occurred more recently.

  15. Stabilization of phenylalanine ammonia lyase against organic solvent mediated deactivation.

    Science.gov (United States)

    Shah, R M; D'mello, Anil P

    2007-02-22

    A potential novel therapy for phenylketonuria involves oral administration of microencapsulated phenylalanine ammonia lyase (PAL), an enzyme that converts phenylalanine to transcinnamic acid. A drawback of this potential therapy is that encapsulated PAL retains only 23% of its activity. Microcapsules are prepared by emulsifying PAL in 10% hemoglobin solution with water-saturated ether (WSE) and subsequent addition of cellulose nitrate dissolved in ether:ethanol (E:E) mixture. The objective of this paper was to determine the contribution of emulsification to the overall loss in activity of encapsulated PAL, and to devise strategies to protect PAL against such loss in activity. Emulsification was simulated by stirring the aqueous phase containing PAL with the organic phase. The mixture was then centrifuged, and the protein content and catalytic activity of PAL in the aqueous phase was measured. Emulsification of PAL solution with WSE caused no loss in activity but resulted in a loss in protein content of aqueous phase. Size exclusion chromatography and gel electrophoresis studies showed that the loss was primarily due to the specific loss of impurities in the PAL sample. Emulsification of PAL solution with E:E resulted in a 50% decrease in its activity. Among the additives, hydroxypropyl-gamma-cyclodextrin and hydroxy propyl-beta-cyclodextrin protected PAL against emulsion mediated loss in activity.

  16. Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington's disease.

    Science.gov (United States)

    Paul, Bindu D; Sbodio, Juan I; Xu, Risheng; Vandiver, M Scott; Cha, Jiyoung Y; Snowman, Adele M; Snyder, Solomon H

    2014-05-01

    Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.

  17. Evolutionary Analysis of Pectin Lyases of the Genus Colletotrichum.

    Science.gov (United States)

    Lara-Márquez, Alicia; Oyama, Ken; Zavala-Páramo, María G; Villa-Rivera, Maria G; Conejo-Saucedo, Ulises; Cano-Camacho, Horacio

    2017-10-01

    Pectin lyases (PNLs) are important enzymes that are involved in plant cell wall degradation during the infection process. Colletotrichum is a diverse genus of fungi, which allows the study of the evolution of PNLs and their possible role in pathogen-host interactions and lifestyle adaptations. The phylogenetic reconstruction of PNLs from Colletotrichum and analysis of selection pressures showed the formation of protein lineages by groups of species with different selection pressures and specific patterns. The analysis of positive selection at individual sites using different methods allowed for the identification of three codons with evidence of positive selection in the oligosaccharide-binding region and two codons on the antiparallel sheet, which may influence the interaction with the substrate. Seven codons on the surface of the protein, mainly in the peripheral helices of the PNLs, could have an important function in evasion of plant defenses, as has been proposed in other enzymes. According to our results, it is possible that events of genetic duplication occurred in ancestral lines, followed by episodes of genetic diversification and gene loss, probably influenced by differences in the composition of the host cell wall. Additionally, different patterns of evolution in Colletotrichum appear to be molded by a strong purifying selection and positive selection episodes that forged the observed evolutionary patterns, possibly influenced by host interaction or substrate specificity. This work represents a starting point for the study of sites that may be important for evasion of plant defenses and biotechnological purposes.

  18. Molecular devices for the regulation of chloroplast ATP synthase

    NARCIS (Netherlands)

    Hisabori, T.; Konno, H.; Ichimura, H.; Strotmann, H.; Bald, D.

    2002-01-01

    In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential

  19. Ocular safety of Viagra, (sildenafil citrate).

    Science.gov (United States)

    Laties, A M; Fraunfelder, F T

    1999-01-01

    To date, sildenafil citrate (Viagra) gives every evidence of being a safe drug for the eye despite a series of expressed concerns. A review of how its ocular safety profile has been identified offers insights into the strengths and weaknesses of present systems and resources for judging the ocular safety of Viagra or, for that matter, of any new drug. Such insights include: The great value of careful, informed assessment of preclinical information gleaned from laboratory experiments. By and large, such assessments point the way toward appropriate clinical evaluation. For Viagra, early in its development it was noted that besides exerting a major inhibitory effect on the intended target, the vascular-associated enzyme phosphodiesterase 5 (PDE5), the drug also exerts a lesser but definite inhibitory effect on the closely related PDE6, located in the retina. For this reason, preclinical evaluation of the drug included electroretinography plus postmortem histology. In addition, an extended eye examination was incorporated into clinical protocols. The often chaotic but invaluable information stream that becomes available once marketing approval has been gained and large populations begin to use a drug. False alarms, misattribution, and erroneous information are the order of the day. Nevertheless, as information accumulates, patterns of response clarify and the true nature of special susceptibility for subpopulations, if any, becomes apparent. A role for the astute clinician remains: Subtle changes or unusual risks for subpopulations can be missed entirely for long periods of time. A manifest need for improvement in evaluation of postmarketing side-effects. This need has led to the establishment of a new discipline: pharmacoepidemiology. In ophthalmology, the National Registry of Drug Induced Ocular Side-Effects maintains a constant and invaluable surveillance. Examples are supplied to illustrate each of these major points: Our presentation will include data gleaned from

  20. Extracellular ATP Induces Calcium Signaling in Odontoblasts.

    Science.gov (United States)

    Lee, B M; Jo, H; Park, G; Kim, Y H; Park, C K; Jung, S J; Chung, G; Oh, S B

    2017-02-01

    Odontoblasts form dentin at the outermost surface of tooth pulp. An increasing level of evidence in recent years, along with their locational advantage, implicates odontoblasts as a secondary role as sensory or immune cells. Extracellular adenosine triphosphate (ATP) is a well-characterized signaling molecule in the neuronal and immune systems, and its potential involvement in interodontoblast communications was recently demonstrated. In an effort to elaborate the ATP-mediated signaling pathway in odontoblasts, the current study performed single-cell reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent detection to investigate the expression of ATP receptors related to calcium signal in odontoblasts from incisal teeth of 8- to 10-wk-old rats, and demonstrated an in vitro response to ATP application via calcium imaging experiments. While whole tissue RT-PCR analysis detected P2Y2, P2Y4, and all 7 subtypes (P2X1 to P2X7) in tooth pulp, single-cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2Y2, P2Y4, P2X2, P2X4, P2X6, and P2X7 expression in only a subset (23% to 47%) of cells tested, with no evidence for P2X1, P2X3, and P2X5 expression. An increase of intracellular Ca(2+) concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin or under the Ca(2+)-free condition, suggested function of both ionotropic and metabotropic ATP receptors in odontoblasts. The enhancement of ATP-induced calcium response by ivermectin and inhibition by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) confirmed a functional P2X4 subtype in odontoblasts. Positive calcium response to 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP suggested P2X2, P2X4, and P2X7 as functional subunits in rat odontoblasts. Single-cell RT-PCR analysis of the cells with confirmed calcium response and immunofluorescent detection further corroborated the expression of P2X

  1. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    Directory of Open Access Journals (Sweden)

    Lenartowicz Malgorzata

    2016-08-01

    Full Text Available Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD or the rare autosomal disorder Wilson disease (WD, respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation.

  2. Redox regulation of ATP sulfurylase in microalgae

    Czech Academy of Sciences Publication Activity Database

    Prioretti, L.; Lebrun, R.; Gontero, B.; Giordano, Mario

    2016-01-01

    Roč. 478, č. 4 (2016), s. 1555-1562 ISSN 0006-291X Institutional support: RVO:61388971 Keywords : ATP sulfurylase * cysteine * Sulfur metabolism Subject RIV: EE - Microbiology, Virology Impact factor: 2.466, year: 2016

  3. Application of Triethyl Citrate to Filters in Virginia Type Cigarettes*

    Directory of Open Access Journals (Sweden)

    Jin Yong

    2015-01-01

    Full Text Available Triethyl citrate (TEC was applied in the preparation of cigarette filter rods by two approaches. One was to utilize TEC as a plasticizer sprayed onto the surface of cellulose acetate (CA fibers. The other was to apply TEC as an additive coated onto the cellulosic paper of acetate-paper dual filters. Three types of cigarette filter rods, namely, triethyl citrate-cellulose acetate (TEC-CA filter rods, triethyl citrate-cellulose acetate-paper (TEC-CA-paper dual filter rods and cellulose acetate-paper-triethyl citrate (CA-paper-TEC dual filter rods, were manufactured. In order to promote the curing of CA rods, high-frequency radiation was introduced into the procedure of filter manufacture. Then Virginia type cigarettes, combined with the three kinds of prepared filter rods were manufactured and the removal efficiency of phenols from the cigarette mainstream smoke was investigated. The results revealed that no matter where the triethyl citrate was applied as plasticizer or coating additive, the content of phenol, o-, m-and p-cresol in cigarette mainstream smoke could be greatly reduced. The optimal removal efficiency for phenol was 50% compared with the control. [Beitr. Tabakforsch. Int. 26 (2014 176-182

  4. Inpatient citrate-based hemodialysis in pediatric patients.

    Science.gov (United States)

    Fajardo, Cecile; Sanchez, Cheryl P; Cutler, Drew; Sahney, Shobha; Sheth, Rita

    2016-10-01

    Citrate-based dialysate is an effective method of hemodialysis (HD) anticoagulation in adults. The objective of this study was to evaluate this therapy as an alternative to heparin anticoagulation in pediatric patients in the inpatient setting requiring HD. We performed a prospective, non-randomized study of citrate-based dialysate HD treatments (N = 119) over a 9-month period in 18 pediatric patients (age range 0-18 years) admitted to hospital. Primary outcome measures were thrombosis incidence rates that resulted in circuit loss, catheter loss or early dialysis termination. Secondary outcome measures were hypocalcemia incidence and heparin use. Data analysis was performed using descriptive and comparative statistics. There was a thrombosis incidence rate of 2.5 % circuit loss, 2.5 % catheter loss and 5.9 % early dialysis termination due to the thrombosis risk. In 64 % of treatments a circuit clot developed but with no circuit loss, and mild asymptomatic hypocalcemia deveoped in 58 % of the monitored HD sessions . No patient required additional heparin during the citrate-based HD treatments, but 11.1 % were subsequently converted to heparin anticoagulation. Our study showed a low percentage of thrombotic episodes resulting in catheter or circuit loss. Hypocalcemia was common but remained mild and asymptomatic. Citrate-based dialysate was well tolerated by our patients. We therefore conclude that citrate-based dialysate is a safe alternative to heparin-based hemodialysis anticoagulation.

  5. Sildenafil citrate (Viagra) enhances vasodilatation in fetal growth restriction.

    Science.gov (United States)

    Wareing, Mark; Myers, Jenny E; O'Hara, Maureen; Baker, Philip N

    2005-05-01

    Fetal growth restriction (FGR) affects up to 8% of all pregnancies and has massive short-term (increased fetal morbidity and mortality) and long-term (increased incidence of cardiovascular disease in adulthood) health implications. Doppler waveform analysis of pregnancies complicated by FGR suggests compromised uteroplacental circulation and placental hypoperfusion. Our aim was to determine whether myometrial small artery function was aberrant in FGR and to assess whether sildenafil citrate could improve vasodilatation in FGR pregnancies. Small arteries dissected from myometrial biopsies obtained at cesarean section from normal pregnant women (n = 27) or women whose pregnancies were complicated by FGR (n = 12) were mounted on wire myographs. Vessels were constricted (with arginine vasopressin or U46619) and relaxed (with bradykinin) before and after incubation with a phosphodiesterase-5 inhibitor, sildenafil citrate. We demonstrated increased myometrial small artery vasoconstriction and decreased endothelium-dependent vasodilatation in vessels from women whose pregnancies were complicated by FGR. Sildenafil citrate significantly reduced vasoconstriction and significantly improved relaxation of FGR small arteries. We conclude that sildenafil citrate improves endothelial function of myometrial vessels from women whose pregnancies are complicated by intrauterine growth restriction. Sildenafil citrate may offer a potential therapeutic strategy to improve uteroplacental blood flow in FGR pregnancies.

  6. The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

    Directory of Open Access Journals (Sweden)

    Alexander Krah

    Full Text Available The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

  7. Solvent Isotope-induced Equilibrium Perturbation for Isocitrate Lyase

    Science.gov (United States)

    Quartararo, Christine E.; Hadi, Timin; Cahill, Sean M.; Blanchard, John S.

    2014-01-01

    Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacteria’s life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage of D2OV = 2.0 ± 0.1 and D2O[V/Kisocitrate] = 2.2 ± 0.3 arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate and succinate prepared in D2O, would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of NMR spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by 1H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed di-deuterated succinate, protiated glyoxylate, and mono-deuterated isocitrate, with the transient appearance and disappearance of mono-deuterated succinate. A model for the equilibrium perturbation of substrate species, and their time-dependent isotopic composition is presented. PMID:24261638

  8. Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

    DEFF Research Database (Denmark)

    Ernst, Heidi; Lo Leggio, Leila; Yu, Shukun

    2005-01-01

    Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very r...

  9. DMSP-LYASE ACTIVITY IN A SPRING PHYTOPLANKTON BLOOM OFF THE DUTCH COAST, RELATED TO PHAEOCYSTIS SP ABUNDANCE

    NARCIS (Netherlands)

    STEFELS, J; DIJKHUIZEN, L; GIESKES, WWC

    An enzyme assay for measuring dimethylsulfoniopropionate (DMSP)-lyase activity was applied to natural sea water samples taken during the 1993 spring bloom off the Dutch coast. When relating the measured DMSP-lyase activity to the most abundant algal species found during the cruise, a significant

  10. Preparation of unnatural amino acids with ammonia-lyases and 2,3-aminomutases.

    Science.gov (United States)

    Poppe, László; Paizs, Csaba; Kovács, Klaudia; Irimie, Florin-Dan; Vértessy, Beáta

    2012-01-01

    Ammonia-lyases catalyze a wide range of processes leading to α,β-unsaturated compounds by elimination of ammonia. In this chapter, ammonia-lyases are reviewed with major emphasis on their synthetic applications in stereoselective preparation of unnatural amino acids. Besides the synthesis of various unnatural α-amino acids with the aid of phenylalanine ammonia-lyases (PALs) utilizing the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic groups, the biotransformations leading to various unnatural β-amino acids with phenylalanine 2,3-aminomutases using the same catalytic MIO prosthetic group are discussed. Cloning, production, purification, and biotransformation protocols for PAL are described in detail.

  11. Enhanced citrate production through gene insertion in Aspergillus niger

    DEFF Research Database (Denmark)

    Jongh, Wian de; Nielsen, Jens

    2007-01-01

    The effect of inserting genes involved in the reductive branch of the tricarboxylic acid (TCA) cycle on citrate production by Aspergillus niger was evaluated. Several different genes were inserted individually and in combination, i.e. malate dehydrogenase (mdh2) from Saccharomyces cerevisiae, two...... truncated, cytosolic targeted, fumarases (Fum1s and FumRs) from S. cerevisiae and Rhizopus oryzae, respectively, and the cytosolic soluble fumarate reductase (Frds1) from S. cerevisiae. Overexpression of these genes in their native strain backgrounds has been reported to lead to alterations...... in the intracellular cytosolic dicarboxylate concentrations. It was found that all the transformant strains had enhanced yield and productivities of citrate compared with the wild-type strain. The transformants also had the ability to produce citrate in trace-manganese-contaminated medium, where the wild type...

  12. Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

    Science.gov (United States)

    Badur, Ahmet H.; Jagtap, Sujit Sadashiv; Yalamanchili, Geethika; Lee, Jung-Kul; Zhao, Huimin

    2015-01-01

    Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (kcat) for alginate of 0.60 ± 0.02 s−1, 3.7 ± 0.3 s−1, 4.5 ± 0.5 s−1, and 7.1 ± 0.2 s−1, respectively. The Km values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers. PMID:25556193

  13. Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells.

    Science.gov (United States)

    Qian, Yanrong; Wang, Xuan; Liu, Yi; Li, Yunsheng; Colvin, Robert A; Tong, Lingying; Wu, Shiyong; Chen, Xiaozhuo

    2014-09-01

    ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 10(4) times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells' resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. Immunocytochemical Localization of Prunasin Hydrolase and Mandelonitrile Lyase in Stems and Leaves of Prunus serotina.

    Science.gov (United States)

    Swain, E.; Poulton, J. E.

    1994-12-01

    In macerates of black cherry (Prunus serotina Ehrh.) leaves and stems, (R)-prunasin is catabolized to HCN, benzaldehyde, and D-glucose by the sequential action of prunasin hydrolase (EC 3.2.1.21) and (R)-(+)-mandelonitrile lyase (EC 4.1.2.10). Immuno-cytochemical techniques have shown that within these organs prunasin hydrolase occurs within the vacuoles of phloem parenchyma cells. In arborescent leaves, mandelonitrile lyase was also located in phloem parenchyma vacuoles, but comparison of serial sections revealed that these two degradative enzymes are usually localized within different cells.

  15. Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention generally relates to the field of biotechnology as it applies to the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity. More particularly, the present invention pertains to polypeptides having tyrosine ammonia lyase activity and high...... substrate specificity towards tyrosine, which makes them particularly suitable in the production of p-coumaric acid and other hydroxycinnamic acids. The present invention thus provides processes for the production of p-coumaric acid and other hydroxycinnamic acids employing these polypeptides as well...

  16. Crystallization and preliminary crystallographic analysis of recombinant hyaluronate lyase from Streptococcus suis.

    Science.gov (United States)

    Khan, Abdul Hamid; Mohamed Omar, Youssef Mohamed; Kakar, Mohammad Azam; Bangulzai, Nasrullah

    2013-06-01

    Hyaluronate lyase is an important surface enzyme of many streptococcal species. The enzyme degrades several biologically important connective tissue components, which facilitates the spreading of the bacteria throughout the host tissues and presumably provides energy and a carbon source for bacterial cells. Recombinant hyaluronate lyase was expressed in Escherichia coli and was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P222(1), with unit-cell parameters a = 58.08, b = 101.32, c = 103.47 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.50 Å resolution.

  17. A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity.

    Science.gov (United States)

    Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

    2015-09-01

    Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2-3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also

  18. CRRT Regional Anticoagulation Using Citrate in the Liver Failure and Liver Transplant Population.

    Science.gov (United States)

    Wonnacott, Rob; Josephs, Brandi; Jamieson, Jill

    2016-01-01

    Regional citrate for continuous renal replacement therapy (CRRT) use in patients with liver failure or post-liver transplant has been considered a contraindication because of the risk of citrate toxicity development. Regional citrate has the benefit of decreased bleeding risks over systemic anticoagulation; therefore, it is of great benefit to the coagulopathic and surgical populations. This article analyzes current empiric data and compares with a case study specifically related to liver failure, liver transplant, and CRRT use. We found that the use of a total serum to ionized calcium ratio was much more reliable in measuring liver function than liver enzyme figures. This when paired with a citrate-reduction guideline based on serum to ionized calcium ratios provided effective, early management of citrate toxicity. Using new measurements to calculate liver metabolism of citrate and using a new citrate-reducing guideline allow the bedside practitioner to use regional citrate anticoagulation in patients with liver failure and liver transplant who require CRRT.

  19. Hemodiafiltration using pre-dilutional on-line citrate dialysate: A new technique for regional citrate anticoagulation: A feasibility study

    Directory of Open Access Journals (Sweden)

    Radhouane Bousselmi

    2015-01-01

    Full Text Available A prospective, observational, feasibility study was carried out on four patients with end-stage renal failure undergoing bicarbonate hemodialysis to study the feasibility of an on-line hemodiafiltration technique using a citrate dialysate with pre-dilutional infusion of citrate as a technique for regional citrate anticoagulation. All patients had contraindication to systemic heparin anticoagulation. The dialysis technique consisted of an on-line hemodiafiltration with a citrate dialysate without calcium using a Fresenius 4008S dialysis machine and Fresenius Polysulfone F60 dialyzers. The infusion solution was procured directly from the dialysate and was infused into the arterial line. To avoid the risk of hypocalcemia, calcium gluconate was infused to the venous return line. The study was carried out in two stages. During the first stage, the citrate infusion rate was 80 mL/min and the calcium infusion rate was 9 mmol/h. At the second stage, the rates were 100 mL/min and 11 mmol/h, respectively. The primary endpoint of this study was the incidence of thrombosis in the extracorporeal blood circuit and/or the dialyzer. A total of 78 sessions were conducted. All the sessions were well tolerated clinically and there were no major incidents in any of the four patients. At the first stage of the study, there were five incidences of small clots in the venous blood chamber, an incidence of extracorporeal blood circuit thrombosis of 12.5%. At the second stage of the study, no cases of extracorporeal blood circuit or dialyzer thrombosis were noted. Hemodiafiltration with on-line citrate dialysate infusion to the arterial line is safe and allows an effective regional anticoagulation of the extracorporeal blood circuit without the need for systemic anticoagulation.

  20. ATP8B1 and ATP11C: Two Lipid Flippases Important for Hepatocyte Function

    NARCIS (Netherlands)

    Naik, Jyoti; de Waart, Dirk R.; Utsunomiya, Karina; Duijst, Suzanne; Mok, Kam Ho; Oude Elferink, Ronald P. J.; Bosma, Piter J.; Paulusma, Coen C.

    2015-01-01

    P4 ATPases are lipid flippases and transport phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. Lipid flipping is important for the biogenesis of transport vesicles. Recently it was shown that loss of the P4 ATPases ATP8B1 and ATP11C are associated with severe

  1. Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion

    DEFF Research Database (Denmark)

    Mortensen, Stefan P.; Gonzalez-Alonso, Jose; Nielsen, Jens Jung

    2009-01-01

    ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanism remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on limb muscle interstitial...... local concentration. Key words: sympathetic nerve activity, vasodilation, endothelium, skeletal muscle....

  2. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

    2006-01-01

    Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this st......Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim...... of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either...... [gamma-(32)P]ATP or (14)C/(3)H-labeled and unlabeled nucleotides as appropriate substrates. Data show that the juice contains the enzyme ecto-nucleoside triphosphate diphosphohydrolase that can hydrolyze both [(14)C]ATP and [(3)H]ADP about equally well, i.e. CD39. Reverse-phase high-performance liquid...

  3. ATP: The crucial component of secretory vesicles.

    Science.gov (United States)

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  4. Citrate anticoagulation for CRRT: don?t always trust the postfilter iCa results!

    OpenAIRE

    Oudemans-van Straaten, Heleen M.; Ostermann, Marlies

    2015-01-01

    Citrate has been recommended as the first-line anticoagulant for continuous renal replacement therapy (CRRT) in critically ill patients. Compared with heparin, citrate anticoagulation is safer and more efficacious. Citrate inhibits the coagulation cascade by lowering the ionized calcium (iCa) concentration in the filter. Monitoring of systemic iCa concentrations is inherent to the protocol, and monitoring of postfilter iCa is recommended to adjust citrate flow and optimize anticoagulation. Wh...

  5. 75 FR 34360 - Listing of Color Additives Exempt From Certification; Bismuth Citrate; Confirmation of Effective...

    Science.gov (United States)

    2010-06-17

    ... Certification; Bismuth Citrate; Confirmation of Effective Date AGENCY: Food and Drug Administration, HHS... level of bismuth citrate as a color additive in cosmetics intended for coloring hair on the scalp. DATES... increasing the permitted use level of bismuth citrate as a color additive in cosmetics intended for coloring...

  6. 75 FR 14491 - Listing of Color Additives Exempt From Certification; Bismuth Citrate

    Science.gov (United States)

    2010-03-26

    ... Certification; Bismuth Citrate AGENCY: Food and Drug Administration, HHS. ACTION: Final rule. SUMMARY: The Food... level of bismuth citrate as a color additive in cosmetics intended for coloring hair on the scalp. This.... 73.2110 Bismuth citrate (21 CFR 73.2110) by increasing the maximum permitted use level of bismuth...

  7. Enzyme Basis for pH Regulation of Citrate and Pyruvate Metabolism by Leuconostoc oenos

    NARCIS (Netherlands)

    Ramos, Ana; Lolkema, Juke S.; Konings, Wilhelmus; Santos, Helena

    Citrate and pyruvate metabolism by nongrowing cells of Leuconostoc oenos was investigated. 13C nuclear magnetic resonance (NMR) spectroscopy was used to elucidate the pathway of citrate breakdown and to probe citrate or pyruvate utilization, noninvasively, in living cell suspensions. The utilization

  8. 77 FR 24461 - Citric Acid and Certain Citrate Salts From Canada: Final Results of Antidumping Duty...

    Science.gov (United States)

    2012-04-24

    ..., which are intermediate products in the production of citric acid, sodium citrate, and potassium citrate... International Trade Administration Citric Acid and Certain Citrate Salts From Canada: Final Results of... the preliminary results of the administrative review of the antidumping duty order on citric acid and...

  9. 78 FR 34338 - Citric Acid and Certain Citrate Salts From Canada: Preliminary Results of Antidumping Duty...

    Science.gov (United States)

    2013-06-07

    ... International Trade Administration Citric Acid and Certain Citrate Salts From Canada: Preliminary Results of... administrative review of the antidumping duty order on citric acid and certain citrate salts (citric acid) from... is citric acid and certain citrate salts. The product is currently classified in the Harmonized...

  10. 78 FR 64914 - Citric Acid and Certain Citrate Salts From Canada: Final Results of Antidumping Duty...

    Science.gov (United States)

    2013-10-30

    ... International Trade Administration Citric Acid and Certain Citrate Salts From Canada: Final Results of... of the antidumping duty order on citric acid and certain citrate salts from Canada.\\1\\ The review... period of review (POR) is May 1, 2011, through April 30, 2012. \\1\\ See Citric Acid and Certain Citrate...

  11. Voltage Dependence of ATP Secretion in Mammalian Taste Cells

    Science.gov (United States)

    Romanov, Roman A.; Rogachevskaja, Olga A.; Khokhlov, Alexander A.; Kolesnikov, Stanislav S.

    2008-01-01

    Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca2+ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels. PMID:19029378

  12. Ovarian capsular drilling in the treatment of clomiphene citrate ...

    African Journals Online (AJOL)

    Background: Laparoscopic Ovarian Drilling (LOD) is an effective and a well evaluated surgical Treatment of Clomiphene Citrate resistant Polycystic ovarian syndrome. Transvaginal hydrolaparoscopy (TVHL) is a relatively new simple method of exploring the pelvis and has recently been introduced as a transvaginal ...

  13. The Effect of Polymer Molecular Weight on Citrate Crosslinked ...

    African Journals Online (AJOL)

    Erah

    The Effect of Polymer Molecular Weight on Citrate. Crosslinked Chitosan Films for Site-Specific Delivery of a Non-Polar Drug. Soheyla Honary*, Behnam Hoseinzadeh and Payman. Shalchian. Mazandaran University of Medical Sciences, School of Pharmacy, Pharmaceutical Sciences Research Center, Sari,. Iran. Abstract.

  14. Acute effect of citrate bath on postdialysis alkalaemia

    Directory of Open Access Journals (Sweden)

    Patricia De Sequera Ortiz

    2015-03-01

    Conclusion: Dialysis with citrate provides better control of postdialysis acid-base balance, decreases/avoids postdialysis alkalaemia, and lowers the increase in Cac and Cai. This finding is of special interest in patients with predisposing factors for arrhythmia and patients with respiratory failure, carbon dioxide retention, calcifications and advanced liver disease.

  15. 21 CFR 520.1803 - Piperazine citrate capsules.

    Science.gov (United States)

    2010-04-01

    ... animal has finished eating the dosed food, the remainder of the food may be given. Dogs and cats may be... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Piperazine citrate capsules. 520.1803 Section 520.1803 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

  16. 21 CFR 520.622a - Diethylcarbamazine citrate tablets.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Diethylcarbamazine citrate tablets. 520.622a Section 520.622a Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... mouth. For the treatment of ascarid infections, repeat in 10 to 20 days to remove immature worms that...

  17. 21 CFR 520.622c - Diethylcarbamazine citrate chewable tablets.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Diethylcarbamazine citrate chewable tablets. 520.622c Section 520.622c Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... should be given in 10 to 20 days to remove immature worms which may enter the intestine from the lungs...

  18. The Effect of Polymer Molecular Weight on Citrate Crosslinked ...

    African Journals Online (AJOL)

    Methods: Films prepared with different molecular weights of chitosan and incorporating indomethacin as a non-polar model drug were obtained by a casting/solvent evaporation method. The chitosan films were crosslinked by dipping in varying concentrations of sodium citrate solution and for different crosslinking times.

  19. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan, E-mail: moonsonlife@yahoo.com; Xian, Shulin; Lu, Yunfei, E-mail: doctorlife@126.com

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. -- Highlights: •Blockage of glycolysis might be a novel way to anticancer. •Both 3-bromopyruvate and sodium citrate could inhibit glycolysis and regulate mitochondrial pathway in cancer cells. •Both 3-bromopyruvate and sodium citrate would be the novel agents on treatment of gastric cancer.

  20. Transcriptional organization of the large and the small ATP synthase operons, atpI/H/F/A and atpB/E, in Arabidopsis thaliana chloroplasts.

    Science.gov (United States)

    Malik Ghulam, Mustafa; Zghidi-Abouzid, Ouafa; Lambert, Emeline; Lerbs-Mache, Silva; Merendino, Livia

    2012-06-01

    The ATP synthase is a ubiquitous enzyme which is found in bacteria and eukaryotic organelles. It is essential in the photosynthetic and respiratory processes, by transforming the electrochemical proton gradient into ATP energy via proton transport across the membranes. In Escherichia coli, the atp genes coding for the subunits of the ATP synthase enzyme are grouped in the same transcriptional unit, while in higher plants the plastid atp genes are organized into a large (atpI/H/F/A) and a small (atpB/E) atp operon. By using the model plant Arabidopsis thaliana, we have investigated the strategy evolved in chloroplasts to overcome the physical separation of the atp gene clusters and to coordinate their transcription. We show that all the identified promoters in the two atp operons are PEP dependent and require sigma factors for specific recognition. Our results indicate that transcription of the two atp operons is initiated by at least one common factor, the essential SIG2 factor. Our data show that SIG3 and SIG6 also participate in transcription initiation of the large and the small atp operon, respectively. We propose that SIG2 might be the factor responsible for coordinating the basal transcription of the plastid atp genes and that SIG3 and SIG6 might serve to modulate plastid atp expression with respect to physiological and environmental conditions. However, we observe that in the sigma mutants (sig2, sig3 and sig6) the deficiency in the recognition of specific atp promoters is largely balanced by mRNA stabilization and/or by activation of otherwise silent promoters, indicating that the rate-limiting step for expression of the atp operons is mostly post-transcriptional.

  1. Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2

    NARCIS (Netherlands)

    Klug-Santner, Barbara G.; Schnitzhofer, Wolfgang; Vrsanska, Maria; Weber, Jörg; Agrawal, Pramod; Nierstrasz, Vincent; Guebitz, Georg M.

    2006-01-01

    An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a

  2. The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis.

    Science.gov (United States)

    Gilbert, H J; Jack, G W

    1981-01-01

    Phenylalanine ammonia-lyase (EC 4.3.1.5) of the yeast Rhodotorula glutinis was rapidly inactivated by duodenal juice. It was susceptible to chymotrypsin and subtilisin and to a lesser extent trypsin. Initial proteolysis of the enzyme by chymotrypsin and trypsin resulted in cleavage of the monomeric subunit (75 000 Mr) into a large (65 000 Mr) and a small (10 000 Mr) peptide. The small peptide was rapidly degraded. The 65 000-Mr fragment was resistant to prolonged incubation with chymotrypsin, but was degraded by trypsin under the same conditions. Phenylalanine ammonia-lyase was cleaved into several polypeptides by subtilisin, the 65 000-Mr peptide being totally absent. The N-terminal region of the enzyme was contained in the 65 000-Mr fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the three proteinases, and peptide-bond cleavage by trypsin and chymotrypsin. Several chemical modifications were performed on phenylalanine ammonia-lyase. Some decreased its antigenicity, and ethyl acetimidate decreased the rate of degradation of the 65 000-Mr peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzymic activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases. PMID:7041889

  3. Alteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based Mutagenesis

    NARCIS (Netherlands)

    Raj, Hans; Weiner, Barbara; Puthan Veetil, Vinod; Reis, Carlos R.; Quax, Wim J.; Janssen, Dick B.; Feringa, Ben L.; Poelarends, Gerrit J.

    2009-01-01

    3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3

  4. Mode of action of pectin lyase A of Aspergillus niger on differently C6-substituted oligogalacturonides

    NARCIS (Netherlands)

    Alebeek, van G.J.W.M.; Christensen, T.M.I.E.; Schols, H.A.; Mikkelsen, J.D.; Voragen, A.G.J.

    2002-01-01

    A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C6-substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of

  5. Production of Alginate Oligosaccharides (AOS as Prebiotic Ingredients through by Alginate lyase enzyme

    Directory of Open Access Journals (Sweden)

    Fahriza Sri Afni

    2017-04-01

    Full Text Available Prebiotics is indigestible foods that can not be digested but can stimulate the growth and activity of bacteria in the digestive tract effecting human health. Alginate oligosaccharides (AOS can be used as a source of prebiotic. That compounds can be produced enzymatically by cutting long chain alginates using alginate lyase. The aim of this study was to produce alginate lyase enzyme then producing Alginate oligosaccharides (AOS as a prebiotic ingredients. The alginate lyase enzyme can be produced from Bacillus megaterium bacteria using a discontinuous fermentor. The enzyme was  optimum temperature of 45°C and an optimum pH of 7.0. Alginate oligosaccharides production was performed with the addition of different enzyme concentrations 25, 50, 75, and 100 U. The result of the addition of enzyme (25, 50,75 U showed that the value of polymerization degrees (DP were between 4-5. However, the addition of enzyme (100 U was in the range of  DP 3-4. Bacterial probiotic growth test results of Bifidobacteria and Lactobacillus showed that 1% added AOS media were able to increase the growth of probiotic bacteria compared to themedia without addition of AOS. The addition Alginate lyase activity of 50 U in AOS production is the best treatment of both probiotic bacteria.

  6. Metabolism of β-valine via a CoA-dependent ammonia lyase pathway

    NARCIS (Netherlands)

    Otzen, Marleen; Crismaru, Ciprian G.; Postema, Christiaan P.; Wijma, Hein J.; Heberling, Matthew M.; Szymanski, Wiktor; de Wildeman, Stefaan; Janssen, Dick B.

    2015-01-01

    Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or

  7. Sugar- and nitrogen-dependent regulation of an Amanita muscaria phenylalanine ammonium lyase gene.

    Science.gov (United States)

    Nehls, U; Ecke, M; Hampp, R

    1999-03-01

    The cDNA of a key enzyme of secondary metabolism, phenylalanine ammonium lyase, was identified for an ectomycorrhizal fungus by differential screening of a mycorrhizal library. The gene was highly expressed in hyphae grown at low external monosaccharide concentrations, but its expression was 30-fold reduced at elevated concentrations. Gene repression was regulated by hexokinase.

  8. Mechanism of Cytochrome P450 17A1-Catalyzed Hydroxylase and Lyase Reactions

    DEFF Research Database (Denmark)

    Bonomo, Silvia; Jorgensen, Flemming Steen; Olsen, Lars

    2017-01-01

    Cytochrome P450 17A1 (CYP17A1) catalyzes C17 hydroxylation of pregnenolone and progesterone and the subsequent C17–C20 bond cleavage (lyase reaction) to form androgen precursors. Compound I (Cpd I) and peroxo anion (POA) are the heme-reactive species underlying the two reactions. We have...

  9. Catalytic Mechanisms and Biocatalytic Applications of Aspartate and Methylaspartate Ammonia Lyases

    NARCIS (Netherlands)

    de Villiers, Marianne; Veetil, Vinod Puthan; Raj, Hans; de Villiers, Jandre; Poelarends, Gerrit J.

    2012-01-01

    Ammonia lyases catalyze the formation of alpha-beta-unsaturated bonds by the elimination of ammonia from their substrates. This conceptually straightforward reaction has been the emphasis of many studies, with the main focus on the catalytic mechanism of these enzymes and/or the use of these enzymes

  10. Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins

    NARCIS (Netherlands)

    Hussain, Z.; Wiedner, R.; Steiner, K.; Hajek, T.; Avi, M.; Hecher, B.; Sessitsch, A.; Schwab, H.

    2012-01-01

    Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity

  11. Hematopoietic sphingosine 1-phosphate lyase deficiency decreases atherosclerotic lesion development in LDL-receptor deficient mice.

    Directory of Open Access Journals (Sweden)

    Martine Bot

    Full Text Available AIMS: Altered sphingosine 1-phosphate (S1P homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/- deficiency on leukocyte subsets relevant to atherosclerosis. METHODS AND RESULTS: LDL receptor deficient mice that were transplanted with Sgpl1(-/- bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/- chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. CONCLUSIONS: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

  12. The Skin Bacterium Propionibacterium acnes Employs Two Variants of Hyaluronate Lyase with Distinct Properties

    DEFF Research Database (Denmark)

    Nazipi, Seven; Stødkilde-Jørgensen, Kristian; Scavenius, Carsten

    2017-01-01

    Hyaluronic acid (HA) and other glycosaminoglycans are extracellular matrix components in the human epidermis and dermis. One of the most prevalent skin microorganisms, Propionibacterium acnes, possesses HA-degrading activity, possibly conferred by the enzyme hyaluronate lyase (HYL). In this study...

  13. Argininosuccinate synthetase and argininosuccinate lyase: two ornithine cycle enzymes from Agaricus bisporus

    NARCIS (Netherlands)

    Wagemaker, M.J.M.; Eastwood, D.C.; Drift, van der C.; Jetten, M.S.M.; Burton, K.; Griensven, van L.J.L.D.; Camp, op den H.J.M.

    2007-01-01

    Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main

  14. Biochemical and structural characterization of a novel bacterial manganese-dependent hydroxynitrile lyase.

    NARCIS (Netherlands)

    Hajnal, I.; Lyskowski, A.; Hanefeld, U.; Gruber, K.; Schwab, H.; Steiner, K.

    2013-01-01

    Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in

  15. Genetic and molecular analyses of Escherichia coli N-acetylneuraminate lyase gene.

    OpenAIRE

    Kawakami, B; Kudo, T; Narahashi, Y; Horikoshi, K

    1986-01-01

    Two plasmids containing the N-acetylneuraminate lyase (NALase) gene (nanA) of Escherichia coli, pNL1 and pNL4, were constructed. Immunoprecipitation analysis indicated that the 35,000-dalton protein encoded in pNL4 was NALase. The synthesis of NALase in E. coli carrying these plasmids was constitutive.

  16. A fluorescent substrate for carbon-phosphorus lyase: towards the pathway for organophosphonate metabolism in bacteria

    DEFF Research Database (Denmark)

    He, Shu-Mei; Lou, Yan; Hove-Jensen, Bjarne

    2009-01-01

    this pathway. In order to detect metabolic intermediates we have synthesized a fluorescently labelled organophosphonate and show that this is a viable substrate for the CP-lyase pathway in Escherichia coli and that the expected product of CP-bond cleavage is formed. The in vivo competence of one potential...

  17. Usefulness of Alginate Lyases Derived from Marine Organisms for the Preparation of Alginate Oligomers with Various Bioactivities.

    Science.gov (United States)

    Takeshita, S; Oda, T

    Alginate-degrading enzyme, alginate lyase, catalyzes the cleavage of glycosidic 1-4 O-linkages between uronic acid residues of alginate by a β-elimination reaction leaving a 4-deoxy-l-erythro-hex-4-ene pyranosyluronate as nonreducing terminal end. The enzymes from a wide variety of sources such as marine molluscs, seaweeds, and marine bacteria have been discovered and studied not only from a point of view of enzymological interest of enzyme itself but also for elucidation of fine chemical structure of alginate, structure-activity relationship of alginate, and biological activities and physicochemical features of the enzymatic digestion products. Based on the substrate specificities, alginate lyases are classified into three groups: poly(β-d-mannuronate) lyase, poly(α-l-guluronate) lyase, and bifunctional alginate lyase, which are specific to mannuronate, guluronate, and both uronic acid residues, respectively. We have studied enzymological aspects of these three types of alginate lyases, and bioactivities of enzymatically digested alginate oligomers. In this chapter, we described the purification and characterization of three types of alginate lyases from different marine origins and overviewed the bioactivities of alginate oligomers. © 2016 Elsevier Inc. All rights reserved.

  18. Overexpression of Phenylalanine Ammonia-Lyase in Transgenic Roots of Coleus blumei Alters Growth and Rosmarinic Acid Synthesis

    Directory of Open Access Journals (Sweden)

    Hrvoje Fulgosi

    2011-01-01

    Full Text Available Most natural phenolic compounds in plants are derived from trans-cinnamic acid, formed by the nonoxidative deamination of L-phenylalanine by phenylalanine ammonia-lyase (EC 4.3.1.24. Although a strict line between primary and secondary metabolism cannot be drawn, phenylalanine ammonia-lyase is considered to play a pivotal role in channelling carbon flux from primary metabolism to phenolic synthesis. The objective of this work is to evaluate the impact of phenylalanine ammonia-lyase activity on phenolic production in Coleus blumei. Transgenic roots of C. blumei, harbouring the Arabidopsis thaliana PAL1 gene, under the control of the CaMV 35S promoter, along with empty vector and wild-type roots, were regenerated. Transgenic root lines had disparate phenylalanine ammonia-lyase activities ranging from 67 to 350 %, compared to wild-type roots. Growth rates significantly differed, with the lowest in transgenic roots exerting augmented phenylalanine ammonia-lyase activity. Transgenic roots with high phenylalanine ammonia-lyase activity had lower growth rates, lower amounts of total phenolics, rosmarinic acid (the major phenolic compound in C. blumei and chlorogenic acid, but increased amounts of caffeic acid. There was no increase in total phenolics and rosmarinic acid content after feeding transgenic roots with casein enzymatic hydrolysate and L-tyrosine. This shows that augmented phenylalanine ammonia-lyase activity inhibits growth and phenolic metabolism, and the probable regulator of these processes is trans-cinnamic acid.

  19. Mayenite Synthesized Using the Citrate Sol-Gel Method

    Energy Technology Data Exchange (ETDEWEB)

    Ude, Sabina N [ORNL; Rawn, Claudia J [ORNL; Meisner, Roberta A [University of Tennessee (UTK) and Oak Ridge National Laboratory (ORNL); Kirkham, Melanie J [ORNL; Jones, Gregory L. [University of Tennessee, Knoxville (UTK); Payzant, E Andrew [ORNL

    2014-01-01

    A citrate sol-gel method has been used to synthesize mayenite (Ca12Al14O33). X-ray powder diffraction data show that the samples synthesized using the citrate sol-gel method contained CaAl2O4 and CaCO3 along with mayenite when fired ex-situ in air at 800 C but were single phase when fired at 900 C and above. Using high temperature x-ray diffraction, data collected in-situ in air at temperatures of 600 C and below showed only amorphous content; however, data collected at higher temperatures indicated the first phase to crystallize is CaCO3. High temperature x-ray diffraction data collected in 4% H2/96% N2 does not show the presence of CaCO3, and Ca12Al14O33 starts to form around 850 C. In comparison, x-ray powder diffraction data collected ex-situ on samples synthesized using traditional solid-state synthesis shows that single phase was not reached until samples were fired at 1350 C. DTA/TGA data collected either in a nitrogen environment or air on samples synthesized using the citrate gel method suggest the complete decomposition of metastable phases and the formation of mayenite at 900 C, although the phase evolution is very different depending on the environment. Brunauer-Emmett-Teller (BET) measurements showed a slightly higher surface area of 7.4 0.1 m2/g in the citrate gel synthesized samples compared to solid-state synthesized sample with a surface area of 1.61 0.02 m2/g. SEM images show a larger particle size for samples synthesized using the solid-state method compared to those synthesized using the citrate gel method.

  20. Molecular characterization of microbial population dynamics during sildenafil citrate degradation.

    Science.gov (United States)

    De Felice, Bruna; Argenziano, Carolina; Guida, Marco; Trifuoggi, Marco; Russo, Francesca; Condorelli, Valerio; Inglese, Mafalda

    2009-02-01

    Little is known about pharmaceutical and personal care products pollutants (PPCPs), but there is a growing interest in how they might impact the environment and microbial communities. The widespread use of Viagra (sildenafil citrate) has attracted great attention because of the high usage rate, the unpredictable disposal and the unknown potential effects on wildlife and the environment. Until now information regarding the impact of Viagra on microbial community in water environment has not been reported. In this research, for the first time, the genetic profile of the microbial community, developing in a Viagra polluted water environment, was evaluated by means of the 16S and 18S rRNA genes, for bacteria and fungi, respectively, amplified by polymerase chain reaction (PCR) and separated using the denaturing gradient gel electrophoresis (DGGE) technique. The DGGE results revealed a complex microbial community structure with most of the population persisting throughout the experimental period. DNA sequences from bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria and fungi found previously mainly in polluted environmental and treating bioreactors. Biotransformation ability of sildenafil citrate by the microbial pool was studied and the capability of these microorganisms to detoxify a polluted water ecosystem was assessed. The bacterial and fungal population was able to degrade sildenafil citrate entirely. Additionally, assays conducted on Daphnia magna, algal growth inhibition assay and cell viability determination on HepG2 human cells showed that biotransformation products obtained from the bacterial growth was not toxic. The higher removal efficiency for sildenafil citrate and the lack of toxicity by the biotransformation products obtained showed that the microbial community identified here represented a composite population that might have biotechnological relevance to

  1. Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism.

    Science.gov (United States)

    Peng, Huan; Cui, Jiangkuan; Long, Haibo; Huang, Wenkun; Kong, Lingan; Liu, Shiming; He, Wenting; Hu, Xianqi; Peng, Deliang

    2016-01-01

    Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests

  2. Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism.

    Directory of Open Access Journals (Sweden)

    Huan Peng

    Full Text Available Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases. In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7. A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7 were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2 and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi. Similarly, this procedure reduced the number of female adults at 40 dpi

  3. Distinct Conformation of ATP Molecule in Solution and on Protein.

    Science.gov (United States)

    Kobayashi, Eri; Yura, Kei; Nagai, Yoshinori

    2013-01-01

    Adenosine triphosphate (ATP) is a versatile molecule used mainly for energy and a phosphate source. The hydrolysis of γ phosphate initiates the reactions and these reactions almost always start when ATP binds to protein. Therefore, there should be a mechanism to prevent spontaneous hydrolysis reaction and a mechanism to lead ATP to a pure energy source or to a phosphate source. To address these questions, we extensively analyzed the effect of protein to ATP conformation based on the sampling of the ATP solution conformations obtained from molecular dynamics simulation and the sampling of ATP structures bound to protein found in a protein structure database. The comparison revealed mainly the following three points; 1) The ribose ring in ATP molecule, which puckers in many ways in solution, tends to assume either C2' exo or C2' endo when it binds to protein. 2) The adenine ring in ATP molecule, which takes open-book motion with the two ring structures, has two distinct structures when ATP binds to protein. 3) The glycosyl-bond and the bond between phosphate and the ribose have unique torsion angles, when ATP binds to protein. The combination of torsion angles found in protein-bound forms is under-represented in ATP molecule in water. These findings suggest that ATP-binding protein exerts forces on ATP molecule to assume a conformation that is rarely found in solution, and that this conformation change should be a trigger for the reactions on ATP molecule.

  4. Electric field driven torque in ATP synthase.

    Directory of Open Access Journals (Sweden)

    John H Miller

    Full Text Available FO-ATP synthase (FO is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the γ-subunit to rotate within the ATP-producing complex (F1 overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring.

  5. Acute effect of citrate bath on postdialysis alkalaemia.

    Science.gov (United States)

    Ortiz, Patricia De Sequera; Ramón, Marta Albalate; Pérez-García, Rafael; Prats, Elena Corchete; Cobo, Patricia Arribas; Arroyo, Roberto Alcázar; Díaz, Maira Ortega; Carretero, Marta Puerta

    2015-01-01

    The correction of metabolic acidosis caused by renal failure is achieved by adding bicarbonate during dialysis. In order to avoid the precipitation of calcium carbonate and magnesium carbonate that takes place in the dialysis fluid (DF) when adding bicarbonate, it is necessary to add an acid, usually acetate, which is not free of side effects. Thus, citrate appears as an advantageous alternative to acetate, despite the fact that its acute effects are not accurately known. To assess the acute effect of a dialysis fluid containing citrate instead of acetate on acid-base balance and calcium-phosphorus metabolism parameters. A prospective crossover study was conducted with twenty-four patients (15 male subjects and 9 female subjects). All patients underwent dialysis with AK-200-Ultra-S monitor with SoftPac® dialysis fluid, made with 3 mmol/L of acetate and SelectBag Citrate®, with 1 mmol/L of citrate and free of acetate. The following were measured before and after dialysis: venous blood gas monitoring, calcium (Ca), ionic calcium (Cai), phosphorus (P) and parathyroid hormone (PTH). Differences (p<0.05) were found when using the citrate bath (C) compared to acetate (A) in the postdialysis values of: pH, C: 7.43 (0.04) vs. A: 7.47 (0.05); bicarbonate, C: 24.7 (2.7) vs. A: 27.3 (2.1) mmol/L; base excess (BEecf), C: 0.4 (3.1) vs. A: 3.7 (2.4) mmol/L; corrected calcium (Cac), C: 9.8 (0.8) vs. A: 10.1 (0.7) mg/dL; and Cai, C: 1.16 (0.05) vs. A: 1.27 (0.06) mmol/L. No differences were found in either of the parameters measured before dialysis. Dialysis with citrate provides better control of postdialysis acid-base balance, decreases/avoids postdialysis alkalaemia, and lowers the increase in Cac and Cai. This finding is of special interest in patients with predisposing factors for arrhythmia and patients with respiratory failure, carbon dioxide retention, calcifications and advanced liver disease. Copyright © 2015. Published by Elsevier España, S.L.U.

  6. Metformin-clomiphene citrate vs. clomiphene citrate alone: Polycystic ovarian syndrome

    Directory of Open Access Journals (Sweden)

    Aqueela Ayaz

    2013-01-01

    Full Text Available Background: Polycystic ovary syndrome (PCOS is the commonest endocrinopathy in women that is associated with reproductive and metabolic disorders. Objectives: We compared the ovulation and conception rates after the treatment with clomiphene citrate (CC alone and in combination with metformin in infertile patients presented with polycystic ovarian syndrome (PCOS. Materials and Methods: This randomized controlled trial of independent cases and controls was conducted at the Department of Obstetrics and Gynecology, Hera General Hospital, Makkah, Saudi Arabia from February 01 to December 31, 2008. The 42 subjects diagnosed as PCOS were divided into group A and B (21 subjects in each for management with CC + metformin and CC alone, respectively. Group A received 500 mg three times a day of metformin continuously from the first cycle for 6 months or till pregnancy was confirmed. In both groups CC was started at a dose of 50 mg from day-2 till day-6 of the menstrual cycle. The dose of CC was increased to 100 mg in second and 150 mg in third cycle, and then remained 150 mg for the remaining three cycles. With ovulation the dose of CC was unaltered in both groups. Data were analyzed using Statistical Package for the Social Sciences (SPSS version 16. Results: More than 50% females in both groups were had body mass index > 25. Group A achieved high rate of regular cycles, ovulation success, and conception than group B (71.4% vs. 38.1%; P = 0.03, (76.2% vs. 38.1%; P = 0.021, and (66.6% vs. 28.6%; P = 0.01, respectively. Conclusion: Management with metformin + CC increased the ovulation and conception rates.

  7. 77 FR 56188 - Citric Acid and Certain Citrate Salts from the People's Republic of China: Notice of Rescission...

    Science.gov (United States)

    2012-09-12

    ... citrate tetrahydrate, which are intermediate products in the production of citric acid, sodium citrate... International Trade Administration Citric Acid and Certain Citrate Salts from the People's Republic of China... of the countervailing duty (CVD) order on citric acid and certain citrate salts from the People's...

  8. Interaction of horse-spleen ferritin with aluminium citrate.

    Science.gov (United States)

    Cochran, M; Chawtur, V

    1988-11-01

    Horse-spleen ferritin was found to bind Al systematically following gel filtration in buffered Al citrate 30 microM, and up to molar ratio 98 when incubated at 37 degrees C with Al citrate, buffered to pH 7.4. Pre-incubation with 3 concentrations of neutral sodium phosphate (0.1, 1.0, 10.0 mM) had no significant effect on binding. Apotransferrin interaction with the Al-ferritin complex to release Fe but not Al. Protein-digestion and EDTA washing procedures showed that the Al was firmly bound to the ferritin, probably to the core. Since ferritin species from different organs are relatively alike, we suggest that in the gut ferritin may scavenge Al followed by its re-excretion into the lumen with the mucosal cells, thus protecting against absorption of the metal.

  9. Biochemical Characteristics and Variable Alginate-Degrading Modes of a Novel Bifunctional Endolytic Alginate Lyase.

    Science.gov (United States)

    Cheng, Yuanyuan; Wang, Dandan; Gu, Jingyan; Li, Junge; Liu, Huihui; Li, Fuchuan; Han, Wenjun

    2017-12-01

    Bifunctional alginate lyases can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G), but their substrate-degrading modes have not been thoroughly elucidated to date. In this study, we present Aly1 as a novel bifunctional endolytic alginate lyase of the genus Flammeovirga The recombinant enzyme showed optimal activity at 50°C and pH 6.0. The enzyme produced unsaturated disaccharide (UDP2) and trisaccharide fractions as the final main alginate digests. Primary substrate preference tests and further structure identification of various size-defined final oligosaccharide products demonstrated that Aly1 is a bifunctional alginate lyase and prefers G to M. Tetrasaccharide-size fractions are the smallest substrates, and M, G, and UDP2 fractions are the minimal product types. Remarkably, Aly1 can vary its substrate-degrading modes in accordance with the terminus types, molecular sizes, and M/G contents of alginate substrates, producing a series of small size-defined saturated oligosaccharide products from the nonreducing ends of single or different saturated sugar chains and yielding unsaturated products in distinct but restricted patterns. The action mode changes can be partially inhibited by fluorescent labeling at the reducing ends of oligosaccharide substrates. Deletion of the noncatalytic region (NCR) of Aly1 caused weak changes of biochemical characteristics but increased the degradation proportions of small size-defined saturated M-enriched oligosaccharide substrates and unsaturated tetrasaccharide fractions without any size changes of degradable oligosaccharides, thereby enhancing the M preference and enzyme activity. Therefore, our results provided insight into the variable action mode of a novel bifunctional endolytic alginate lyase to inform accurate enzyme use.IMPORTANCE The elucidated endolytic alginate lyases usually degrade substrates into various size-defined unsaturated oligosaccharide products (≥UDP2), and exolytic enzymes

  10. Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family.

    Science.gov (United States)

    Shevchik, V E; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1997-12-01

    Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified. We cloned the pelI gene, encoding a ninth pectate lyase of E. chrysanthemi 3937. The pelI reading frame is 1,035 bases long, corresponding to a protein of 344 amino acids including a typical amino-terminal signal sequence of 19 amino acids. The purified mature PelI protein has an isoelectric point of about 9 and an apparent molecular mass of 34 kDa. PelI has a preference for partially methyl esterified pectin and presents an endo-cleaving activity with an alkaline pH optimum and an absolute requirement for Ca2+ ions. PelI is an extracellular protein secreted by the Out secretory pathway of E. chrysanthemi. The PelI protein is very active in the maceration of plant tissues. A pelI mutant displayed reduced pathogenicity on chicory leaves, but its virulence did not appear to be affected on potato tubers or Saintpaulia ionantha plants. The pelI gene constitutes an independent transcriptional unit. As shown for the other pel genes, the transcription of pelI is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, temperature, nitrogen starvation, and catabolite repression. Regulation of pelI expression appeared to be dependent on the three repressors of pectinase synthesis, KdgR, PecS, and PecT, and on the global activator of sugar catabolism, cyclic AMP receptor protein. A functional KdgR binding site was identified close to the putative pelI promoter. Analysis of the amino acid sequence of PelI revealed high homology with a pectate lyase from Erwinia carotovora subsp. carotovora (65% identity) and low homology with pectate lyases of the phytopathogenic fungus Nectria haematococca (Fusarium solani). This finding indicates that PelI belongs to pectate lyase class

  11. Renal Localization of {sup 67}Ga Citrate in Noninfectious Nephritis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kang Wook; Jeong, Min Soo; Rhee, Sunn Kgoo; Kim, Sam Yong; Shin, Young Tai; Ro, Heung Kyu [Chungnam University College of Medicine, Deajeon (Korea, Republic of)

    1992-07-15

    {sup 67}Ga citrate scan has been requested for detection or follow-up of inflammatory or neoplastic disease. Visualization of {sup 67}Ga citrate in the kidneys at 48 and 72 hr post injection is usually interpreted as evidence of renal pathology. But precise mechanisms of abnormal {sup 67}Ga uptake in kidneys were unknown. We undertook a study to determine the clinical value of {sup 67}Ga citrate imaging of the kidneys in 68 patients with primary or secondary nephropathy confirmed by renal biopsy and 66 control patients without renal disease. Renal uptake in 48 to 72 hr images was graded as follows: Grade 0=background activity;1=faint uptake greater than background; 2=definite uptake, but less than lumbar vertebrae;3 same uptake as lumbar vertebrae, but less than liver; 4=same or higher uptake than liver. The results were as follows. 1) 42 of 68(62%) patients with noninfectious nephritis showed grade 2 or higher {sup 67}Ga renal uptake but only 10 percent of control patients showed similar uptake. 2) In 14 patients with systemic lupus erythematosus, 8 of 9 (89%) patients with lupus nephritis exhibited marked renal uptake. 3) 36 of 41 patients (88%) with combined nephrotic syndrome showed Grade 2 or higher renal uptake. 4) Renal {sup 67}Ga uptake was correlated with clinical severity of nephrotic syndrome determined by serum albumin level, 24 hr urine protein excretion and serum lipid levels. 5) After complete remission of nephrotic syndrome, renal uptake in all 8 patients who were initially Grade 3 or 4, decreased to Grade 1 or 0. In conclusion, we think that the mechanism of renal {sup 67}Ga uptake in nephrotic syndrome might be related to the pathogenesis of nephrotic syndrome. In systemic lupus erythematosus, {sup 67}Ga citrate scan is useful in predicting renal involvement.

  12. Acute effect of citrate bath on postdialysis alkalaemia

    OpenAIRE

    De Sequera Ortiz, Patricia; Albalate Ramón, Marta; Pérez-García, Rafael; Corchete Prats, Elena; Arribas Cobo, Patricia; Alcázar Arroyo, Roberto; Ortega Díaz, Maira; Puerta Carretero, Marta

    2015-01-01

    Introduction: The correction of metabolic acidosis caused by renal failure is achieved by adding bicarbonate during dialysis. In order to avoid the precipitation of calcium carbonate and magnesium carbonate that takes place in the dialysis fluid (DF) when adding bicarbonate, it is necessary to add an acid, usually acetate, which is not free of side effects. Thus, citrate appears as an advantageous alternative to acetate, despite the fact that its acute effects are not accurately known. Obj...

  13. Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

    Science.gov (United States)

    Denis, María Florencia Leal; Incicco, J. Jeremías; Espelt, María Victoria; Verstraeten, Sandra V.; Pignataro, Omar P.; Lazarowski, Eduardo R.; Schwarzbaum, Pablo J.

    2014-01-01

    SUMMARY Background The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). Methods Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. Results Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. Conclusions MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General Significance Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation. PMID:23742824

  14. A randomized clinical trial of treatment of clomiphene citrate-resistant anovulation with the use of oral contraceptive pill suppression and repeat clomiphene citrate treatment.

    Science.gov (United States)

    Branigan, Emmett F; Estes, M Antoinette

    2003-06-01

    The purpose of this study was to evaluate the effectiveness and endocrine response of oral contraceptive ovarian suppression followed by clomiphene citrate in patients who previously were clomiphene citrate resistant. Forty-eight patients from a private tertiary infertility clinic were assigned randomly prospectively to either group 1 (oral contraceptive/clomiphene citrate), which received continuous oral contraceptives followed by clomiphene citrate, or to group 2 (control) received no treatment in the cycle before clomiphene citrate treatment. On day 3, 17 beta-estradiol, follicle-stimulating hormone, luteinizing hormone, and androgens were assayed before and after treatment. Follicle growth, ovulation, and pregnancy were evaluated. The Student t test and analysis of variance were used for statistical significance. The oral contraceptive/clomiphene citrate group had a significantly higher percentage of patients who ovulated and of ovulatory cycles and pregnancies. Significantly lower levels of 17 beta-estradiol, luteinizing hormone, and androgen levels were seen in the oral contraceptive/clomiphene citrate group, with no significant changes in group 2. Suppression of the ovary with oral contraceptives results in excellent rates of ovulation and pregnancy in patients who previously were resistant to clomiphene citrate. The decreases in ovarian androgens, luteinizing hormone, and 17 beta-estradiol may be responsible for the improved response.

  15. Transport of citrate catalyzed by the sodium-dependent citrate carrier of Klebsiella pneumoniae is obligatorily coupled to the transport of two sodium ions

    NARCIS (Netherlands)

    Lolkema, Juke S.; Enequist, Hans; Rest, Michel E. van der

    1994-01-01

    Aerobically grown Escherichia coli GM48 harboring plasmid pKScitS that codes for the sodium-dependent citrate carrier from Klebsiella pneumoniae (CitS) allows initial-rate measurements of citrate uptake in whole cells. The cation stoichiometry and selectivity of CitS was studied using this

  16. The crystal structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides malyl-CoA lyases and comparison with CitE-like superfamily enzymes and malate synthases.

    Science.gov (United States)

    Zarzycki, Jan; Kerfeld, Cheryl A

    2013-11-09

    Malyl-CoA lyase (MCL) is a promiscuous carbon-carbon bond lyase that catalyzes the reversible cleavage of structurally related Coenzyme A (CoA) thioesters. This enzyme plays a crucial, multifunctional role in the 3-hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus. A second, phylogenetically distinct MCL from Rhodobacter sphaeroides is involved in the ethylmalonyl-CoA pathway for acetate assimilation. Both MCLs belong to the large superfamily of CitE-like enzymes, which includes the name-giving β-subunit of citrate lyase (CitE), malyl-CoA thioesterases and other enzymes of unknown physiological function. The CitE-like enzyme superfamily also bears sequence and structural resemblance to the malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or both (the malate synthases). Here we report the first crystal structures of MCLs from two different phylogenetic subgroups in apo- and substrate-bound forms. Both the C. aurantiacus and the R. sphaeroides MCL contain elaborations on the canonical β8/α8 TIM barrel fold and form hexameric assemblies. Upon ligand binding, changes in the C-terminal domains of the MCLs result in closing of the active site, with the C-terminal domain of one monomer forming a lid over and contributing side chains to the active site of the adjacent monomer. The distinctive features of the two MCL subgroups were compared to known structures of other CitE-like superfamily enzymes and to malate synthases, providing insight into the structural subtleties that underlie the functional versatility of these enzymes. Although the C. aurantiacus and the R. sphaeroides MCLs have divergent primary structures (~37% identical), their tertiary and quaternary structures are very similar. It can be assumed that the C-C bond formation catalyzed by the MCLs occurs as proposed for

  17. One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm.

    Science.gov (United States)

    Ghadam, Parinaz; Akhlaghi, Fatemeh; Ali, Ahya Abdi

    2017-05-01

    Pseudomonas aeruginosa is a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl) is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated. The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR. Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1. In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.

  18. Effect of Potassium Citrate on Calcium Phosphate Stones in a Model of Hypercalciuria

    Science.gov (United States)

    Asplin, John R.; Frick, Kevin K.; Granja, Ignacio; Culbertson, Christopher D.; Ng, Adeline; Grynpas, Marc D.; Bushinsky, David A.

    2015-01-01

    Potassium citrate is prescribed to decrease stone recurrence in patients with calcium nephrolithiasis. Citrate binds intestinal and urine calcium and increases urine pH. Citrate, metabolized to bicarbonate, should decrease calcium excretion by reducing bone resorption and increasing renal calcium reabsorption. However, citrate binding to intestinal calcium may increase absorption and renal excretion of both phosphate and oxalate. Thus, the effect of potassium citrate on urine calcium oxalate and calcium phosphate supersaturation and stone formation is complex and difficult to predict. To study the effects of potassium citrate on urine supersaturation and stone formation, we utilized 95th-generation inbred genetic hypercalciuric stone-forming rats. Rats were fed a fixed amount of a normal calcium (1.2%) diet supplemented with potassium citrate or potassium chloride (each 4 mmol/d) for 18 weeks. Urine was collected at 6, 12, and 18 weeks. At 18 weeks, stone formation was visualized by radiography. Urine citrate, phosphate, oxalate, and pH levels were higher and urine calcium level was lower in rats fed potassium citrate. Furthermore, calcium oxalate and calcium phosphate supersaturation were higher with potassium citrate; however, uric acid supersaturation was lower. Both groups had similar numbers of exclusively calcium phosphate stones. Thus, potassium citrate effectively raises urine citrate levels and lowers urine calcium levels; however, the increases in urine pH, oxalate, and phosphate levels lead to increased calcium oxalate and calcium phosphate supersaturation. Potassium citrate induces complex changes in urine chemistries and resultant supersaturation, which may not be beneficial in preventing calcium phosphate stone formation. PMID:25855777

  19. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are

  20. ATP release, generation and hydrolysis in exocrine pancreatic duct cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

    2015-01-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, ou...

  1. Limitations of ATP as a measure of microbial biomass | Stuart ...

    African Journals Online (AJOL)

    Estimates of the total living biomass of micro-organisms on decomposing kelp detritus, calculated indirectly from the concentration of ATP, were compared with those obtained directly from cell numbers and volumes. Large overestimates in biomass were obtained from ATP x 250, and C:ATP ratios varied considerably with ...

  2. ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Masaki Nakano

    2017-08-01

    Full Text Available Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances, which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease.

  3. Discovery of a novel (R-selective bacterial hydroxynitrile lyase from Acidobacterium capsulatum

    Directory of Open Access Journals (Sweden)

    Romana Wiedner

    2014-06-01

    Full Text Available Hydroxynitrile lyases (HNLs are powerful carbon–carbon bond forming enzymes. The reverse of their natural reaction – the stereoselective addition of hydrogen cyanide (HCN to carbonyls – yields chiral cyanohydrins, versatile building blocks for the pharmaceutical and chemical industry. Recently, bacterial HNLs have been discovered, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (e.g. high yield recombinant expression in Escherichia coli, the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases in the ongoing search for HNLs with improved activity, enantioselectivity, stability and substrate scope. In this study, database mining revealed a novel cupin HNL from Acidobacterium capsulatum ATCC 51196 (AcHNL, which was able to catalyse the (R-selective synthesis of mandelonitrile with significantly better conversion (97% and enantioselectivity (96.7% than other cupin HNLs.

  4. Discovery of a novel (R)-selective bacterial hydroxynitrile lyase from Acidobacterium capsulatum.

    Science.gov (United States)

    Wiedner, Romana; Gruber-Khadjawi, Mandana; Schwab, Helmut; Steiner, Kerstin

    2014-06-01

    Hydroxynitrile lyases (HNLs) are powerful carbon-carbon bond forming enzymes. The reverse of their natural reaction - the stereoselective addition of hydrogen cyanide (HCN) to carbonyls - yields chiral cyanohydrins, versatile building blocks for the pharmaceutical and chemical industry. Recently, bacterial HNLs have been discovered, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (e.g. high yield recombinant expression in Escherichia coli), the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases in the ongoing search for HNLs with improved activity, enantioselectivity, stability and substrate scope. In this study, database mining revealed a novel cupin HNL from Acidobacterium capsulatum ATCC 51196 (AcHNL), which was able to catalyse the (R)-selective synthesis of mandelonitrile with significantly better conversion (97%) and enantioselectivity (96.7%) than other cupin HNLs.

  5. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    Science.gov (United States)

    Brand, M D; Lehninger, A L

    1977-01-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems. PMID:17116

  6. Probing reversible chemistry in coenzyme B12-dependent ethanolamine ammonia lyase with kinetic isotope effects

    OpenAIRE

    Jones, Alex R; Rentergent, Julius; Scrutton, NS; Hay, S

    2015-01-01

    Coenzyme B12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the ?-carbon of the 5?-deoxyadenosyl moiety of the intrinsic coenzyme B12, it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5?-deoxyadenosyl radical and substrate during si...

  7. Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins.

    Science.gov (United States)

    Hussain, Zahid; Wiedner, Romana; Steiner, Kerstin; Hajek, Tanja; Avi, Manuela; Hecher, Bianca; Sessitsch, Angela; Schwab, Helmut

    2012-03-01

    Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity.

  8. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

    OpenAIRE

    Lima,Juliana de Oliveira; Pereira, Jorge Fernando; Araújo,Elza Fernandes; Queiroz, Marisa Vieira de

    2017-01-01

    Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also stud...

  9. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

    OpenAIRE

    Lima,Juliana de Oliveira; Pereira, Jorge Fernando; Araújo,Elza Fernandes; Queiroz, Marisa Vieira de

    2017-01-01

    Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were ...

  10. Study on optimal production of 3-ketovalidoxylamine A C-N lyase ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... diabetes increasing, these chemicals are receiving considerable attention. G3DH and ... 2 g K2HPO4 and 0.2 g MgSO4. Seed medium was contained (per litre) 5 g beef extract, 10 g peptone, 5 g ... lyase activity was defined as the amount of enzyme that caused the production of 1 µmol of p-nitroaniline per ...

  11. Identification of Bacterial Cell Wall Lyases via Pseudo Amino Acid Composition

    Directory of Open Access Journals (Sweden)

    Xin-Xin Chen

    2016-01-01

    Full Text Available Owing to the abuse of antibiotics, drug resistance of pathogenic bacteria becomes more and more serious. Therefore, it is interesting to develop a more reasonable way to solve this issue. Because they can destroy the bacterial cell structure and then kill the infectious bacterium, the bacterial cell wall lyases are suitable candidates of antibacteria sources. Thus, it is urgent to develop an accurate and efficient computational method to predict the lyases. Based on the consideration, in this paper, a set of objective and rigorous data was collected by searching through the Universal Protein Resource (the UniProt database, whereafter a feature selection technique based on the analysis of variance (ANOVA was used to acquire optimal feature subset. Finally, the support vector machine (SVM was used to perform prediction. The jackknife cross-validated results showed that the optimal average accuracy of 84.82% was achieved with the sensitivity of 76.47% and the specificity of 93.16%. For the convenience of other scholars, we built a free online server called Lypred. We believe that Lypred will become a practical tool for the research of cell wall lyases and development of antimicrobial agents.

  12. Characterization of a Long-Lived Alginate Lyase Derived from Shewanella Species YH1

    Directory of Open Access Journals (Sweden)

    Hisashi Yagi

    2017-12-01

    Full Text Available Polysaccharides from seaweeds are widely used in various fields, including the food, biomedical material, cosmetic, and biofuel industries. Alginate, which is a major polysaccharide in brown algae, and the products of its degradation (oligosaccharides have been used in stabilizers, thickeners, and gelling agents, especially in the food industry. Discovering novel alginate lyases with unique characteristics for the efficient production of oligosaccharides may be relevant for the food and pharmaceutical fields. In this study, we identified a unique alginate lyase derived from an alginate-utilizing bacterium, Shewanella species YH1. The recombinant enzyme (rAlgSV1-PL7 was produced in an Escherichia coli system and it was classified in the Polysaccharide Lyase family 7. The optimal temperature and pH for rAlgSV1-PL7 activity were around 45 °C and 8, respectively. Interestingly, we observed that rAlgSV1-PL7 retained over 80% of its enzyme activity after incubation at 30 °C for at least 20 days, indicating that rAlgSV1-PL7 is a long-lived enzyme. Moreover, the degradation of alginate by rAlgSV1-PL7 produced one to four sugars because of the broad substrate specificity of this enzyme. Our findings suggest that rAlgSV1-PL7 may represent a new commercially useful enzyme.

  13. Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011

    Directory of Open Access Journals (Sweden)

    Shiva Kumar Vasanth V

    2002-03-01

    Full Text Available Abstract Background Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP. DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP. Results S. typhimurium PU011, a non-virulent bacterial strain isolated in our lab, was found to produce DAP ammonia lyase enzyme when grown in minimal medium containing DAP. There was a direct correlation between biomass yield and enzyme activity, until 16 h post inoculation in minimal medium containing DAP. Following ammonium sulphate precipitation and passing through Sephadex G100, CM-Sephadex and DEAE-Sephacel for crude enzyme extract preparation, about 68-fold enzyme purity was obtained. The purified enzyme gave maximum activity at pH 8.0 and was stable up to 45 degrees C. The Km value for the substrate was found to be 0.685mM, calculated from a Line Weaver Burk plot. Conclusion A new bacterial strain, S.typhimurium PU 011, which is capable of producing DAP ammonia lyase, was isolated.

  14. Upregulation of ABC transporters contributes to chemoresistance of sphingosine 1-phosphate lyase-deficient fibroblasts.

    Science.gov (United States)

    Ihlefeld, Katja; Vienken, Hans; Claas, Ralf Frederik; Blankenbach, Kira; Rudowski, Agnes; ter Braak, Michael; Koch, Alexander; Van Veldhoven, Paul P; Pfeilschifter, Josef; Meyer zu Heringdorf, Dagmar

    2015-01-01

    Sphingosine 1-phosphate (S1P) is an extra- and intracellular mediator that regulates cell growth, survival, migration, and adhesion in many cell types. S1P lyase is the enzyme that irreversibly cleaves S1P and thereby constitutes the ultimate step in sphingolipid catabolism. It has been reported previously that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1(-/-)-MEFs) are resistant to chemotherapy-induced apoptosis through upregulation of B cell lymphoma 2 (Bcl-2) and Bcl-2-like 1 (Bcl-xL). Here, we demonstrate that the transporter proteins Abcc1/MRP1, Abcb1/MDR1, Abca1, and spinster-2 are upregulated in Sgpl1(-/-)-MEFs. Furthermore, the cells efficiently sequestered the substrates of Abcc1 and Abcb1, fluo-4 and doxorubicin, in subcellular compartments. In line with this, Abcb1 was localized mainly at intracellular vesicular structures. After 16 h of incubation, wild-type MEFs had small apoptotic nuclei containing doxorubicin, whereas the nuclei of Sgpl1(-/-)-MEFs appeared unchanged and free of doxorubicin. A combined treatment with the inhibitors of Abcb1 and Abcc1, zosuquidar and MK571, respectively, reversed the compartmentalization of doxorubicin and rendered the cells sensitive to doxorubicin-induced apoptosis. It is concluded that upregulation of multidrug resistance transporters contributes to the chemoresistance of S1P lyase-deficient MEFs. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  15. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    Science.gov (United States)

    Chappelle, E. W.; Picciolo, G. L.

    1974-01-01

    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  16. ATP synthases from archaea: the beauty of a molecular motor.

    Science.gov (United States)

    Grüber, Gerhard; Manimekalai, Malathy Sony Subramanian; Mayer, Florian; Müller, Volker

    2014-06-01

    Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Ferric Citrate Hydrate as a Phosphate Binder and Risk of Aluminum Toxicity

    OpenAIRE

    Ajay Gupta

    2014-01-01

    Ferric citrate hydrate was recently approved in Japan as an oral phosphate binder to be taken with food for the control of hyperphosphatemia in patients with chronic kidney disease (CKD). The daily therapeutic dose is about 3 to 6 g, which comprises about 2 to 4 g of citrate. Oral citrate solubilizes aluminum that is present in food and drinking water, and opens the tight junctions in the intestinal epithelium, thereby increasing aluminum absorption and urinary excretion. In healthy animals d...

  18. Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937.

    Science.gov (United States)

    Lojkowska, E; Masclaux, C; Boccara, M; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1995-06-01

    Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes. We cloned the pelL gene, encoding one of these secondary pectate lyases of E. chrysanthemi 3937, from a genomic bank of a strain deleted of the five major pel genes. The nucleotide sequence of the region containing the pelL gene was determined. The pelL reading frame is 1275 bases long, corresponding to a protein of 425 amino acids including a typical amino-terminal signal sequence of 25 amino acids. Comparison of the amino acid sequences of PelL and the exo-pectate lyase PelX of E. chrysanthemi EC16 revealed a low homology, limited to 220 residues of the central part of the proteins. No homology was detected with other bacterial pectinolytic enzymes. Regulation of pelL transcription was analysed using gene fusion. As shown for the other pel genes, the transcription of pelL is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, temperature, iron starvation, osmolarity, anaerobiosis, nitrogen starvation and catabolite repression. Regulation of pelL expression appeared to be independent of the KdgR repressor, which controls all the steps of pectin catabolism. In contrast, the pecS gene, which is involved in regulation of the synthesis of the major pectate lyases and of cellulase, also appeared to be involved in pelL expression. The PelL protein is able to macerate plant tissue. This enzyme has a basic isoelectric point, presents an endo-cleaving activity on polygalacturonate or partially methylated pectin, with a basic pH optimum and an absolute requirement for Ca2+. The pelL mutant displayed a reduced virulence on potato tubers and Saintpaulia ionantha plants, demonstrating the important role of this enzyme in soft-rot disease.

  19. Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937.

    Science.gov (United States)

    Shevchik, V E; Kester, H C; Benen, J A; Visser, J; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1999-03-01

    Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by

  20. Citrate versus heparin anticoagulation in continuous renal replacement therapy in small children.

    Science.gov (United States)

    Raymakers-Janssen, Paulien A M A; Lilien, Marc; van Kessel, Ingrid A; Veldhoen, Esther S; Wösten-van Asperen, Roelie M; van Gestel, Josephus P J

    2017-10-01

    Citrate is preferred over heparin as an anticoagulant in adult continuous renal replacement therapy (CRRT). However, its potential adverse effects and data on use in CRRT in infants and toddlers is limited. We conducted a prospective study on using citrate in CRRT in critically ill small children. Children who underwent CRRT with the smallest filter in our PICU between November 2011 and November 2016 were included. Both heparin and citrate were applied according to a strict protocol. Our primary outcome was circuit survival time. Secondary outcomes were alkalosis, citrate toxicity, and number of red blood cell transfusions. Heparin was used in six patients (121 circuits, total CRRT time 3723 h). Citrate was used in 14 patients (105 circuits, total CRRT time 4530 h). Median circuit survival time with heparin was 21 h (IQR 14.5-27.5) compared to 45.2 h (IQR 37.5-52.8) with citrate (p CRRT regimes. In the heparin group, a median of 6.5 units of red blood cells (IQR 1.5-23.8) were given during CRRT, compared to three in the citrate group (IQR 2.0-5.0, p = 0.12). Use of regional citrate significantly prolongs circuit survival time and thereby should increase CRRT efficiency when compared to heparin. In addition, citrate appears safe for CRRT in critically ill small children.

  1. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations.

    Directory of Open Access Journals (Sweden)

    Jennifer C Ewald

    Full Text Available Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET. We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.

  2. Photochemistry of iron citrates initiated by UV-VIS light

    Science.gov (United States)

    Corral Arroyo, Pablo; Dou, Jing; Alpert, Peter; Krieger, Ulrich; Ammann, Markus

    2017-04-01

    Aerosol aging refers to the multitude of physical and chemical transformation atmospheric particles undergo, which play an important role in the impact of aerosols on climate, air quality and health. Aging processes may be started by chromophores, which act as photocatalysts that induce the oxidation of non-absorbing molecules [1]. Iron (Fe(III)) carboxylate complexes absorb light below about 500 nm, which is followed by ligand to metal charge transfer (LMCT) resulting in the reduction of iron to Fe(II) and oxidation of the carboxylate ligands, a process that represents an important sink of organic acids in the troposphere [2]. Our goal is to investigate how these photochemical processes contribute to the change of chemical and physical properties of the aerosol particles. To achieve this scope, we carry out coated wall flow tube experiments, exposing films with iron citrate to UV light, which will give information about the radical and LVOC production (connecting the CWFT to a Chemiluminescent Detector or PTR-TOF-MS respectively). From extracting and analyzing the films after irradiation with UV light, we obtain a profile of low-volatility products evolving from the photochemistry of iron citrates. By Scanning Transmission X-Ray Microspectroscopy (STXM) we analyze changes in the C K-edge and Fe L-edge in particles loaded with iron citrate upon exposure to light and follow their chemical and structural evolution upon photochemical oxidation in situ to investigate the degradation kinetics under varying environmental conditions. [1] George G., Ammann M., D'Anna B., Donaldson D. J., Nizkorodov S. A., Heterogeneous photochemistry in the Atmosphere, Chem. Rev., 2015, 115 (10), pp 4218-4258 [2] Weller, C., Horn, S., and Herrmann, H.: Photolysis of Fe(III) carboxylate complexes: Fe(II) quantum yields and reaction mechanisms, Photochemistry and Photobiology A: Chemistry, 268, 24-36, 2013.

  3. Methodology of citrate-based biomaterial development and application

    Science.gov (United States)

    Tran, M. Richard

    Biomaterials play central roles in modern strategies of regenerative medicine and tissue engineering. Attempts to find tissue-engineered solutions to cure various injuries or diseases have led to an enormous increase in the number of polymeric biomaterials over the past decade. The breadth of new materials arises from the multiplicity of anatomical locations, cell types, and mode of application, which all place application-specific requirements on the biomaterial. Unfortunately, many of the currently available biodegradable polymers are limited in their versatility to meet the wide range of requirements for tissue engineering. Therefore, a methodology of biomaterial development, which is able to address a broad spectrum of requirements, would be beneficial to the biomaterial field. This work presents a methodology of citrate-based biomaterial design and application to meet the multifaceted needs of tissue engineering. We hypothesize that (1) citric acid, a non-toxic metabolic product of the body (Krebs Cycle), can be exploited as a universal multifunctional monomer and reacted with various diols to produce a new class of soft biodegradable elastomers with the flexibility to tune the material properties of the resulting material to meet a wide range of requirements; (2) the newly developed citrate-based polymers can be used as platform biomaterials for the design of novel tissue engineering scaffolding; and (3) microengineering approaches in the form thin scaffold sheets, microchannels, and a new porogen design can be used to generate complex cell-cell and cell-microenvironment interactions to mimic tissue complexity and architecture. To test these hypotheses, we first developed a methodology of citrate-based biomaterial development through the synthesis and characterization of a family of in situ crosslinkable and urethane-doped elastomers, which are synthesized using simple, cost-effective strategies and offer a variety methods to tailor the material properties to

  4. Citrate anticoagulation for continuous renal replacement therapy in small children

    OpenAIRE

    Soltysiak, Jolanta; Warzywoda, Alfred; Kociński, Bartłomiej; Ostalska-Nowicka, Danuta; Benedyk, Anna; Silska-Dittmar, Magdalena; Zachwieja, Jacek

    2013-01-01

    Background Regional citrate anticoagulation (RCA) is one of the methods used to prevent clotting in continuous renal replacement therapy (CRRT). The aim of this study was to describe the outcomes and complications of RCA-CRRT in comparison to heparin anticoagulation (HA)-CRRT in critically ill children. Methods This study was a retrospective review of 30 critically ill children (16 on RCA- and 14 on HA-CRRT) who underwent at least 24 h of CRRT. The mean body weight of the children was 8.69 ± ...

  5. Bilateral cerebral hemispheric infarction associated with sildenafil citrate (Viagra) use.

    Science.gov (United States)

    Kim, K-K; Kim, D G; Ku, Y H; Lee, Y J; Kim, W-C; Kim, O J; Kim, H S

    2008-03-01

    Sildenafil citrate (Viagra) is one of the frequently prescribed drugs for men with erectile dysfunction. We describe a 52-year-old man with bilateral middle cerebral artery (MCA) territory infarction after sildenafil use. He ingested 100 mg of sildenafil and about 1 h later, he complained of chest discomfort, palpitation and dizziness followed by mental obtundation, global aphasia and left hemiparesis. Brain magnetic resonance imaging documented acute bilateral hemispheric infarction, and cerebral angiography showed occluded bilateral MCA. Despite significant bilateral MCA stenosis and cerebral infarction, systemic hypotension persisted for a day. We presume that cerebral infarction was caused by cardioembolism with sildenafil use.

  6. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Gruenhagen, Jason Alan [Iowa State Univ., Ames, IA (United States)

    2003-01-01

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K+ and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol

  7. A taste for ATP: neurotransmission in taste buds

    Science.gov (United States)

    Kinnamon, Sue C.; Finger, Thomas E.

    2013-01-01

    Not only is ATP a ubiquitous source of energy but it is also used widely as an intercellular signal. For example, keratinocytes release ATP in response to numerous external stimuli including pressure, heat, and chemical insult. The released ATP activates purinergic receptors on nerve fibers to generate nociceptive signals. The importance of an ATP signal in epithelial-to-neuronal signaling is nowhere more evident than in the taste system. The receptor cells of taste buds release ATP in response to appropriate stimulation by tastants and the released ATP then activates P2X2 and P2X3 receptors on the taste nerves. Genetic ablation of the relevant P2X receptors leaves an animal without the ability to taste any primary taste quality. Of interest is that release of ATP by taste receptor cells occurs in a non-vesicular fashion, apparently via gated membrane channels. Further, in keeping with the crucial role of ATP as a neurotransmitter in this system, a subset of taste cells expresses a specific ectoATPase, NTPDase2, necessary to clear extracellular ATP which otherwise will desensitize the P2X receptors on the taste nerves. The unique utilization of ATP as a key neurotransmitter in the taste system may reflect the epithelial rather than neuronal origins of the receptor cells. PMID:24385952

  8. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

    Science.gov (United States)

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

    2013-01-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  9. Insertion Mutation in HMG-CoA Lyase Increases the Production Yield of MPA through Agrobacterium tumefaciens-Mediated Transformation.

    Science.gov (United States)

    Dong, Yuguo; Zhang, Jian; Xu, Rui; Lv, Xinxin; Wang, Lihua; Sun, Aiyou; Wei, Dongzhi

    2016-11-28

    Mycophenolic acid (MPA) is an antibiotic produced by Penicillium brevicompactum. MPA has antifungal, antineoplastic, and immunosuppressive functions, among others. β-Hydroxy-β-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the bypass metabolic pathway. The inhibitory activity of HMG-CoA lyase increases the MPA biosynthetic flux by reducing the generation of by-products. In this study, we cloned the P. brevicompactum HMG-CoA lyase gene using the thermal asymmetric interlaced polymerase chain reaction and gene walking technology. Agrobacterium tumefaciens-mediated transformation (ATMT) was used to insert a mutated HMG-CoA lyase gene into P. brevicompactum. Successful insertion of the HMG-CoA lyase gene was confirmed by hygromycin screening, PCR, Southern blot analysis, and enzyme content assay. The maximum MPA production by transformants was 2.94 g/l. This was 71% higher than wild-type ATCC 16024. Our results demonstrate that ATMT may be an alternative practical genetic tool for directional transformation of P. brevicompactum.

  10. Assessing the survival of MRC5 and a549 cell lines upon exposure to pyruvic Acid, sodium citrate and sodium bicarbonate - biomed 2013.

    Science.gov (United States)

    Farah, Ibrahim O; Lewis, Veshell L; Ayensu, Wellington K; Cameron, Joseph A

    2013-01-01

    Lung cancer is among the most prevalent and deadly cancers in United States. In general, cancer cells are known to exhibit higher rates of glycolysis in comparison to normal cells. In attempting to exploit this unique cancer-dependent ATP generation phenomenon, it was our hypothesis that upon exposure to organic inhibitors of glycolysis, cancer cells would not survive normally and that their growth and viability would be vastly decreased; essential glycolytic ATP production will be exhausted to the point of collapsing energy utilization. Furthermore, we hypothesize that no negative effect would be seen with exposures to organic inhibitors for normal lung cells. The human lung fibroblast MRC-5 and the human A549 alveolar epithelial cell lines were used as in vitro models of normal lung and lung cancers respectively. Using standard methods, both cell lines were maintained and exposed to pyruvic acid, sodium citrate and sodium bicarbonate reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging for parallel morphological displays of any changes in the course of their vitality and metabolic activities. Our results indicate that exposure of both cell lines to these organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Pyruvic acid, sodium citrate and sodium bicarbonate showed statistically significant (p<0.05) differential negative effects on the A549 cell line in comparison to its unexposed control as well as to their effects on the MRC-5 cell line, presenting a potential promise for their use as cancer biotherapeutics.

  11. Formulation, Characterization and Physicochemical Evaluation of Potassium Citrate Effervescent Tablets

    Directory of Open Access Journals (Sweden)

    Fatemeh Fattahi

    2013-02-01

    Full Text Available Purpose: The aim of this study was to design and formulation of potassium citrate effervescent tablet for reduction of calcium oxalate and urate kidney stones in patients suffering from kidney stones. Methods: In this study, 13 formulations were prepared from potassium citrate and effervescent base in different concentration. The flowability of powders and granules was studied. Then effervescent tablets were prepared by direct compression, fusion and wet granulation methods. The prepared tablets were evaluated for hardness, friability, effervescent time, pH, content uniformity. To amend taste of formulations, different flavoring agents were used and then panel test was done by using Latin Square method by 30 volunteers. Results: Formulations obtained from direct compression and fusion methods had good flow but low hardness. Wet granulation improves flowability and other physicochemical properties such as acceptable hardness, effervescence time ≤3 minutes, pH<6, friability < 1%, water percentage < 0.5% and accurate content uniformity. In panel test, both of combination flavors; (orange - lemon and (strawberry - raspberry had good acceptability. Conclusion: The prepared tablets by wet granulation method using PVP solution had more tablet hardness. It is a reproducible process and suitable to produce granules that are compressed into effervescent tablets due to larger agglomerates.

  12. Formulation, characterization and physicochemical evaluation of potassium citrate effervescent tablets.

    Science.gov (United States)

    Aslani, Abolfazl; Fattahi, Fatemeh

    2013-01-01

    The aim of this study was to design and formulation of potassium citrate effervescent tablet for reduction of calcium oxalate and urate kidney stones in patients suffering from kidney stones. In this study, 13 formulations were prepared from potassium citrate and effervescent base in different concentration. The flowability of powders and granules was studied. Then effervescent tablets were prepared by direct compression, fusion and wet granulation methods. The prepared tablets were evaluated for hardness, friability, effervescent time, pH, content uniformity. To amend taste of formulations, different flavoring agents were used and then panel test was done by using Latin Square method by 30 volunteers. Formulations obtained from direct compression and fusion methods had good flow but low hardness. Wet granulation improves flowability and other physicochemical properties such as acceptable hardness, effervescence time ≤3 minutes, pHtablets by wet granulation method using PVP solution had more tablet hardness. It is a reproducible process and suitable to produce granules that are compressed into effervescent tablets due to larger agglomerates.

  13. Evaluating the efficacy of citrate anticoagulation during CRRT in cardiac patients

    Directory of Open Access Journals (Sweden)

    А. М. Караськов

    2015-10-01

    Full Text Available Systemic anticoagulation during renal replacement therapy in cardiac patients increases the risk of postoperative complications. Citrate anticoagulation is a promising alternative. The objective of our study was to evaluate the efficacy of citrate anticoagulation and its influence on the parameters of hemostasis and complications.

  14. Bioavailability of zinc and phosphorus in calcareous soils as affected by citrate exudation

    NARCIS (Netherlands)

    Duffner, A.; Hoffland, E.; Temminghoff, E.J.M.

    2012-01-01

    Aims Zinc (Zn) and phosphorus (P) deficiency often occurs at the same time and limits crop production in many soils. It has been suggested that citrate root exudation is a response of plants to both deficiencies. We used white lupin (Lupinus albus L.) as a model plant to clarify if citrate exuded by

  15. Citrate versus heparin anticoagulation in continuous renal replacement therapy in small children

    NARCIS (Netherlands)

    Raymakers-Janssen, Paulien A M; Lilien, Marc; van Kessel, Ingrid A.; Veldhoen, Esther S.; Wösten-van Asperen, Roelie M.; van Gestel, Josephus P.J.

    2017-01-01

    Background: Citrate is preferred over heparin as an anticoagulant in adult continuous renal replacement therapy (CRRT). However, its potential adverse effects and data on use in CRRT in infants and toddlers is limited. We conducted a prospective study on using citrate in CRRT in critically ill small

  16. 76 FR 34044 - Citric Acid and Certain Citrate Salts From Canada: Final Results of Antidumping Duty...

    Science.gov (United States)

    2011-06-10

    ... tricalcium citrate tetrahydrate, which are intermediate products in the production of citric acid, sodium... International Trade Administration Citric Acid and Certain Citrate Salts From Canada: Final Results of... preliminary results of the first administrative review of the antidumping duty order on citric acid and...

  17. 78 FR 34648 - Citric Acid and Certain Citrate Salts: Preliminary Results of Countervailing Duty Administrative...

    Science.gov (United States)

    2013-06-10

    ... International Trade Administration Citric Acid and Certain Citrate Salts: Preliminary Results of Countervailing... review of the countervailing duty (CVD) order on citric acid and citrate salts from the People's Republic... (202) 482-1503. Scope of the Order The merchandise subject to the order is citric acid and certain...

  18. Effects of citrate on hexavalent chromium reduction by structural Fe(II) in nontronite.

    Science.gov (United States)

    Liu, Xiaolei; Dong, Hailiang; Yang, Xuewei; Kovarik, Libor; Chen, Yu; Zeng, Qiang

    2018-02-05

    Previous studies have shown that organic ligands could influence Cr(VI) reduction by aqueous Fe(2+) and pyrite. In this study, the effects of citrate on Cr(VI) reduction by structural Fe(II) in nontronite (NAu-2) were investigated at pH 6. Our results showed that the presence of citrate decreased the rate but increased the amount of Cr(VI) reduction. The decreased rate was likely due to competitive sorption of citrate and anionic dichromate (Cr2O7(-)) to NAu-2 surface sites, because sorption of dichromate appeared to be the first step for subsequent Cr(VI) reduction. The increased amount of Cr(VI) reduction was likely because citrate served as an additional electron donor to reduce Cr(VI) through ligand-metal electron transfer in the presence of soluble Fe(3+), which was possibly derived from dissolution of reduced NAu-2. Soluble Cr(III)-citrate complex was a possible form of reduced Cr(VI) when citrate was present. Without citrate, nanometer-sized Cr2O3 particles were the product of Cr(VI) reduction. Our study highlights the importance of citrate on Cr(VI) reduction and immobilization when iron-rich smectite is applied to treat Cr(VI) contaminant in organic carbon rich environments. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

    Directory of Open Access Journals (Sweden)

    Mathias J Gerl

    Full Text Available Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1 HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.

  20. Characterization of chondroitin sulfate lyase ABC from Bacteroides thetaiotaomicron WAL2926.

    Science.gov (United States)

    Shaya, David; Hahn, Bum-Soo; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong Shik; Cygler, Miroslaw

    2008-06-24

    Chondroitin sulfate ABC lyase (ChonABC) is an enzyme with broad specificity that depolymerizes via beta-elimination chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs). ChonABC eliminates the glycosidic bond of its GAG substrates on the nonreducing end of their uronic acid component. This lyase possesses the unusual ability to act on both epimers of uronic acid, either glucuronic acid present in CS or iduronic acid in DS. Recently, we cloned, purified, and determined the three-dimensional structure of a broad specificity chondroitin sulfate ABC lyase from Bacteroides thetaiotaomicron (BactnABC) and identified two sets of catalytic residues. Here, we report the detailed biochemical characterization of BactnABC together with extensive site-directed mutagenesis resulting in characterization of the previously identified active site residues. BactnABC's catalysis is stimulated by Ca(2+) and Mg(2+) cations, particularly against DS. It displays extremely low activity toward hyaluronic acid and no activity toward heparin/heparan sulfate. Degradation of CS and DS by BactnABC yields only disaccharide products, pointing to an exolytic mode of action. The kinetic evaluations of the active-site mutants indicate that CS and DS substrates bind in the same active site, which is accompanied by a conformational change bringing the two sets of active site residues together. Conservative replacements of key residues suggest that His345 plays the role of a general base, initiating the degradation by abstracting the C5 bound proton from DS substrates, whereas either Tyr461 or His454 perform the equivalent role for CS substrates. Tyr461 is proposed, as well, to serve as general acid, completing the degradation of both CS and DS by protonating the leaving group.

  1. Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

    Energy Technology Data Exchange (ETDEWEB)

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M. (Catholic Univ of Korea); (NUST); (McGill); (Nat); (Natural Products Res Inst, Korea)

    2008-03-19

    Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a {beta}-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: ({alpha}/{alpha}){sub 5} for CS (chondroitin lyases AC) and {beta}-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located {approx}12 {angstrom} from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

  2. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  3. MRT letter: Expression of ATP sensor protein in Caenorhabditis elegans.

    Science.gov (United States)

    Kishikawa, Jun-ichi; Fujikawa, Makoto; Imamura, Hiromi; Yasuda, Kayo; Noji, Hiroyuki; Ishii, Naoaki; Mitani, Shohei; Yokoyama, Ken

    2012-01-01

    Adenosine 5'-triphosphate (ATP) is the major energy currency and is involved in many biological processes. The ATP-monitoring system for cells in animals can be helpful to study the relationship between energy metabolism and biological processes. The fluorescent ATP biosensor ATeam (ATP indicator based on Epsilon subunit for Analytical Measurements), which has been reported to monitor ATP levels in cultured cells on the basis of fluorescence resonance energy transfer (FRET), was introduced into nematodes by microinjection and UV-irradiation method. To confirm whether ATeam functions as an ATP sensor in nematode cells, the authors measured FRET of ATeam in cells of transgenic nematode. The ATeam was expressed in target cells in nematode. In vulva cells, ATP levels in the cytosol were higher than those in mitochondria. ATeam also sensed ATP level change in cultured cells from the transgenic nematode. These experiments indicated that ATeam is available for detection of changes in ATP levels in nematode cells. Copyright © 2011 Wiley Periodicals, Inc.

  4. Pyruvate formate lyase (PFL) and PFL activating enzyme in the chytrid fungus Neocallimastix frontalis: a free-radical enzyme system conserved across divergent eukaryotic lineages.

    Science.gov (United States)

    Gelius-Dietrich, Gabriel; Henze, Katrin

    2004-01-01

    Fermentative formate production involves the activity of pyruvate formate lyase, an oxygen-sensitive enzyme that employs a glycyl radical in its reaction mechanism. While common among anaerobic prokaryotes, this enzyme has so far been found in only two distantly related eukaryotic lineages, anaerobic chytridiomycetes and chlorophytes. Sequence comparisons of homologues from the chytridiomycetes Piromyces and Neocallimastix, the chlorophyte Chlamydomonas, and numerous prokaryotes suggest a single, eubacterial origin of eukaryotic pyruvate formate lyases. Pyruvate formate lyase activating enzyme introduces the glycyl radical into the pyruvate formate lyase protein chain. We discovered this enzyme, which had not previously been reported from eukaryotes, in the same two eukaryotic lineages and show that it shares a similar evolutionary history to pyruvate formate lyase. Sequences with high homology to pyruvate formate lyase activating enzyme were identified in the genomes of the anaerobic protozoan parasites Trichomonas vaginalis, Entamoeba histolytica, and Giardia intestinalis. While the occurrence of pyruvate formate lyase activating enzyme together with pyruvate formate lyase in fungi and chlorophytes was to be expected, the target protein of a glycyl radical enzyme-activating enzyme in these protozoa remains to be identified.

  5. Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

    DEFF Research Database (Denmark)

    da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm

    2014-01-01

    Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 °C and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing...... acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects. © 2014 Springer-Verlag Berlin Heidelberg....

  6. Pectinolytic bacteria and their secreted pectate lyases: agents for the maceration and solubilization of phytomass for fuels production

    Energy Technology Data Exchange (ETDEWEB)

    Preston, J.F. III; Rice, J.D.; Chow, M.C. (Florida Univ., Gainesville, FL (United States). Dept. of Microbiology and Cell Science)

    1993-01-01

    The objectives of this research have been to identify the pectinolytic enzymes secreted by bacteria and apply these towards the enhanced maceration and solubilization of plant material, focusing on the pectate lyases secreted by the phytopathogenic strains of Erwinia chrysanthemi, the ruminant resident Lachnospira multiparus, and the wood digestor isolate, Clostridium populeti. An HPLC approach has been developed that permits the kinetic analysis of each enzyme with respect to the formation of individual products during the pectate depolymerization process. This approach has demonstrated that each of these organisms secretes a nonrandom trimer-generating pectate lyase with a combination of endolytic and exolytic depolymerizing mechanisms. Two different strains of E. chrysanthemi secrete a battery of pectate lyases that include random endolytic as well as nonrandom dimer - and nonrandom trimer-generating endolytic/exolytic mechanisms. (author)

  7. Biosynthesis of 15NL-phenylalanine by phenylalanine ammonia-lyase from Rhodotorula glutinis.

    Science.gov (United States)

    Wang, Wenya; Yue, Haiyan; Yuan, Qipeng; Wang, Wenchuan

    2009-02-01

    Catalyzed by phenylalanine ammonia-lyase from Rhodotorula glutinis, 2% trans-cinnamic acid and 0.5 mol/l (15NH4)2SO4 was bioconverted to 15NL-phenylalanine. The yield and the purity of 15NL-phenylalanine reached 71 and 99.3%, respectively. The results showed that 96% of 15N was labeled on the L-phenylalanine and 88% of (15NH4)2SO4 was recovered. The present paper provides a new and economic way for biosynthesis of 15NL-phenylalanine.

  8. Cinnamaldehyde inhibits phenylalanine ammonia-lyase and enzymatic browning of cut lettuce.

    Science.gov (United States)

    Fujita, Narumi; Tanaka, Eriko; Murata, Masatsune

    2006-03-01

    Stored cut lettuce gradually turns brown on the cut section after several days of storage, because cutting induces phenylalanine ammonia-lyase (PAL) activity, the biosynthesis of polyphenol is promoted, and the polyphenols are oxidized by polyphenol oxidase. In this study, we screened for inhibitors of PAL derived from fermented broths of microbes and from foods and found that a cinnamon extract definitely inhibited PLA of cut lettuce. An active component was isolated by chromatographic procedures and was identified as trans-cinnamaldehyde. Browning of cut lettuce immersed in a solution containing trans-cinnamaldehyde was definitely repressed.

  9. One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm

    Directory of Open Access Journals (Sweden)

    Parinaz Ghadam

    2017-05-01

    Full Text Available Objective(s: Pseudomonas aeruginosais a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated. Materials and Methods: The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR. Results: Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1. Conclusion: In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.

  10. Modelling the ATP production in mitochondria

    CERN Document Server

    Saa, Alberto

    2012-01-01

    We revisit here the mathematical model for ATP production in mitochondria introduced recently by Bertram, Pedersen, Luciani, and Sherman (BPLS) as a simplification of the more complete but intricate Magnus and Keizer's model. We correct some inaccuracies in the BPLS original approximations and then analyze some of the dynamical properties of the model. We infer from exhaustive numerical explorations that the enhanced BPLS equations have a unique attractor fixed point for physiologically acceptable ranges of mitochondrial variables and respiration inputs. We determine, in the stationary regime, the dependence of the mitochondrial variables on the respiration inputs, namely the cytosolic concentration of calcium ${\\rm Ca}_{\\rm c}$ and the substrate fructose 1,6-bisphosphate FBP. The same effect of calcium saturation reported for the original BPLS model is observed here. We find out, however, an interesting non-stationary effect: the inertia of the model tends to increase considerably for high concentrations of ...

  11. Reduced Cystathionine γ-Lyase and Increased miR-21 Expression Are Associated with Increased Vascular Resistance in Growth-Restricted Pregnancies

    Science.gov (United States)

    Cindrova-Davies, Tereza; Herrera, Emilio A.; Niu, Youguo; Kingdom, John; Giussani, Dino A.; Burton, Graham J.

    2013-01-01

    Increased vascular impedance in the fetoplacental circulation is associated with fetal hypoxia and growth restriction. We sought to investigate the role of hydrogen sulfide (H2S) in regulating vasomotor tone in the fetoplacental vasculature. H2S is produced endogenously by catalytic activity of cystathionine β-synthase and cystathionine γ-lyase (CSE). Immunohistochemical analysis localized CSE to smooth muscle cells encircling arteries in stem villi. Immunoreactivity was reduced in placentas from pregnancies with severe early-onset growth-restriction and preeclampsia displaying abnormal umbilical artery Doppler waveforms compared with preeclamptic placentas with normal waveforms and controls. These findings were confirmed at the protein and mRNA levels. MicroRNA-21, which negatively regulates CSE expression, was increased in placentas with abnormal Doppler waveforms. Exposure of villus explants to hypoxia-reoxygenation significantly reduced CSE protein and mRNA and increased microRNA-21 expression. No changes were observed in cystathionine β-synthase expression, immunolocalized principally to the trophoblast, in pathologic placentas or in vitro. Finally, perfusion of normal placentas with an H2S donor, after preconstriction with a thromboxane mimetic, resulted in dose-dependent vasorelaxation. Glibenclamide and NG-nitro-l-arginine methyl ester partially blocked the effect, indicating that H2S acts through ATP-sensitive K+ channels and nitric oxide synthesis. These results demonstrate that H2S is a powerful vasodilator of the placental vasculature and that expression of CSE is reduced in placentas associated with increased vascular resistance. PMID:23410520

  12. Properties of R-Citramalyl-Coenzyme A Lyase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus▿

    Science.gov (United States)

    Friedmann, Silke; Alber, Birgit E.; Fuchs, Georg

    2007-01-01

    The autotrophic CO2 fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO2 acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO2) to β-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/β-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO2 fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to β-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg2 or Mn2+ ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO2 fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the γ-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process. PMID:17259315

  13. Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    Science.gov (United States)

    Friedmann, Silke; Alber, Birgit E; Fuchs, Georg

    2007-04-01

    The autotrophic CO(2) fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO(2) acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO(2)) to beta-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/beta-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO(2) fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg(2) or Mn(2+) ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO(2) fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the gamma-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.

  14. Dynamics of the metal binding domains and regulation of the human copper transporters ATP7B and ATP7A.

    Science.gov (United States)

    Yu, Corey H; Dolgova, Natalia V; Dmitriev, Oleg Y

    2017-04-01

    Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  15. 76 FR 77206 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Final Results of...

    Science.gov (United States)

    2011-12-12

    ... intermediate products in the production of citric acid, sodium citrate, and potassium citrate. The scope of the... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China...'') has completed its administrative review of the countervailing duty (``CVD'') order on citric acid and...

  16. 77 FR 9891 - Citric Acid and Certain Citrate Salts from the People's Republic of China: Amended Final Results...

    Science.gov (United States)

    2012-02-21

    ... International Trade Administration Citric Acid and Certain Citrate Salts from the People's Republic of China... antidumping duty order on citric acid and certain citrate salts (``citric acid'') from the People's Republic... Act of 1930, as amended (``the Act''). \\1\\ See Citric Acid and Certain Citrate Salts from the People's...

  17. 76 FR 17835 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Extension of Time...

    Science.gov (United States)

    2011-03-31

    ... International Trade Administration A-570-937] Citric Acid and Certain Citrate Salts From the People's Republic... order on citric acid and certain citrate salts (``citric acid'') from the People's Republic of China.... See Citric Acid and Certain Citrate Salts from the People's Republic of China: Notice of Extension of...

  18. Presence of Fe3+ and Zn2+ promoted biotransformation of Cd-citrate complex and removal of metals from solutions.

    Science.gov (United States)

    Qian, Jun-Wei; Tao, Yong; Zhang, Wen-Jie; He, Xiao-Hong; Gao, Ping; Li, Da-Ping

    2013-12-15

    The promotion to Cd-citrate complex biotransformation via addition of Fe(3+) and Zn(2+) was investigated. Single Fe(III)- or Zn-citrate complex was completely degraded by Pseudomonas sp. MBR, Cd-citrate complex was not. In the Cd-citrate media with molar ratio of 1:2 and 1:3, pH increase obtained from the metabolism of excess citrate slightly promoted the biotransformation of Cd-citrate complex, Cd remained in solutions. The presence of Fe(3+) and Zn(2+) resulted in complete biotransformation of Cd-citrate complex in the 1:1:2 Fe:Cd:citrate and Zn:Cd:citrate and 1:1:1:3 Fe:Zn:Cd:citrate media. Alkaline pH obtained from biotransformation of metal-citrate complexes caused almost complete removal of metals (>98%) through precipitation and co-precipitation. Pseudomonas sp. MBR potentially could be used to treat wastewater containing mixed citrate complexes of Fe(III), Zn and Cd. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Effect of Nb on barium titanate prepared from citrate solutions

    Directory of Open Access Journals (Sweden)

    Stojanović Biljana D.

    2002-01-01

    Full Text Available The influence of the addition of dopants on the microstructure development and electrical properties of BaTiO3 doped with 0.2, 0.4, 0.6, 0.8 mol% of Nb and 0.01 mol% of Mn based compounds was studied. Doped barium titanate was prepared using the polymeric precursor method from citrate solutions. The powders calcined at 700°C for 4 hours were analysed by infrared (IR spectroscopy to verify the presence of carbonates, and by X-ray diffraction (XRD for phase formation. The phase composition, microstructure and dielectric properties show a strong dependence on the amount of added niobium.

  20. Use of Blemaren citrate formula in gout patients with nephrolithiasis

    Directory of Open Access Journals (Sweden)

    M S Eliseev

    2008-01-01

    Results. After completion of a course of Blemaren therapy, there was an 8% reduction in the mean serum UA levels, which correlated with an increase in its daily excretion (by an average of 20%. The highest increase in UA excretion was observed in 20 patients with baseline hypoex-cretion (<700 mg/day: from 226,3 (range 201,6-436,8 to 635,0 (range 272,2-705,6 mg/day (p = 0,01. UA excretion substantially unchanged in patients with normal uricosuria (>700 mg/day. Side effects that could cause the agent to be discontinued were absent. Conclusion. The Blemaren citrate formula used in gout patients with nephrolithiasis causes a significant increase in the renal excretion of UA (p = 0,01, normalizes its metabolic parameters, and shows a high safety, without worsening hepatic and renal functions and electrolyte metabolism.

  1. Usefulness of gallium-67 citrate scanning in testicular seminoma

    Energy Technology Data Exchange (ETDEWEB)

    Willan, B.D.; Penney, H.; Castor, W.R.; McGowan, D.G.

    1987-10-01

    An analysis of 77 consecutive patients with a histologic diagnosis of seminoma testis, assessed and treated at the Cross Cancer Institute between 1977 and 1982, is presented. Ga-67 citrate was first used in the assessment of patients with malignant testicular tumors in 1973. Following three years of study that supported the observation of the gallium-avid nature of seminoma, gallium scans became routine in the initial staging assessment and were used also when recurrence was suspected. From 1977 through 1982, 72 patients with biopsy-proven seminoma testis were assessed initially for extent of disease by Ga-67 scanning. Comparison with intravenous pyelography and bipedal lymphography was possible for accuracy of tumor assessment. The scan sensitivity was 83%, and the specificity was 95%. During the same period, gallium was studied in nonseminomatous testicular tumors but the results were disappointing and its use was discontinued. The gallium-avid nature of seminoma testis may be useful in determining the extent of disease.

  2. Internet-ordered viagra (sildenafil citrate) is rarely genuine.

    Science.gov (United States)

    Campbell, Neil; Clark, John P; Stecher, Vera J; Goldstein, Irwin

    2012-11-01

    Counterfeit medication is a growing problem. This study assessed the requirement for prescription, cost, origin, and content of medications sold via the Internet and purporting to be the phosphodiesterase type 5 inhibitor Viagra (sildenafil citrate). Pfizer monitored top search results for the query "buy Viagra" on the two leading Internet search engines in March 2011. Orders were placed from 22 unique Web sites claiming to sell Viagra manufactured by Pfizer. Tablets received were assessed for chemical composition. No Web site examined required a prescription for purchase or a health screening survey; 90% offered illegal "generic Viagra." Cost per tablet ranged from $3.28-$33.00. Shipment origins of purchases were Hong Kong (N = 11), the United States (N = 6), and the United Kingdom (N = 2) as well as Canada, China, and India (N = 1 each). Notably, the four Internet pharmacies claiming to be Canadian did not ship medication from a Canadian address. Of 22 sample tablets examined, 17 (77%) were counterfeit, 4 (18%) were authentic, and 1 (5%) was an illegal generic. Counterfeit tablets were analyzed for sildenafil citrate, the active pharmaceutical ingredient (API) of Viagra, and contents varied between 30% and 50% of the label claim. Counterfeits lacked product information leaflets, including appropriate safety warnings, and genuine Viagra formulations. Internet sites claiming to sell authentic Viagra shipped counterfeit medication 77% of the time; counterfeits usually came from non-U.S. addresses and had 30% to 50% of the labeled API claim. Caution is warranted when purchasing Viagra via the Internet. © 2012 International Society for Sexual Medicine.

  3. Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase.

    Science.gov (United States)

    Zhu, Yongqun; Li, Hongtao; Long, Chengzu; Hu, Liyan; Xu, Hao; Liu, Liping; Chen, She; Wang, Da-Cheng; Shao, Feng

    2007-12-14

    The OspF family of phosphothreonine lyase, including SpvC from Salmonella, irreversibly inactivates the dual-phosphorylated host MAPKs (pT-X-pY) through beta elimination. We determined crystal structures of SpvC and its complex with a phosphopeptide substrate. SpvC adopts a unique fold of alpha/beta type. The disordered N terminus harbors a canonical D motif for MAPK substrate docking. The enzyme-substrate complex structure indicates that recognition of the phosphotyrosine followed by insertion of the threonine phosphate into an arginine pocket places the phosphothreonine into the enzyme active site. This requires the conformational flexibility of pT-X-pY, which suggests that p38 (pT-G-pY) is likely the preferred physiological substrate. Structure-based biochemical and enzymatic analysis allows us to propose a general acid/base mechanism for beta elimination reaction catalyzed by the phosphothreonine lyase. The mechanism described here provides a structural understanding of MAPK inactivation by a family of pathogenic effectors conserved in plant and animal systems and may also open a new route for biological catalysis.

  4. Immunocytochemical Localization of Mandelonitrile Lyase in Mature Black Cherry (Prunus serotina Ehrh.) Seeds.

    Science.gov (United States)

    Wu, H C; Poulton, J E

    1991-08-01

    Mandelonitrile lyase (MDL, EC 4.1.2.10), which catalyzes the reversible dissociation of (R)-(+)-mandelonitrile to benzaldehyde and hydrogen cyanide, was purified to apparent homogeneity from mature black cherry (Prunus serotina Ehrh.) seeds by conventional protein purification techniques. This flavoprotein is monomeric with a subunit molecular mass of 57 kilodaltons. Glycoprotein character was shown by its binding to the affinity matrix concanavalin A-Sepharose 4B with subsequent elution by alpha-methyl-d-glucoside. Upon chemical deglycosylation by trifluoromethanesulfonic acid, the molecular mass was reduced to 50.9 kilodaltons. Two-dimensional gel analysis of deglycosylated MDL revealed the presence of several subunit isoforms of similar molecular mass but differing slightly in isoelectric point. Polyclonal antibodies were raised in New Zealand white rabbits against deglycosylated and untreated MDL. Antibody titers were determined by enzyme linked immunosorbent and dot immunobinding assays, while their specificities were assessed by Western immunoblot analysis. Antibodies raised against untreated lyase recognized several proteins in addition to MDL. In contrast, antisera raised against deglycosylated MDL were monospecific and were utilized for developmental and immunocytochemical localization studies. SDS-PAGE and immunoblotting analysis of seed proteins during fruit maturation showed that MDL first appeared in seeds shortly after cotyledons began development. In cotyledon cells of mature seeds, MDL was localized primarily in the cell wall with lesser amounts in the protein bodies, whereas in endosperm cells, this labeling pattern was reversed. N-terminal sequence data was gathered for future molecular approaches to the question of MDL microheterogeneity.

  5. Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

    Science.gov (United States)

    Zechel, David L.; Jochimsen, Bjarne

    2014-01-01

    SUMMARY After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses. PMID:24600043

  6. Expression and Bioinformatics Analysis of Pectate Lyase Gene from Bacillus subtilis521

    Science.gov (United States)

    Xiao, Jing; Lu, Fu-Ping; Li, Yu; Li, Jin-Ting

    In order to exploit new genetic resources, Pectate lyase(PEL) gene was amplified by PCR using the genome DNA from an alkaline Bacillus subtilis521. The PCR product was inserted into pET22b(+) vector. The recombinant plasmids were cloned in E.coli DH5α and then expressed in E.coli BL21. When cultured in the optimized medium, the positive clones E.coli BL21(pET22b(+)pel)showed intracellular pectate lyase activity of 90.0 U/mL. It was indicated that we had obtained the correct PEL gene. The pel has an open reading frame of 1263 nucleotides and codes for a product of 420 amino acids with a calculated molecular mass of 45.5 kD. Based on computer assisted analysis, a signal peptides and two conserved domains were revealed. The sequence analysis for PEL showed that it shares 26-82% homology with other strains in GenBank. In addition, the advanced structure of PEL were also predicted and analysed. This study will help to the experimental design of PEL fermentation and production purification and enzyme evolution.

  7. Biochemical and structural characterization of a novel bacterial manganese-dependent hydroxynitrile lyase.

    Science.gov (United States)

    Hajnal, Ivan; Lyskowski, Andrzej; Hanefeld, Ulf; Gruber, Karl; Schwab, Helmut; Steiner, Kerstin

    2013-11-01

    Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal-dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg(-1) at pH 6 with (R)-mandelonitrile, and synthesized (R)-mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether. © 2013 FEBS.

  8. Abundance and genetic diversity of microbial polygalacturonase and pectate lyase in the sheep rumen ecosystem.

    Directory of Open Access Journals (Sweden)

    Peng Yuan

    Full Text Available Efficient degradation of pectin in the rumen is necessary for plant-based feed utilization. The objective of this study was to characterize the diversity, abundance, and functions of pectinases from microorganisms in the sheep rumen.A total of 103 unique fragments of polygalacturonase (PF00295 and pectate lyase (PF00544 and PF09492 genes were retrieved from microbial DNA in the rumen of a Small Tail Han sheep, and 66% of the sequences of these fragments had low identities (<65% with known sequences. Phylogenetic tree building separated the PF00295, PF00544, and PF09492 sequences into five, three, and three clades, respectively. Cellulolytic and noncellulolytic Butyrivibrio, Prevotella, and Fibrobacter species were the major sources of the pectinases. The two most abundant pectate lyase genes were cloned, and their protein products, expressed in Escherichia coli, were characterized. Both enzymes probably act extracellularly as their nucleotide sequences contained signal sequences, and they had optimal activities at the ruminal physiological temperature and complementary pH-dependent activity profiles.This study reveals the specificity, diversity, and abundance of pectinases in the rumen ecosystem and provides two additional ruminal pectinases for potential industrial use under physiological conditions.

  9. Sugar-cane juice induces pectin lyase and polygalacturonase in Penicillium griseoroseum

    Directory of Open Access Journals (Sweden)

    Minussi Rosana Cristina

    1998-01-01

    Full Text Available The use of other inducers as substitutes for pectin was studied aiming to reduce the production costs of pectic enzymes. The effects of sugar-cane juice on the production of pectin lyase (PL and polygalacturonase (PG by Penicillium griseoroseum were investigated. The fungus was cultured in a mineral medium (pH 6.3 in a rotary shaker (150 rpm for 48 h at 25oC. Culture media were supplemented with yeast extract and sucrose or sugar-cane juice. Sugar-cane juice added singly to the medium promoted higher PL activity and mycelial dry weight when compared to pectin and the use of sugar-cane juice and yeast extract yielded levels of PG activity that were similar to those obtained with sucrose-yeast extract or pectin. The results indicated that, even at low concentrations, sugar-cane juice was capable of inducing pectin lyase and polygalacturonase with no cellulase activity in P. griseoroseum.

  10. Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus.

    Directory of Open Access Journals (Sweden)

    Klaudia Kovács

    Full Text Available The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24 of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD studies showed that RxPAL is associated with an extensive α-helical character (far UV CD and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia.

  11. Expression, purification and crystallization of l-methionine γ-lyase 2 from Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Dan [Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Yamagata, Wataru; Kamei, Kaeko [Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Nozaki, Tomoyoshi [Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Harada, Shigeharu, E-mail: harada@kit.ac.jp [Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    2006-10-01

    l-Methionine γ-lyase 2 from E. histolytica, a key enzyme in sulfur-containing amino-acid degradation in this protozoan parasite, has been crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 Å. The crystal diffracted to a resolution of 2.0 Å. The presence of a tetramer in the asymmetric unit (4 × 43.1 kDa) gives a Matthews coefficient of 2.2 Å{sup 3} Da{sup −1}. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  12. Novel alginate lyases from marine bacterium Alteromonas sp. strain H-4.

    Science.gov (United States)

    Sawabe, T; Ohtsuka, M; Ezura, Y

    1997-10-28

    A bacterium Alteromonas sp. strain H-4 isolated from Laminaria fronds produced extra- and intra-cellular alginate lyases and utilized alginate as its sole carbon source. An extracellular alginate lyase was purified from the culture supernatant of the strain and its substrate specificity was characterized. The estimated molecular mass of the enzyme was 32 kDa and the isoelectric point was 4.7. Both polyM and polyG block degrading activities were observed using the substrate-containing gel overlay technique after isoelectric focusing of the enzyme. By analyzing the reaction products from the polyM block, polyG block, MG random block and intact alginate, three major peaks containing unsaturated tri-uronide through octa-uronide were detected for each substrate. The results indicate that the enzyme of Alteromonas sp. H-4 can degrade both polyM and polyG blocks with a K(m) in mg/mL 20-times higher for the polyM block.

  13. Structural Basis for Streptogramin B Resistance in Staphylococcus aureus by Virginiamycin B Lyase

    Energy Technology Data Exchange (ETDEWEB)

    Korczynska,M.; Mukhtar, T.; Wright, G.; Berghuis, A.

    2007-01-01

    The streptogramin combination therapy of quinupristin-dalfopristin (Synercid) is used to treat infections caused by bacterial pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. However, the effectiveness of this therapy is being compromised because of an increased incidence of streptogramin resistance. One of the clinically observed mechanisms of resistance is enzymatic inactivation of the type B streptogramins, such as quinupristin, by a streptogramin B lyase, i.e., virginiamycin B lyase (Vgb). The enzyme catalyzes the linearization of the cyclic antibiotic via a cleavage that requires a divalent metal ion. Here, we present crystal structures of Vgb from S. aureus in its apoenzyme form and in complex with quinupristin and Mg{sup 2+} at 1.65- and 2.8-{angstrom} resolution, respectively. The fold of the enzyme is that of a seven-bladed {beta}-propeller, although the sequence reveals no similarity to other known members of this structural family. Quinupristin binds to a large depression on the surface of the enzyme, where it predominantly forms van der Waals interactions. Validated by site-directed mutagenesis studies, a reaction mechanism is proposed in which the initial abstraction of a proton is facilitated by a Mg{sup 2+}-linked conjugated system. Analysis of the Vgb-quinupristin structure and comparison with the complex between quinupristin and its natural target, the 50S ribosomal subunit, reveals features that can be exploited for developing streptogramins that are impervious to Vgb-mediated resistance.

  14. Characterization of a neutral pectin lyase produced by Oidiodendron echinulatum MTCC 1356 in solid state fermentation.

    Science.gov (United States)

    Yadav, Sangeeta; Dubey, Amit Kumar; Anand, Gautam; Yadav, Dinesh

    2012-12-01

    A neutral pectin lyase produced by a new fungal strain Oidiodendron echinulatum MTCC 1356 under solid state fermentation using wheat bran as agro waste has been studied. The enzyme was purified by ammonium sulphate precipitation (30-60%), DEAE anion exchange and Sephadex G-100 column chromatographies. The SDS-PAGE and native PAGE revealed two bands of sizes 42 and 47 kDa. The enzyme was purified 37 fold with specific activity of 4.5 U/mg and 2.25% yield. The K(m) and V(max) values determined using citrus pectin were 1.2 mg/ml and 0.36 IU/min respectively. The pH and temperature optima were pH 7.0 and 50 °C, respectively. The pH stability was around 5.0 for 24 h at 20 °C. The purified enzyme retained maximum activity for 30 min upto 50 °C. The activation energy for thermal denaturation of the purified enzyme was found to be 60.0 kJ/Mol. The effects of various metal ions and protein inhibitors on enzyme activity have revealed total inhibition of the enzyme activity in the presence of Ag(+) and Cu(+) and KMnO(4) at 1 mM. The neutral pectin lyase showed retting of Crotalaria juncea fibre in the presence of EDTA. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Purification and characterization of pectin lyase secreted by Aspergillus flavus MTCC 10938.

    Science.gov (United States)

    Yadav, S; Dubey, A K; Anand, G; Yadav, D

    2013-01-01

    An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the K(m) and k(cat) values of the enzyme lyase were obtained as 1.7 mg/mL and 66 s(-1), respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55 degrees C and it was completely stable up to 40 degrees C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres.

  16. Exon duplications in the ATP7A gene

    DEFF Research Database (Denmark)

    Mogensen, Mie; Skjørringe, Tina; Kodama, Hiroko

    2011-01-01

    BACKGROUND: Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene. ME...

  17. K ATP channels in pig and human intracranial arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Sørensen, Mette Aaskov; Strøbech, Lotte

    2008-01-01

    Clinical trials suggest that synthetic ATP-sensitive K(+) (K(ATP)) channel openers may cause headache and migraine by dilating cerebral and meningeal arteries. We studied the mRNA expression profile of K(ATP) channel subunits in the pig and human middle meningeal artery (MMA) and in the pig middle...... cerebral artery (MCA). We determined the order of potency of four K(ATP) channel openers when applied to isolated pig MMA and MCA, and we examined the potential inhibitory effects of the Kir6.1 subunit specific K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the isolated...... pig MMA and MCA. Using conventional RT-PCR, we detected the mRNA transcripts of the K(ATP) channel subunits Kir6.1 and SUR2B in all the examined pig and human intracranial arteries. Application of K(ATP) channel openers to isolated pig MMA and MCA in myographs caused a concentration...

  18. ATP release and purinergic signaling in NLRP3 inflammasome activation

    Directory of Open Access Journals (Sweden)

    Isabelle eCOUILLIN

    2013-01-01

    Full Text Available The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns. One step- or two step- models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers ATP release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP (eATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1β through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key proinflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

  19. Cellular ATP release in the lung and airway

    Directory of Open Access Journals (Sweden)

    Satoru Ito

    2016-11-01

    Full Text Available Adenosine triphosphate (ATP is a universal energy source synthesized by mitochondrial oxidative phosphorylation and cytosolic glycolysis and transported by the vesicular nucleotide transporter for storage in secretory vesicles. Extracellular ATP regulates physiological functions and homeostasis of the respiratory system and is associated with pathogenesis of respiratory diseases. Thus, modulation of ATP and purinergic signaling may be a novel therapeutic approach to pulmonary disease. ATP is released from alveolar epithelial cells, airway epithelial cells, airway smooth muscle cells, fibroblasts and endothelial cells in response to various chemical and mechanical stimuli. In addition to conductive pathways such as connexins and pannexins, vesicular exocytosis is involved in the mechanisms of ATP release from the cells. Imaging approaches enable us to visualize ATP release from not only cultured cells but also lung tissue ex vivo. Extracellular vesicles, exosomes and membrane-derived microvesicles, containing cytoplasmic proteins, mRNA and microRNA, represent important mediators of cell-to-cell communication and the intercellular microenvironment. However, it is not known whether extracellular vesicles contain ATP as an intercellular messenger. Future studies are necessary to elucidate the mechanisms of cellular ATP release and purinergic signaling in the respiratory system.

  20. Renal epithelial cells can release ATP by vesicular fusion

    Directory of Open Access Journals (Sweden)

    Randi G Bjaelde

    2013-09-01

    Full Text Available Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30, which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1 cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin reduced both the spontaneous and hypotonically (80%-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1 and vesicular transport (nocodazole. These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ∼90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50% or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8% and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

  1. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway.

    Science.gov (United States)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  2. The Role of ATP in the Regulation of NCAM Function

    DEFF Research Database (Denmark)

    Hübschmann, Martin; Skladchikova, Galina

    2008-01-01

    Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a dire...... shedding, possibly affecting the structural plasticity associated with learning and memory.......Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a direct...... ATP-NCAM interaction and discuss its functional implications. The ectodomain of NCAM contains the ATP binding Walker motif A and has intrinsic ATPase activity, which could modulate NCAM-dependent signaling processes. NCAM interacts directly with and signals through FGFR. The NCAM binding site to ATP...

  3. Time-limited hydrotubation combined with clomiphene citrate treatment for unexplained infertility.

    Science.gov (United States)

    Yapca, O E; Delibas, I B; Karaca, I; Kumtepe, Y; Kadanali, S

    2015-01-01

    To evaluate time-limited hydrotubation combined with clomiphene citrate as treatment for unexplained infertility. In this unblinded, randomized controlled trial of patients who had unexplained infertility, 40 patients were treated with time-limited hydrotubation (saline, 20 ml; flushed within 20 to 30 seconds) and clomphene citrate (total, 70 cycles) and 40 patients were treated with clomiphene citrate alone (total, 74 cycles). All women underwent an ovulation induction protocol with clomiphene citrate (100 mg/d orally for five days, from day 3 to day 7 of the cycle). Hydrotubation was performed after detection of the dominant follicle. There were 15 pregnancies in the 80 patients (19%) (total, 144 stimulated cycles; 10% pregnancies per cycle). The frequency of clinical pregnancy per cycle was significantly greater in patients who were treated with hydrotubation and clomiphene citrate (nine pregnancies per cycle [13%]) than those treated with clomiphene citrate alone (two pregnancies per cycle [3%]; odds ratio, 5.3; 95% confidence interval, 1.1 to 25.5; p ≤ 0.05). The frequency of pregnancy per patient (total, clinical, or chemical) was similar for the two treatment groups. The frequency of live birth or abortion per cycle or patient was similar between the two treatment groups. Time-limited hydrotubation and clomiphene citrate may increase the frequency of clinical pregnancy per cycle in women who have unexplained infertility.

  4. Dexamethasone Enhances ATP-Induced Inflammatory Responses in Endothelial Cells

    Science.gov (United States)

    Ding, Yi; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2010-01-01

    The purinergic nucleotide ATP is released from stressed cells and is implicated in vascular inflammation. Glucocorticoids are essential to stress responses and are used therapeutically, yet little information is available that describes the effects of glucocorticoids on ATP-induced inflammation. In a human microvascular endothelial cell line, extracellular ATP-induced interleukin (IL)-6 secretion in a dose- and time-dependent manner. When cells were pretreated with dexamethasone, a prototypic glucocorticoid, ATP-induced IL-6 production was enhanced in a time- and dose-dependent manner. Mifepristone, a glucocorticoid receptor antagonist, blocked these effects. ATP-induced IL-6 release was significantly inhibited by a phospholipase C inhibitor [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] (63.2 ± 3%, p dexamethasone induced mRNA expression of the purinergic P2Y2 receptor (P2Y2R) 1.8- ± 0.1-fold and, when stimulated with ATP, enhanced Ca2+ release and augmented IL-6 mRNA expression. Silencing of the P2Y2R by its small interfering RNA decreased ATP-induced IL-6 production by 81 ± 1% (p Dexamethasone enhanced the transcription rate of P2Y2R mRNA and induced a dose-related increase in the activity of the P2Y2R promoter. Furthermore, dexamethasone-enhanced ATP induction of adhesion molecule transcription and augmented the release of IL-8. Dexamethasone leads to an unanticipated enhancement of endothelial inflammatory mediator production by extracellular ATP via a P2Y2R-dependent mechanism. These data define a novel positive feedback loop of glucocorticoids and ATP-induced endothelial inflammation. PMID:20826566

  5. Expression of ATP7B in normal human liver

    Directory of Open Access Journals (Sweden)

    D Fanni

    2009-06-01

    Full Text Available ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.

  6. Nanomolar ambient ATP decelerates P2X3 receptor kinetics.

    Science.gov (United States)

    Grote, Alexander; Hans, Michael; Boldogkoi, Zsolt; Zimmer, Andreas; Steinhäuser, Christian; Jabs, Ronald

    2008-12-01

    Homomeric P2X receptors differ in their electrophysiological and pharmacological profiles. The rapidly activating and desensitizing P2X3 receptors are known for their involvement in pain signalling pathways. Modulatory effects on P2X3 receptors have been reported for low concentrations of ATP ([ATP]). This includes both, enhancement and reduction of receptor currents. The first has been reported to be mediated by activation of ectoprotein kinases and high affinity desensitization (HAD), respectively. Both processes influence receptor current amplitudes. Here we describe a new phenomenon, the modulatory influence of ambient low [ATP] on P2X3 receptor kinetics. First, we studied in HEK cells whether persistent ATP affects current decay. To this end, P2X3 receptor mediated currents, elicited by pressure application of saturating [ATP], were analyzed after pre-application of low [ATP]. Second, UV-flash photolysis of ATP was employed to investigate whether submicromolar [ATP] affects receptor activation. Finally we confirmed the action of nanomolar [ATP] on native P2X3 receptors of neurons freshly isolated from rat dorsal root ganglia. We found that persistent low [ATP] caused pronounced deceleration of receptor current activation and decay. This priming effect indicates a mechanism different from HAD. It could be explained by a pre-opening receptor isomerization, induced by the occupation of a high affinity binding site already at the resting state. The observed modulation of the receptor kinetics could be considered as a physiological fine tuning mechanism of the nociceptive system, driven by the actual ambient agonist concentration.

  7. Combined oral administration of bovine collagen peptides with calcium citrate inhibits bone loss in ovariectomized rats.

    Directory of Open Access Journals (Sweden)

    JunLi Liu

    Full Text Available Collagen peptides (CPs and calcium citrate are commonly used as bone health supplements for treating osteoporosis. However, it remains unknown whether the combination of oral bovine CPs with calcium citrate is more effective than administration of either agent alone.Forty 12-week-old Sprague-Dawley rats were randomly divided into five groups (n = 8 for once-daily intragastric administration of different treatments for 3 months at 3 months after ovariectomy (OVX as follows: sham + vehicle; OVX + vehicle; OVX + 750 mg/kg CP; OVX + CP-calcium citrate (75 mg/kg; OVX + calcium citrate (75 mg/kg. After euthanasia, the femurs were removed and analyzed by dual energy X-ray absorptiometry and micro-computed tomography, and serum samples were analyzed for bone metabolic markers.OVX rats supplemented with CPs or CP-calcium citrate showed osteoprotective effects, with reductions in the OVX-induced decreases in their femoral bone mineral density. Moreover, CP-calcium citrate prevented trabecular bone loss, improved the microarchitecture of the distal femur, and significantly inhibited bone loss with increased bone volume, connectivity density, and trabecular number compared with OVX control rats. CP or CP-calcium citrate administration significantly increased serum procollagen type I N-terminal propeptide levels and reduced serum bone-specific alkaline phosphatase, osteocalcin, and C-telopeptide of type I collagen levels.Our data indicate that combined oral administration of bovine CPs with calcium citrate inhibits bone loss in OVX rats. The present findings suggest that combined oral administration of bovine CPs with calcium citrate is a promising alternative for reducing bone loss in osteopenic postmenopausal women.

  8. RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity

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    Elias Abdou

    2017-05-01

    Full Text Available For aerobic human pathogens, adaptation to hypoxia is a critical factor for the establishment of persistent infections, as oxygen availability is low inside the host. The two-component system RegB/A of Brucella suis plays a central role in the control of respiratory systems adapted to oxygen deficiency, and in persistence in vivo. Using an original “in vitro model of persistence” consisting in gradual oxygen depletion, we compared transcriptomes and proteomes of wild-type and ΔregA strains to identify the RegA-regulon potentially involved in the set-up of persistence. Consecutive to oxygen consumption resulting in growth arrest, 12% of the genes in B. suis were potentially controlled directly or indirectly by RegA, among which numerous transcriptional regulators were up-regulated. In contrast, genes or proteins involved in envelope biogenesis and in cellular division were repressed, suggesting a possible role for RegA in the set-up of a non-proliferative persistence state. Importantly, the greatest number of the RegA-repressed genes and proteins, including aceA encoding the functional IsoCitrate Lyase (ICL, were involved in energy production. A potential consequence of this RegA impact may be the slowing-down of the central metabolism as B. suis progressively enters into persistence. Moreover, ICL is an essential determinant of pathogenesis and long-term interactions with the host, as demonstrated by the strict dependence of B. suis on ICL activity for multiplication and persistence during in vivo infection. RegA regulates gene or protein expression of all functional groups, which is why RegA is a key regulator of B. suis in adaptation to oxygen depletion. This function may contribute to the constraint of bacterial growth, typical of chronic infection. Oxygen-dependent activation of two-component systems that control persistence regulons, shared by several aerobic human pathogens, has not been studied in Brucella sp. before. This work

  9. ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease.

    Science.gov (United States)

    Nakano, Masaki; Imamura, Hiromi; Sasaoka, Norio; Yamamoto, Masamichi; Uemura, Norihito; Shudo, Toshiyuki; Fuchigami, Tomohiro; Takahashi, Ryosuke; Kakizuka, Akira

    2017-08-01

    Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin.

    Science.gov (United States)

    Liu, Xiao-Yan; Chi, Zhe; Liu, Guang-Lei; Madzak, Catherine; Chi, Zhen-Ming

    2013-02-01

    In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.

  11. Continuous venovenous hemodialysis with regional citrate anticoagulation in patients with liver failure: a prospective observational study

    Science.gov (United States)

    2012-01-01

    Introduction Liver failure patients might be at risk for citrate accumulation during continuous venovenous hemodialysis (CVVHD) with regional citrate anticoagulation. The aim of this study was to investigate the predictive capability of baseline liver function parameters regarding citrate accumulation, expressed as an increase in the calcium total/calcium ionized (Catot/Caion) ratio ≥2.5, and to describe the feasibility of citrate CVVHD in liver failure patients. Methods We conducted a prospective observational study in medical ICU patients treated in a German university hospital. We performed 43 CVVHD runs using citrate for regional anticoagulation in 28 critically ill patients with decompensated liver cirrhosis or acute liver failure (maximum of two CVVHD runs per patient). Liver function was characterized before CVVHD using laboratory parameters, calculation of Child-Pugh and Model of End-stage Liver Disease scores, and determination of the plasma disappearance rate of indocyanine green. In addition to blood gas analysis, we measured total calcium and citrate in serum at baseline and after definitive time points for each CVVHD run. Results Accumulation of citrate in serum correlated with an increase in the Catot/Caion ratio. Although the critical upper threshold of Catot/Caion ratio ≥2.5 was exceeded 10 times in seven different CVVHD runs, equalization of initial metabolic acidosis was possible without major disturbances of acid-base and electrolyte status. Standard laboratory liver function parameters showed poor predictive capabilities regarding citrate accumulation in terms of an elevated Catot/Caion ratio ≥2.5. In contrast, serum lactate ≥3.4 mmol/l and prothrombin time ≤26% predicted an increase in the Catot/Caion ratio ≥2.5 with high sensitivity (86% for both lactate and prothrombin time) and specificity (86% for lactate, 92% for prothrombin time). Conclusions Despite substantial accumulation of citrate in serum, CVVHD with regional citrate

  12. [Dissolution of pancreatic calcifications with oral citrates in a woman with chronic idiopathic pancreatitis].

    Science.gov (United States)

    Uscanga, L; Galván Guerra, E; Robles-Díaz, G; Campuzano Fernández, M

    1992-01-01

    Pain control is one of the most important objectives in the medical treatment of patients with chronic calcifying pancreatitis (CCP). It is generally accepted that pain in CCP is secondary to the obstruction of the pancreatic duct by calcifications, protein plugs and stenotic areas. Experimental studies have shown that calculi and protein plugs are soluble in citrates. We report here the case of a young non alcoholic woman with idiopathic CCP who after a pancreatojejunostomy developed again pain and pancreatic calcifications which were dissolved with oral citrates. The case confirms previous observations and illustrates the potential use of citrates in subjects with CCP.

  13. Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

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    Ip Virginia

    2010-09-01

    Full Text Available Abstract Background ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg or drug vehicle twice weekly for 8 weeks. Results In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H. High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals. Conclusions In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non

  14. A Process-Based Model of TCA Cycle Functioning to Analyze Citrate Accumulation in Pre- and Post-Harvest Fruits.

    Directory of Open Access Journals (Sweden)

    Audrey Etienne

    Full Text Available Citrate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. The regulation of citrate accumulation throughout fruit development, and the origins of the phenotypic variability of the citrate concentration within fruit species remain to be clarified. In the present study, we developed a process-based model of citrate accumulation based on a simplified representation of the TCA cycle to predict citrate concentration in fruit pulp during the pre- and post-harvest stages. Banana fruit was taken as a reference because it has the particularity of having post-harvest ripening, during which citrate concentration undergoes substantial changes. The model was calibrated and validated on the two stages, using data sets from three contrasting cultivars in terms of citrate accumulation, and incorporated different fruit load, potassium supply, and harvest dates. The model predicted the pre and post-harvest dynamics of citrate concentration with fairly good accuracy for the three cultivars. The model suggested major differences in TCA cycle functioning among cultivars during post-harvest ripening of banana, and pointed to a potential role for NAD-malic enzyme and mitochondrial malate carriers in the genotypic variability of citrate concentration. The sensitivity of citrate accumulation to growth parameters and temperature differed among cultivars during post-harvest ripening. Finally, the model can be used as a conceptual basis to study citrate accumulation in fleshy fruits and may be a powerful tool to improve our understanding of fruit acidity.

  15. A Process-Based Model of TCA Cycle Functioning to Analyze Citrate Accumulation in Pre- and Post-Harvest Fruits.

    Science.gov (United States)

    Etienne, Audrey; Génard, Michel; Bugaud, Christophe

    2015-01-01

    Citrate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. The regulation of citrate accumulation throughout fruit development, and the origins of the phenotypic variability of the citrate concentration within fruit species remain to be clarified. In the present study, we developed a process-based model of citrate accumulation based on a simplified representation of the TCA cycle to predict citrate concentration in fruit pulp during the pre- and post-harvest stages. Banana fruit was taken as a reference because it has the particularity of having post-harvest ripening, during which citrate concentration undergoes substantial changes. The model was calibrated and validated on the two stages, using data sets from three contrasting cultivars in terms of citrate accumulation, and incorporated different fruit load, potassium supply, and harvest dates. The model predicted the pre and post-harvest dynamics of citrate concentration with fairly good accuracy for the three cultivars. The model suggested major differences in TCA cycle functioning among cultivars during post-harvest ripening of banana, and pointed to a potential role for NAD-malic enzyme and mitochondrial malate carriers in the genotypic variability of citrate concentration. The sensitivity of citrate accumulation to growth parameters and temperature differed among cultivars during post-harvest ripening. Finally, the model can be used as a conceptual basis to study citrate accumulation in fleshy fruits and may be a powerful tool to improve our understanding of fruit acidity.

  16. A randomized controlled trial of clomifene citrate, metformin, and pioglitazone versus letrozole, metformin, and pioglitazone for clomifene-citrate-resistant polycystic ovary syndrome.

    Science.gov (United States)

    El-khayat, Waleed; Abdel Moety, Ghada; Al Mohammady, Maged; Hamed, Dalia

    2016-02-01

    To examine the efficacy of clomifene citrate, metformin, and pioglitazone versus letrozole, metformin, and pioglitazone among women with polycystic ovary syndrome (PCOS) resistant to clomifene citrate. A prospective double-blind randomized controlled trial of women younger than 40 years who had primary/secondary infertility associated with PCOS and had not ovulated in response to clomifene citrate regimens previously was conducted at a center in Cairo, Egypt, between August 1, 2013, and December 31, 2014. Computer-generated random number tables and opaque envelopes were used to assign participants to group A or group B. Participants allocated to group A received 100mg clomifene citrate daily for 5 days from the third day of the menstrual cycle, whereas those in group B received 5mg letrozole daily in the same regimen. All patients received 850 mg metformin and 15 mg pioglitazone for 10 days from the first day of the menstrual cycle. The primary outcome was cumulative ovulation rate. Analyses were by intention to treat. Fifty women were assigned to each group. Ovulation occurred in 108 (92.3%) of 117 cycles in group A and 93 (86.9%) of 107 cycles in Group B (P=0.184). Combined treatment with letrozole, metformin, and pioglitazone was efficacious among women with PCOS resistant to clomifene citrate. ClinicalTrials.gov: NCT01909141. Copyright © 2015 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

  17. Molecular and Functional Analyses of the metC Gene of Lactococcus lactis, Encoding Cystathionine β-Lyase

    NARCIS (Netherlands)

    Fernández, María; Doesburg, Wim van; Rutten, Ger A.M.; Marugg, Joey D.; Alting, Arno C.; Kranenburg, Richard van; Kuipers, Oscar P.

    2000-01-01

    The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine β-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an α,γ elimination. With methionine as a substrate, it

  18. Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation

    Directory of Open Access Journals (Sweden)

    Danielle Biscaro Pedrolli

    2014-01-01

    Full Text Available A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking.

  19. Untreated Congenital Adrenal Hyperplasia with 17-α Hydroxylase/17,20-Lyase Deficiency Presenting as Massive Adrenocortical Tumor

    Science.gov (United States)

    Lee, Su Jin; Song, Je Eun; Hwang, Sena; Lee, Ji-Yeon; Park, Hye-Sun; Han, Seunghee

    2015-01-01

    Congenital adrenal hyperplasia (CAH) with 17α-hydroxylase/17,20-lyase deficiency is usually characterized by hypertension and primary amenorrhea, sexual infantilism in women, and pseudohermaphroditism in men. hypertension, and sexual infantilism in women and pseudohermaphroditism in men. In rare cases, a huge adrenal gland tumor can present as a clinical manifestation in untreated CAH. Adrenal cortical adenoma is an even more rare phenotype in CAH with 17α-hydroxylase/17,20-lyase deficiency. A 36-year-old female presented with hypertension and abdominal pain caused by a huge adrenal mass. Due to mass size and symptoms, left adrenalectomy was performed. After adrenalectomy, blood pressure remained high. Based on hormonal and genetic evaluation, the patient was diagnosed as CAH with 17α-hydroxylase/17,20-lyase deficiency. The possibility of a tumorous change in the adrenal gland due to untreated CAH should be considered. It is important that untreated CAH not be misdiagnosed as primary adrenal tumor as these conditions require different treatments. Adequate suppression of adrenocorticotropic hormone (ACTH) in CAH is also important to treat and to prevent the tumorous changes in the adrenal gland. Herein, we report a case of untreated CAH with 17α-hydroxylase/17,20-lyase deficiency presenting with large adrenal cortical adenoma and discuss the progression of adrenal gland hyperplasia due to inappropriate suppression of ACTH secretion. PMID:26248854

  20. Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp YM55-1

    NARCIS (Netherlands)

    Veetil, Vinod Puthan; Raj, Hans; Quax, Wim J.; Janssen, Dick B.; Poelarends, Gerrit J.

    Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of l-aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-aspartate to form an enzyme-stabilized enediolate intermediate.

  1. Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli.

    Science.gov (United States)

    Hounsa, C G; Aubry, J M; Dubourguier, H C; Hornez, J P

    1996-07-01

    The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2(4) factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 gl-1 to 1.9 gl-1 (dry weight) and from 10 units ml-1 to 210 units ml-1 within 24 h at 30 degrees C in shake flasks.

  2. The ketogenic diet is well tolerated and can be effective in patients with argininosuccinate lyase deficiency and refractory epilepsy

    NARCIS (Netherlands)

    Peuscher, Rosanne; Dijsselhof, Monique E.; Abeling, Nico G.; van Rijn, Margreet; van Spronsen, Francjan J.; Bosch, Annet M.

    2012-01-01

    Argininosuccinate lyase (ASL) deficiency (MIM 608310, McKusick 207900) is a rare disorder of the urea cycle, which leads to a deficiency of arginine and hyperammonemia. Epilepsy is a frequent complication of this disorder. A ketogenic diet (KD) can be a very effective therapy for refractory

  3. Structure of PhnP: a phosphodiesterase of the carbon-phosphorous lyase pathway for phosphonate degradation

    DEFF Research Database (Denmark)

    Podzelinska, Kateryna; He, Shu-Mei; Wathier, Matthew

    2009-01-01

    Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent ...

  4. Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Lolle, Signe; McSorley, Fern R.

    2011-01-01

    Organophosphonate utilization by Escherichia coli requires the 14 cistrons of the phnCDEFGHIJKLMNOP operon, of which the carbon-phosphorus lyase has been postulated to consist of the seven polypeptides specified by phnG to phnM. A 5,660-bp DNA fragment encompassing phnGHIJKLM is cloned, followed...

  5. Analysis of extracellular alginate lyase and its gene from a marine bacterial strain, Pseudoalteromonas atlantica AR06.

    Science.gov (United States)

    Matsushima, Ryoji; Danno, Hiroko; Uchida, Motoharu; Ishihara, Kenji; Suzuki, Toshiyuki; Kaneniwa, Masaki; Ohtsubo, Yoshiyuki; Nagata, Yuji; Tsuda, Masataka

    2010-03-01

    Pseudoalteromonas atlantica AR06 is a marine bacterial strain that can utilize alginate as a sole source of carbon and energy. The extracellular protein fraction prepared from the AR06 cultivation media exhibited alginate lyase activity to depolymerize the alginate molecules having homopolymeric and heteropolymeric forms of mannuronate and guluronate so as to mainly convert into the dimer to tetramer. A DNA fragment encoding a portion of alginate lyase was amplified from AR06 genomic DNA by PCR using a set of degenerated primers, and then the whole alginate lyase gene, named alyA, and its flanking regions were obtained from a cosmid library of AR06 genomic DNA. The alyA mutant of AR06 showed (1) the loss of alginate depolymerization activity on alginate agar plate and (2) significant growth defects in alginate minimal medium; these defects were complemented by the introduction of the alyA gene. Furthermore, zymography and biochemical analyses revealed that three extracellular protein bands of AR06 had alginate lyase activities and that all three protein bands were derived from the nascent alyA gene product. These results clearly indicated that the alyA gene greatly contributes to the assimilation of alginate in AR06. The transcription of the alyA gene was induced by the presence of alginate in minimal medium, but its obvious induction was not observed in rich medium even in the presence of alginate.

  6. A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

    KAUST Repository

    La, Honggui

    2011-09-06

    DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counter-act transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

  7. Activities of methionine-γ-lyase in the acidophilic archaeon “Ferroplasma acidarmanus” strain fer1

    Directory of Open Access Journals (Sweden)

    Khan MA

    2013-04-01

    Full Text Available M A Khan,1 Madeline M López-Muñoz,2 Charles W Kaspar,3 Kai F Hung1 1Department of Biological Sciences, Eastern Illinois University, Charleston, IL, USA; 2Department of Biology, Universidad de Puerto Rico, Mayaguez, Puerto Rico; 3Bacteriology Department, University of Wisconsin, Madison, WI, USA Abstract: Biogeochemical processes on exposed pyrite ores result in extremely high levels of sulfuric acid at these locations. Acidophiles that thrive in these conditions must overcome significant challenges, including an environment with proton concentrations at pH 3 or below. The role of sulfur metabolism in the archaeon “Ferroplasma acidarmanus” strain fer1's ability to thrive in this environment was investigated due to its growth-dependent production of methanethiol, a volatile organic sulfur compound. Two putative sequences for methionine-γ-lyase (EC 4.4.1.11, an enzyme known to carry out α, γ-elimination on L-methionine to produce methanethiol, were identified in fer1. Bioinformatic analyses identified a conserved pyridoxal-5'-phosphate (PLP binding domain and a partially conserved catalytic domain in both putative sequences. Detection of PLP-dependent and L-methionine-dependent production of α-keto compounds and thiol groups in fer1 confirmed the presence of methionine-γ-lyase activity. Further, fer1 lysate was capable of processing related substrates, including D-methionine, L-cysteine, L-cystathionine, and L/D-homocysteine. When the two putative fer1 methionine-γ-lyase gene-coded proteins were expressed in Escherichia coli cells, one sequence demonstrated an ability to carry out α, γ-elimination activity, while the other exhibited γ-replacement activity. These fer1 methionine-γ-lyases also exhibited optimum pH, substrate specificity, and catalytic preferences that are different from methionine-γ-lyases from other organisms. These differences are discussed in the context of molecular phylogeny constructed using a maximum

  8. The impact of citrate introduction at UK syringe exchange programmes: a retrospective cohort study in Cheshire and Merseyside, UK

    Directory of Open Access Journals (Sweden)

    Wareing Michelle

    2007-12-01

    Full Text Available Abstract Background In 2003, it became legal in the UK for syringe exchange programmes (SEPs to provide citrate to injecting drug users to solubilise heroin. Little work has been undertaken on the effect of policy change on SEP function. Here, we examine whether the introduction of citrate in Cheshire and Merseyside SEPs has altered the number of heroin/crack injectors accessing SEPs, the frequency at which heroin/crack injectors visited SEPs and the number of syringes dispensed. Methods Eleven SEPs in Cheshire and Merseyside commenced citrate provision in 2003. SEP-specific data for the six months before and six months after citrate was introduced were extracted from routine monitoring systems relating to heroin and crack injectors. Analyses compared all individuals attending pre and post citrate and matched analyses only those individuals attending in both periods (defined as 'longitudinal attenders'. Non-parametric tests were used throughout. Results Neither new (first seen in either six months period nor established clients visited SEPs more frequently post citrate. New clients collected significantly less syringes per visit post citrate, than pre citrate (14.5,10.0; z = 1.992, P Conclusion The introduction of citrate did not negatively affect SEP attendance. 'Longitudinal attenders' visited SEPs more frequently post citrate, providing staff with greater opportunity for intervention and referral. As the number of syringes they collected each visit remained unchanged the total number of clean syringes made available to this group of injectors increased very slightly between the pre and post citrate periods. However, new clients collected significantly less syringes post citrate than pre citrate, possibly due to staff concerns regarding the amount of citrate (and thus syringes to dispense safely to new clients. These concerns should not be allowed to negatively impact on the number of syringes dispensed.

  9. Active site proton delivery and the lyase activity of human CYP17A1

    Energy Technology Data Exchange (ETDEWEB)

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G., E-mail: s-sligar@illinois.edu

    2014-01-03

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b{sub 5} augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing

  10. Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW--A Mononuclear Iron-Dependent DMSP Lyase.

    Directory of Open Access Journals (Sweden)

    Adam E Brummett

    Full Text Available The osmolyte dimethylsulfoniopropionate (DMSP is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS, a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121. Measurements of metal binding affinity and catalytic activity indicate that Fe(II is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II per monomer. Electronic absorption and electron paramagnetic resonance (EPR studies show an interaction between NO and Fe(II-DddW, with NO binding to the EPR silent Fe(II site giving rise to an EPR active species (g = 4.29, 3.95, 2.00. The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.

  11. Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW—A Mononuclear Iron-Dependent DMSP Lyase

    Science.gov (United States)

    Brummett, Adam E.; Schnicker, Nicholas J.; Crider, Alexander; Todd, Jonathan D.; Dey, Mishtu

    2015-01-01

    The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW. PMID:25993446

  12. Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW--A Mononuclear Iron-Dependent DMSP Lyase.

    Science.gov (United States)

    Brummett, Adam E; Schnicker, Nicholas J; Crider, Alexander; Todd, Jonathan D; Dey, Mishtu

    2015-01-01

    The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.

  13. Structure-function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism.

    Science.gov (United States)

    Ulaganathan, ThirumalaiSelvi; Helbert, William; Kopel, Moran; Banin, Ehud; Cygler, Miroslaw

    2018-01-30

    Ulvan is a major cell wall component of green algae of the genus Ulva and some marine bacteria encode enzymes that can degrade this polysaccharide. The first ulvan degrading lyases have been recently characterized and several putative ulvan lyases have been recombinantly expressed, confirmed as ulvan lyases and partially characterized. Two families of ulvan degrading lyases, PL24 and PL25, have recently been established. The PL24 lyase LOR_107 from the bacterial Alteromonadales sp. strain LOR degrades ulvan endolytically, cleaving the bond at the C4 of a glucuronic acid. However, the mechanism and LOR_107 structural features involved are unknown. We present here the crystal structure of LOR_107, representing the first PL24 family structure. We found that LOR_107 adopts a seven-bladed β-propeller fold with a deep canyon on one side of the protein. Comparative sequence analysis revealed a cluster of conserved residues within this canyon, and site-directed mutagenesis disclosed several residues essential for catalysis. We also found that LOR_107 uses the His/Tyr catalytic mechanism, common to several PL families. We captured a tetrasaccharide substrate in the structures of two inactive mutants, which indicated a two-step binding event, with the first substrate interaction near the top of the canyon coordinated by Arg-320, followed by sliding of the substrate into the canyon toward the active-site residues. Surprisingly, the LOR_107 structure was very similar to that of PL25 family PLSV_3936, despite only ~14% sequence identity between the two enzymes. On the basis of our structural and mutational analyses, we propose a catalytic mechanism for LOR_107 that differs from the typical His/Tyr mechanism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  14. A discrete neutral transition-metal citrate cubane with an M₄O₄ core; coordinative versatility of the [M(II)₄(citrate)₄]⁸⁻ fragment.

    Science.gov (United States)

    Falvello, Larry R; Forcén-Vázquez, Elena; Palacio, Fernando; Sanz, Sergio; Tomás, Milagros

    2014-07-28

    The neutral cluster [Mn(II)8(citrate)4(H2O)18] is formed by the [M4(citrate)4](8-) fragment, with an Mn4O4 cubane core, which bonds four peripheral aquomanganese units--two [Mn(H2O)4](2+) and two [Mn(H2O)5](2+)--through a total of six metal-ligand bonds, giving a discrete neutral compound. The compound presents a unique coordination mode in which the citrate cubane acts as a chelate to each of the two peripheral [Mn(H2O)4](2+) (tetra-aquo) units. A detailed analysis of the central and peripheral geometries is given in terms of the tetrahedral distortions of key structural features. A reversible dehydration-rehydration process has been observed in a polycrystalline sample of the complex, whose structure lacks pores or channels.

  15. Tamoxifen citrate: a glimmer of hope for silicosis.

    Science.gov (United States)

    Yoldas, Omer; Karaca, Turgut; Bilgin, Bulent Caglar; Yilmaz, Omer Hinc; Simsek, Gulcin Guler; Alici, Ibrahim Onur; Uzdogan, Andaç; Karaca, Nihal; Akin, Tezcan; Yoldas, Suna; Akbiyik, Filiz

    2015-01-01

    Inhalation of crystalline silica nanoparticles causes pulmonary damage resulting in progressive lung fibrosis. Currently, there is no effective treatment for silicosis. Tamoxifen citrate is a selective estrogen receptor modulator, which is one of the adjuvant treatment choices for breast cancer. It is also known with its inhibitory effect on the production of transforming growth factor-beta (TGF-β) and studied for the anti-fibrotic effect in some fibrotic diseases. The aim of the study was to determine the effect of tamoxifen citrate on the prevention of pulmonary fibrosis and the treatment of silicosis. A total of 100 adult female Wistar Albino rats (200-250 g) were used in this study. The rats were divided into five groups including 20 rats in each. Rats were exposed to silica for 84 d in all groups. In group 1, rats were sacrificed on the day 84 without receiving treatment. In group 2, rats received 1 mg/kg tamoxifen (tmx1 + 1), from the first day of the study for the whole 114 d of the study. In group 3, (tmx10 + 10) rats were given 10 mg/kg tamoxifen from the first day of the study for the whole 114 d of the study. In group 4 (tmx1), rats were started 1 mg/kg of tamoxifen on day 84 and were given until day 114. In group 5 (tmx10), rats were fed with 10 mg/kg tamoxifen starting from day 84 to day 114. All rats except group 1 were sacrificed on 114 day of the study. Lung inflammation and fibrosis scores, serum TGF β levels, lung smooth muscle antigen and tissue transforming growth factor β (t-TGF-β) antibody staining levels, and number of silicotic rats were compared between groups. Silicosis was caused successfully in all rats in group 1. There were six silicotic rats in group 3 and it was the lowest number of all groups. Plasma TGF-ß levels and fibrosis score were significantly lower in all groups when compared with the control group. Tamoxifen could have preventive or treating effects in silicosis and found that lung fibrosis score was

  16. Closed circuit recovery of copper, lead and iron from electronic waste with citrate solutions.

    Science.gov (United States)

    Torres, Robinson; Lapidus, Gretchen T

    2017-02-01

    An integral closed circuit hydrometallurgical process is presented for base metal recovery from electronic waste. The leaching medium consists of a sodium citrate solution, from which base metals are retrieved by direct electrowinning, and the barren solution is recycled back to the leaching stage. This leaching-electrowinning cycle was repeated four times. The redox properties of the fresh citrate solution, as well as the leach liquors, were characterized by cyclic voltammetry to determine adequate conditions for metal reduction, as well as to limit citrate degradation. The leaching efficiency of electronic waste, employing the same solution after four complete cycles was 71, 83 and 94% for copper, iron and lead, respectively, compared to the original leach with fresh citrate solution. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Intranasal Microemulsion of Sildenafil Citrate: In Vitro Evaluation and In Vivo Pharmacokinetic Study in Rabbits

    National Research Council Canada - National Science Library

    Elshafeey, Ahmed H; Bendas, Ehab R; Mohamed, Osama H

    2009-01-01

    The purpose of the present study was to prepare intranasal delivery system of sildenafil citrate and estimate its relative bioavailability after nasal administration in rabbits to attain rapid onset...

  18. Electrodeposition and characterisation of Ni/Cu nanostructured multilayers from citrate solutions

    CERN Document Server

    Meuleman, W R A

    2002-01-01

    A study of the effect of chemical and electrochemical parameters such as solution composition, pH, and current and potential waveforms on magnetic metal multi-layers plated from citrate electrolytes was carried out. Until now, magnetic multilayers have usually been electrodeposited mainly form sulfamate electrolytes; far less information is available on Cu-Ni multilayers obtained from citrate electrolytes. Since copper is deposited at its diffusion limiting current during multilayer deposition from citrate electrolytes, a rotating disc electrode study was carried out. It was found that the apparent diffusion coefficient changes significantly depending on the citrate ion concentration and pH, indicating the importance of metal speciation. In order to identify the rate controlling species, speciation calculations were carried out in order to model the dependence of the limiting current on the solution composition. The model is based on the assumption that complexes in solution are either labile or inert. A vert...

  19. Atomic force microscopy reveals hydroxyapatite-citrate interfacial structure at the atomic level.

    Science.gov (United States)

    Jiang, Wenge; Pan, Haihua; Cai, Yurong; Tao, Jinhui; Liu, Peng; Xu, Xurong; Tang, Ruikang

    2008-11-04

    An approach to organic-inorganic interfacial structure at the atomic level is a great challenge in the studies of biomineralization. We demonstrate that atomic force microscopy (AFM) is powerful tool to discover the biomineral interface in detail. By using a model system of (100) hydroxyapatite (HAP) face and citrate, it reveals experimentally that only a side carboxylate and a surface calcium ion are involved in the binding effect during the citrate adsorption, which is against the previous understandings by using Langmuir adsorption and computer simulation. Furthermore, the adsorbed citrate molecules can use their free carboxylate and hydroxyl groups to be self-assembled on the HAP surface. AFM examination also finds that the presence of citrate molecules on the HAP crystal faces can enhance the adhesion force of the HAP surface. We suggest that the established AFM method can be used for a precise and direct understanding of biointerfaces at the atomic level.

  20. Effect of temperature on copper, iron and lead leaching from e-waste using citrate solutions.

    Science.gov (United States)

    Torres, Robinson; Segura-Bailón, Brenda; Lapidus, Gretchen T

    2018-01-01

    E-waste is a potential source of large quantities of metals. The ability of citrate solutions to recover base metals from these materials has been demonstrated. In the present study, the effect of the temperature on base metal leaching capacity by the citrate solutions is determined. The material employed consisted of a mechanically prepared, gravity concentrated e-waste, with a metallic content greater than 90%. The leaching conditions were selected based on previous research performed by the authors (0.5 M sodium citrate, pH 4.5 and 20 g per liter e-waste concentrate). Leaching tests were performed at temperatures between 0° and 70 °C. The initial leaching rates for the three metals increased with temperature. However, these tapered off with time for temperatures above 30 °C, which can be associated to citrate destruction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. DnaK protein alleviates toxicity induced by citrate-coated gold nanoparticles in Escherichia coli

    National Research Council Canada - National Science Library

    Makumire, Stanley; Revaprasadu, Neerish; Shonhai, Addmore

    2015-01-01

    ... (called DnaK in prokaryotes) is one of the most prominent molecular chaperones. Since gold nanoparticles exhibit chaperone-like function in vitro, we investigated the effect of citrate-coated gold nanoparticles on the growth of E...

  2. Citrate salts for preventing and treating calcium containing kidney stones in adults.

    Science.gov (United States)

    Phillips, Rebecca; Hanchanale, Vishwanath S; Myatt, Andy; Somani, Bhaskar; Nabi, Ghulam; Biyani, C Shekhar

    2015-10-06

    Kidney stones affect people worldwide and have a high rate of recurrence even with treatment. Recurrences are particularly prevalent in people with low urinary citrate levels. These people have a higher incidence of calcium phosphate and calcium oxalate stones. Oral citrate therapy increases the urinary citrate levels, which in turn binds with calcium and inhibits the crystallisation thus reduces stone formation. Despite the widespread use of oral citrate therapy for prevention and treatment of calcium oxalate stones, the evidence to support its clinical efficacy remains uncertain. The objective of this review was to determine the efficacy and adverse events associated with citrate salts for the treatment and prevention of calcium containing kidney stones. We searched the Cochrane Kidney and Transplant Specialised Register to 29 July 2015 through contact with the Trials' Search Co-ordinator using search terms relevant to this review. We included randomised controlled trials (RCTs) that assessed the efficacy and adverse events associated with citrate salts for the treatment and prevention of calcium containing kidney stones in adults treated for a minimum of six months. Two authors assessed studies for inclusion in this review. Data were extracted according to predetermined criteria. Summary estimates of effect were obtained using a random-effects model, and results were expressed as risk ratios (RR) and their 95% confidence intervals (CI) for dichotomous outcomes, and mean difference (MD) and 95% CI for continuous outcomes. We included seven studies that included a total of 477 participants, most of whom had oxalate stones. Of these, three studies (247 participants) compared potassium citrate with placebo or no intervention; three (166 participants) compared potassium-sodium citrate with no intervention; and one (64 participants) compared potassium-magnesium citrate with placebo. Overall, quality of the reporting of the included studies was considered moderate to

  3. Iron transport in Mycobacterium smegmatis: uptake of iron from ferric citrate

    Energy Technology Data Exchange (ETDEWEB)

    Messenger, A.J.M.; Ratledge, C.

    1982-01-01

    In mycobacterial growth medium 40 to 400 ..mu..M citrate was required to solubilize 2 ..mu..M /sup 55/Fe. This solubilized /sup 55/Fe was taken up into both iron-deficient and iron-sufficient washed cell suspensions of Mycobacterium smegmatis and Mycobacterium bovis BCG. Although the /sup 55/Fe was taken up into the cell, the citrate was not. The uptake system with M. smegmatis was not inhibited by electron transport inhibitors, uncouplers of oxidative phosphorylation, or thiol reagents and was saturable with iron at approximately 35 ..mu..M. The system was independent of the iron transport systems already known to exist in M. smegmatis: i.e., the two exochelin routes of assimilation as well as the mycobactin-salicylate system. It was not induced by the presence of 400 ..mu..M citrate in the growth medium, nor did the presence of citrate in the medium affect the production of either exochelin or mycobactin.

  4. Effect of sildenafil citrate (Viagra) on coronary flow in normal subjects.

    Science.gov (United States)

    Ishikura, Fuminobu; Beppu, Shintaro; Ueda, Hiroaki; Nehra, Ajay; Khandheria, Bijoy K

    2008-01-01

    The purpose of this study was to evaluate the effect of sildenafil citrate (Viagra) on coronary function in normal subjects. The study assessed mean blood pressure, left anterior descending coronary artery (LAD) flow, and echocardiographic variables before and 30 and 60 minutes after taking 50 mg of sildenafil citrate. The mean velocity of LAD flow was assessed with Doppler flow imaging. The study subjects were 6 healthy male volunteers (mean age 37 years). The mean velocity of LAD flow increased 60 minutes after taking sildenafil citrate, but there were no other changes. Two volunteers felt mild flashing and one had mild headache during the study. Sildenafil citrate caused vasodilatation in a normal coronary artery without systemic pressure drops. These results suggest that the agent itself did not have negative effects on the heart in normal subjects.

  5. Study on the Antimicrobial Properties of Citrate-Based Biodegradable Polymers

    OpenAIRE

    Lee-Chun eSu; Zhiwei eXie; Yi eZhang; Kytai Truong Nguyen; Jian eYang

    2014-01-01

    Citrate-based polymers possess unique advantages for various biomedical applications since citric acid is a natural metabolism product, which is biocompatible and antimicrobial. In polymer synthesis, citric acid also provides multiple functional groups to control the crosslinking of polymers and active binding sites for further conjugation of biomolecules. Our group recently developed a number of citrate-based polymers for various biomedical applications by taking advantage of their controlla...

  6. Citrate anticoagulation for CRRT: don't always trust the postfilter iCa results!

    Science.gov (United States)

    Oudemans-van Straaten, Heleen M; Ostermann, Marlies

    2015-12-04

    Citrate has been recommended as the first-line anticoagulant for continuous renal replacement therapy (CRRT) in critically ill patients. Compared with heparin, citrate anticoagulation is safer and more efficacious. Citrate inhibits the coagulation cascade by lowering the ionized calcium (iCa) concentration in the filter. Monitoring of systemic iCa concentrations is inherent to the protocol, and monitoring of postfilter iCa is recommended to adjust citrate flow and optimize anticoagulation. While systemic iCa targets are in the physiological range, postfilter iCa concentrations are targeted between 0.20 and 0.35 mmol/l. In a previous issue of Critical Care, Schwarzer et al. compared systemic and postfilter iCa measurements of patients receiving citrate-based CRRT between six devices. They highlight the unreliability of iCa concentrations in the postfilter range, because the instruments cannot be validated in the low iCa range. The maximum mean difference between two instruments was as high as 0.33 mmol/l (range 0.21-0.50 mmol/l). The authors call for dialysis companies to revise their protocols. However, the first implication of their study is that the accuracy of blood gas analyzers to measure iCa in the low range needs to improve; and, secondly, clinicians using citrate anticoagulation need to be aware that the postfilter iCa result may be falsely high or low. This is particularly relevant when frequent premature filter clotting is observed despite postfilter iCa results in the seemingly target range. In these situations, citrate flow can be safely increased up to 4 mmol/l blood flow under monitoring of signs of citrate accumulation.

  7. Testosterone restoration using enclomiphene citrate in men with secondary hypogonadism: a pharmacodynamic and pharmacokinetic study

    OpenAIRE

    Wiehle, Ronald; Cunningham, Glenn R; Pitteloud, Nelly; Wike, Jenny; Hsu, Kuang; Fontenot, Gregory K; Rosner, Michele; Dwyer, Andrew; Podolski, Joseph

    2013-01-01

    Objectives To determine the pharmacodynamic profile of serum total testosterone and luteinizing hormone (LH) levels in men with secondary hypogonadism after initial and chronic daily oral doses of enclomiphene citrate vs transdermal testosterone. To determine the effects of daily oral doses of enclomiphene citrate in comparison with transdermal testosterone on other hormones and markers in men with secondary hypogonadism. Patients and Methods This was a randomized, single-blind, two-centre, p...

  8. Comparison of Elaeagnus angustifolia Extract and Sildenafil Citrate on Female Orgasmic Disorders: A Randomized Clinical Trial

    OpenAIRE

    Akbarzadeh, Marzieh; Zeinalzadeh, Sanaz; Zolghadri, Jaleh; Mohagheghzadeh, Abdolali; Faridi, Pouya; Sayadi, Mehrab

    2014-01-01

    Background Orgasmic disorder can create a feeling of deprivation and failure and provide mental problems, incompatibility and marital discord. This study aimed to compare the effects of Elaeagnus angustifolia flower extract and sildenafil citrate on female orgasmic disorder in women in 2013. Methods In this randomized clinical trial, 125 women between 18-40 years old who suffered from orgasmic disorder were divided into three E. angustifolia, sildenafil citrate and control groups. The data we...

  9. Effects of citrate on hexavalent chromium reduction by structural Fe(II) in nontronite

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xiaolei; Dong, Hailiang; Yang, Xuewei; Kovarik, Libor; Chen, Yu; Zeng, Qiang

    2018-02-01

    Iron-bearing clay minerals and organic matter are two important components in natural environments that influence hexavalent chromium (Cr(VI)) reduction. Previous studies have shown that organic ligands could influence Cr(VI) reduction by aqueous Fe2+ and pyrite. However, the effects of organic ligands on Cr(VI) reduction by structural Fe(II) in clays are not well understood. In this study, the effects of citrate on Cr(VI) reduction by nontronite (NAu-2) were investigated under near neutral pH condition (pH=6). Our results showed that the presence of citrate decreased the rate but increased the amount of Cr(VI) reduction by structural Fe(II) in NAu-2. The decreased reaction rate was likely due to competitive sorption of citrate and polyanionic dichromate (Cr2O7- ), because sorption of dichromate appeared to be the first step for subsequent Cr(VI) reduction. The increased amount of Cr(VI) reduction in the presence of citrate was likely because citrate provided additional reducing power through ligand-metal electron transfer in the presence of soluble Fe 3+ derived from dissolution of reduced NAu-2. Soluble Cr(III)-citrate complex was the possible form of reduced chromium when citrate was present. In contrast, nanometer-sized Cr2O3 particles were the product of Cr(VI) reduction by reduced NAu-2 without citrate. Our study highlights the importance of organic ligands on Cr(VI) reduction and immobilization when iron-bearing clay minerals are applied to treat Cr(VI) contaminant in organic matter rich environments.

  10. The variability in DMSP content and DMSP lyase activity in marine dinoflagellates

    Science.gov (United States)

    Caruana, Amandine M. N.; Malin, Gill

    2014-01-01

    More than 20 years ago Maureen Keller and co-workers published a study that identified dinoflagellates as an important marine phytoplankton group with respect to the production of dimethylsulphoniopropionate (DMSP). Here, we present a synthesis and analysis of all the DMSP and DMSP lyase activity (DLA) measurements currently available for dinoflagellates. The data cover 110 species and strains and reveal over 6 orders of magnitude variability in intracellular DMSP concentrations and substantial variations in DLA in 23 strains. Inter-specific variability was explored with reference to a range of biological characteristics. The presence of a theca did not appear to be related to DMSP concentration but there was a potential relationship with toxicity (P = 0.06) and bioluminescent species produced significantly lower concentrations (P knowledge.

  11. The phosphothreonine lyase activity of a bacterial type III effector family.

    Science.gov (United States)

    Li, Hongtao; Xu, Hao; Zhou, Yan; Zhang, Jie; Long, Chengzu; Li, Shuqin; Chen, She; Zhou, Jian-Min; Shao, Feng

    2007-02-16

    Pathogenic bacteria use the type III secretion system to deliver effector proteins into host cells to modulate the host signaling pathways. In this study, the Shigella type III effector OspF was shown to inactivate mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinases 1 and 2 (Erk1/2), c-Jun N-terminal kinase, and p38]. OspF irreversibly removed phosphate groups from the phosphothreonine but not from the phosphotyrosine residue in the activation loop of MAPKs. Mass spectrometry revealed a mass loss of 98 daltons in p-Erk2, due to the abstraction of the alpha proton concomitant with cleavage of the C-OP bond in the phosphothreonine residue. This unexpected enzymatic activity, termed phosphothreonine lyase, appeared specific for MAPKs and was shared by other OspF family members.

  12. Stabilization of Phenylalanine Ammonia Lyase from Rhodotorula glutinis by Encapsulation in Polyethyleneimine-Mediated Biomimetic Silica.

    Science.gov (United States)

    Cui, Jiandong; Liang, Longhao; Han, Cong; Lin Liu, Rong

    2015-06-01

    Phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis was encapsulated within polyethyleneimine-mediated biomimetic silica. The main factors in the preparation of biomimetic silica were optimized by response surface methodology (RSM). Compared to free PAL (about 2 U), the encapsulated PAL retained more than 43 % of their initial activity after 1 h of incubation time at 60 °C, whereas free PAL lost most of activity in the same conditions. It was clearly indicated that the thermal stability of PAL was improved by encapsulation. Moreover, the encapsulated PAL exhibited the excellent stability of the enzyme against denaturants and storage stability, and pH stability was improved by encapsulation. Operational stability of 7 reaction cycles showed that the encapsulated PAL was stable. Nevertheless, the K m value of encapsulated PAL in biomimetic silica was higher than that of the free PAL due to lower total surface area and increased mass transfer resistance.

  13. Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis.

    Science.gov (United States)

    Zhu, Longbao; Cui, Wenjing; Fang, Yueqin; Liu, Yi; Gao, Xinxing; Zhou, Zhemin

    2013-05-01

    The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.

  14. Structure and Function of CutC Choline Lyase from Human Microbiota Bacterium Klebsiella pneumoniae.

    Science.gov (United States)

    Kalnins, Gints; Kuka, Janis; Grinberga, Solveiga; Makrecka-Kuka, Marina; Liepinsh, Edgars; Dambrova, Maija; Tars, Kaspars

    2015-08-28

    CutC choline trimethylamine-lyase is an anaerobic bacterial glycyl radical enzyme (GRE) that cleaves choline to produce trimethylamine (TMA) and acetaldehyde. In humans, TMA is produced exclusively by the intestinal microbiota, and its metabolite, trimethylamine oxide, has been associated with a higher risk of cardiovascular diseases. Therefore, information about the three-dimensional structures of TMA-producing enzymes is important for microbiota-targeted drug discovery. We have cloned, expressed, and purified the CutC GRE and the activating enzyme CutD from Klebsiella pneumoniae, a representative of the human microbiota. We have determined the first crystal structures of both the choline-bound and choline-free forms of CutC and have discovered that binding of choline at the ligand-binding site triggers conformational changes in the enzyme structure, a feature that has not been observed for any other characterized GRE. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Structure and Function of CutC Choline Lyase from Human Microbiota Bacterium Klebsiella pneumoniae*

    Science.gov (United States)

    Kalnins, Gints; Kuka, Janis; Grinberga, Solveiga; Makrecka-Kuka, Marina; Liepinsh, Edgars; Dambrova, Maija; Tars, Kaspars

    2015-01-01

    CutC choline trimethylamine-lyase is an anaerobic bacterial glycyl radical enzyme (GRE) that cleaves choline to produce trimethylamine (TMA) and acetaldehyde. In humans, TMA is produced exclusively by the intestinal microbiota, and its metabolite, trimethylamine oxide, has been associated with a higher risk of cardiovascular diseases. Therefore, information about the three-dimensional structures of TMA-producing enzymes is important for microbiota-targeted drug discovery. We have cloned, expressed, and purified the CutC GRE and the activating enzyme CutD from Klebsiella pneumoniae, a representative of the human microbiota. We have determined the first crystal structures of both the choline-bound and choline-free forms of CutC and have discovered that binding of choline at the ligand-binding site triggers conformational changes in the enzyme structure, a feature that has not been observed for any other characterized GRE. PMID:26187464

  16. Mammalian O-phosphorylethanolamine phospho-lyase activity and its inhibition.

    Science.gov (United States)

    Grøn, I H

    1978-04-01

    The activity of the enzyme O-phosphorylethanolamine phospho-lyase, metabolizing O-phosphorylethanolamine to acetaldehyde, orthophosphate, and ammonia in vitro, was studied in human liver biopsy and autopsy material, and leucocytes. Only in the liver biopsies enzyme activity towards O-phosphorylethanolamine could be found, and in amounts corresponding to one tenth of the activity found in rat liver examined under identical conditions. The enzyme activity of the liver biopsies was confined to the post-microsomal fraction, the activity amounting to 35 +/- 7 (SD) micromicron/mg protein. The results suggest the presence of an inhibiting factor of protein character. Inhibition was not due to competition from alkaline phosphatase (E.C. 3.1.3.1.) or O-phosphorylethanolamine cytidylyl-transferase (E.C. 2.7.7.14).

  17. Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

    Science.gov (United States)

    Lanfranchi, Elisa; Pavkov-Keller, Tea; Koehler, Eva-Maria; Diepold, Matthias; Steiner, Kerstin; Darnhofer, Barbara; Hartler, Jürgen; van den Bergh, Tom; Joosten, Henk-Jan; Gruber-Khadjawi, Mandana; Thallinger, Gerhard G.; Birner-Gruenberger, Ruth; Gruber, Karl; Winkler, Margit; Glieder, Anton

    2017-05-01

    Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

  18. Ethylene-controlled Induction of Phenylalanine Ammonia-lyase in Citrus Fruit Peel 1

    Science.gov (United States)

    Riov, J.; Monselise, S. P.; Kahan, R. S.

    1969-01-01

    l-Phenylalanine ammonia-lyase (PAL) activity is low in the external layers (flavedo) of intact mature grapefruit peel. Flavedo discs evince upon incubation increasing PAL activity and ethylene production. Light has no effect in enhancing PAL activity in discs. Exogenous ethylene stimulates PAL activity in the flavedo of intact mature grapefruits (half maximum stimulation at 15 ppm); such activity rapidly decreases when fruit is removed from the ethylene containing atmosphere. Carbon dioxide inhibits both ethylene production and PAL activity of discs; exogenous ethylene only partly relieves PAL inhibition. Cycloheximide inhibits both PAL activity and ethylene production by flavedo discs. The same concentration of cycloheximide also inhibits PAL activity of discs in the presence of exogenous ethylene. Protein synthesis seems therefore to be needed at both levels of ethylene evolution and enhancement of PAL activity. PMID:16657113

  19. Immunocytochemical Localization of Mandelonitrile Lyase in Mature Black Cherry (Prunus serotina Ehrh.) Seeds 1

    Science.gov (United States)

    Wu, Hua-Cheng; Poulton, Jonathan E.

    1991-01-01

    Mandelonitrile lyase (MDL, EC 4.1.2.10), which catalyzes the reversible dissociation of (R)-(+)-mandelonitrile to benzaldehyde and hydrogen cyanide, was purified to apparent homogeneity from mature black cherry (Prunus serotina Ehrh.) seeds by conventional protein purification techniques. This flavoprotein is monomeric with a subunit molecular mass of 57 kilodaltons. Glycoprotein character was shown by its binding to the affinity matrix concanavalin A-Sepharose 4B with subsequent elution by α-methyl-d-glucoside. Upon chemical deglycosylation by trifluoromethanesulfonic acid, the molecular mass was reduced to 50.9 kilodaltons. Two-dimensional gel analysis of deglycosylated MDL revealed the presence of several subunit isoforms of similar molecular mass but differing slightly in isoelectric point. Polyclonal antibodies were raised in New Zealand white rabbits against deglycosylated and untreated MDL. Antibody titers were determined by enzyme linked immunosorbent and dot immunobinding assays, while their specificities were assessed by Western immunoblot analysis. Antibodies raised against untreated lyase recognized several proteins in addition to MDL. In contrast, antisera raised against deglycosylated MDL were monospecific and were utilized for developmental and immunocytochemical localization studies. SDS-PAGE and immunoblotting analysis of seed proteins during fruit maturation showed that MDL first appeared in seeds shortly after cotyledons began development. In cotyledon cells of mature seeds, MDL was localized primarily in the cell wall with lesser amounts in the protein bodies, whereas in endosperm cells, this labeling pattern was reversed. N-terminal sequence data was gathered for future molecular approaches to the question of MDL microheterogeneity. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:16668338

  20. Lysine succinylation of Mycobacterium tuberculosis isocitrate lyase (ICL) fine-tunes the microbial resistance to antibiotics.

    Science.gov (United States)

    Zhou, Mingliang; Xie, Longxiang; Yang, Zhaozhen; Zhou, Jiahai; Xie, Jianping

    2017-04-01

    Lysine succinylation (Ksucc) is a newly identified protein posttranslational modification (PTM), which may play an important role in cellular physiology. However, the role of lysine succinylation in antibiotic resistance remains elusive. Isocitrate lyase (ICL) is crucial for broad-spectrum antibiotics tolerance in Mycobacterium tuberculosis (Mtb). We previously found that MtbICL (Rv0467) has at least three succinylated lysine residues, namely K189, K322, and K334.To explore the effect of succinylation on the activity of MtbICL, mutants' mimicry of the lysine succinylation were generated by site-directed mutagenesis. ICL-K189E mutant strain is more sensitive than the wild-type to rifampicin and streptomycin, but not isoniazid. For the in vitro activity of the purified isocitrate lyase, only K189E mutant showed significantly decreased activity. Crystal structure analysis showed that Lys189 Glu dramatically increased the pKa of Glu188 and decreased the pKa of Lys190, whereas had negligible effect on other residues within 5 Å as well as disruption of the electrostatic interaction between Lys189 and Glu182, which might prevent the closure of the active site loop and cause severe reduction of the enzyme activity. Considering the genetic, biochemical, and crystallographical evidences together, the succinylation of specific ICL residue can fine-tune the bacterial resistance to selected antibiotics. The decreased enzymatic activity resulting from the succinylation-changed electrostatic interaction might underlie this phenotype. This study provided the first insight into the link between lysine succinylation and antibiotic resistance.

  1. Clomiphene citrate treatment for late onset hypogonadism: rise and fall.

    Science.gov (United States)

    Marconi, Marcelo; Souper, Renato; Hartmann, Jonathan; Alvarez, Matías; Fuentes, Ignacio; Guarda, Francisco J

    2016-01-01

    Previous series have demonstrated that Clomiphene Citrate (CC) is an effective treatment to increase Total Testosterone (TT) in Late Onset Hypogonadism (LOH) patients. However, what happens to TT levels after ending CC treatment is still debatable. The objective of this study is to evaluate TT levels 3 months after the discontinuation of CC in patients with LOH who were previously successfully treated with the same drug. Twenty-seven patients with LOH that were successfully treated (achieved TT levels >11nmol/l) with CC 50mgs daily for 50 days were prospectively recruited in our Andrological outpatient clinic. CC was then stopped for 3 months and TT levels were measured at the end of this period. Mean TT level before discontinuation of CC was 22.7±8.1nmol/L (mean±SD). Three months after discontinuation, mean TT level significantly decreased in all pa¬tients, 10.2±3.9nmol/l (p < 0.01). Twenty-one patients (78%) decreased TT levels under 11nmol/L. Six patients (22%) had TT levels that remained within the normal recommended range (≥11nmol/l). No statistical significant differences were observed between both groups. In the short term LOH does not seem to be a reversible condition in most patients after CC treatment. More studies with longer follow-up are needed to evaluate the kinetics of TT in LOH. Copyright® by the International Brazilian Journal of Urology.

  2. Anticaries effect of dentifrices with calcium citrate and sodium trimetaphosphate

    Directory of Open Access Journals (Sweden)

    Alberto Carlos Botazzo Delbem

    2012-02-01

    Full Text Available Because of the growing concerns regarding fluoride ingestion by young children and dental fluorosis, it is necessary to develop new dentifrices. OBJECTIVE: The aim of this study was to evaluate the effect of dentifrices with calcium citrate (Cacit and sodium trimetaphosphate (TMP on enamel demineralization. MATERIAL AND METHODS: Enamel blocks (n=70, previously selected through surface hardness analysis, were submitted to daily treatment with dentifrices diluted in artificial saliva and to a pH-cycling model. The fluoride concentration in dentifrices was 0, 250, 450, 550, 1,000 and 1,100 µg F/g. CrestTM was used as a positive control (1,100 mg F/g. Cacit (0.25% and TMP (0.25% were added to dentifrices with 450 and 1,000 µg F/g. Surface hardness was measured again and integrated loss of subsurface hardness and fluoride concentration in enamel were calculated. Parametric and correlation tests were used to determine difference (p0.05. CONCLUSIONS: Dentifrices with 450 and 1,000 µg F/g, Cacit and TMP were as effective as a gold standard one.

  3. Pseudohypernatremia secondary to trisodium citrate (Citra-LockTM).

    Science.gov (United States)

    Milliere, Janice; Corriveau, Daryl; Parmar, Malvinder S

    2016-01-01

    Hypernatremia is common among hospitalized patients especially in the intensive care units and presents an independent risk factor for mortality. Mild hypernatremia is often asymptomatic but severe hypernatremia causes central nervous system dysfunction with initial non-specific symptoms of encephalopathy that may progress to seizures, coma and death, if left untreated. Severe hypernatremia is a medical emergency and requires emergent medical attention. A haemodialysis patient who arrived for his scheduled haemodialysis treatment had monthly blood work drawn and was reported to have severe hypernatremia with serum sodium concentration of 183 mmol/L. The possibility of technique or laboratory error was considered and systematically evaluated. The serum sodium measurement using another analyser showed similar value of 182 mmolL. A repeat serum sodium level on a sample drawn 2 h later showed normal value of 139-140 mmol/L. A step-wise evaluation of the complete procedure from blood collection to analysis of the sample revealed this to be spuriously elevated serum sodium concentration secondary to contamination of the sample during sample collection with trisodium citrate, a catheter-lock solution, commonly used in dialysis units to maintain patency of dialysis catheters. Spuriously elevated plasma sodium concentration (pseudohypernatremia) of mild degree is common but severe pseudohypernatremia is rare and the possibility of sample contaminations or laboratory error should be considered. Vigilance is required by both the medical and the laboratory staff to resolve such issues in a timely fashion to avoid unintended consequences.

  4. Preparation and Quality Control of 68Ga-Citrate for PET Applications

    Directory of Open Access Journals (Sweden)

    Ayuob Aghanejad

    2015-07-01

    Full Text Available Objective(s: In nuclear medicine studies, gallium-68 (68Ga citrate has been recently known as a suitable infection agent in positron emission tomography (PET. In this study, by applying an in-house produced 68Ge/68Ga generator, a simple technique for the synthesis and quality control of 68Ga-citrate was introduced; followed by preliminary animal studies. Methods: 68GaCl3 eluted from the generator was studied in terms of quality control factors including radiochemical purity (assessed by HPLC and RTLC, chemical purity (assessed by ICP-EOS, radionuclide purity (evaluated by HPGe, and breakthrough. 68Ga-citrate was prepared from eluted 68GaCl3 and sodium citrate under various reaction conditions. Stability of the complex was evaluated in human serum for 2 h at 370C, followed by biodistribution studies in rats for 120 min. Results: 68Ga-citrate was prepared with acceptable radiochemical purity (>97 ITLC and >98% HPLC, specific activity (4-6 GBq/mM, chemical purity (Sn, FeConclusion: This study demonstrated the possible in-house preparation and quality control of 68Ga-citrate, using a commercially available 68Ge/68Ga generator for PET imaging throughout the country.

  5. Transport of citrate across renal brush border membrane: effects of dietary acid and alkali loading

    Energy Technology Data Exchange (ETDEWEB)

    Jenkins, A.D.; Dousa, T.P.; Smith, L.H.

    1985-10-01

    Dietary acid or alkali loading was given to rats by providing 150 mM NH4Cl or 150 mM NaHCO3 in place of drinking water for 6 days; control animals received 150 mM NaCl. After 6 days, the citrate clearance was 0.04 +/- 0.01 ml/min (mean +/- SE) in the acid-loaded group, 0.9 +/- 0.1 ml/min in the control group, and 2.5 +/- 0.2 ml/min in the alkali-loaded group. At the end of the experiment, the rats were killed, and the Na gradient-dependent citrate uptake was measured in brush border membrane (BBM) vesicles prepared from each group. At 0.3 min, the ( UC)citrate uptake was 198 +/- 8 pmol/mg protein (mean +/- SE) in the acid-loaded group, 94 +/- 16 pmol/mg protein in the control group, and 94 +/- 13 pmol/mg protein in the alkali-loaded group. The rate of Na -independent (NaCl in medium replaced by KCl) ( UC)-citrate uptake by BBM vesicles was the same for acid-loaded, control, and alkali-loaded animals. Thus, the increased capacity of the proximal tubular BBM to transport citrate from the tubular lumen into the cell interior may be an important factor that contributes to decreased urinary citrate in the presence of metabolic acidosis induced by chronic dietary acid loading.

  6. Is it safe to prescribe clomiphene citrate without ultrasound monitoring facilities?

    LENUS (Irish Health Repository)

    Coughlan, C

    2010-05-01

    The majority of triplet and higher order multiple pregnancies now result from ovulation induction\\/superovulation rather than in vitro fertilisation. However, clomiphene citrate is still widely prescribed by gynaecologists and general practitioners who do not have access to ultrasound monitoring. The objective of our study was to determine the prevalence of multifollicular development with different doses of clomiphene citrate. A retrospective review of transvaginal ultrasound monitoring of 425 cycles in 182 women receiving clomiphene citrate from January 2002 to December 2003, was studied. Three or more follicles of >or= 14 mm were identified in 58 cycles (14%). Patients received 50 mg of clomiphene citrate in 52 of these 58 cycles and 25 mg in the remaining six. One patient was noted to have developed five follicles and 10 patients developed four follicles. One patient developed six follicles, despite receiving only 25 mg clomiphene citrate daily. It was concluded that a significant number of women (14%) developed three or more follicles, despite receiving low doses of clomiphene citrate.

  7. Preparation and Quality Control of (68)Ga-Citrate for PET Applications.

    Science.gov (United States)

    Aghanejad, Ayuob; Jalilian, Amir Reza; Ardaneh, Khosro; Bolourinovin, Fatemeh; Yousefnia, Hassan; Samani, Ali Bahrami

    2015-01-01

    In nuclear medicine studies, gallium-68 ((8)Ga) citrate has been recently known as a suitable infection agent in positron emission tomography (PET). In this study, by applying an in-house produced (68)Ge/(68)Ga generator, a simple technique for the synthesis and quality control of (68)Ga-citrate was introduced; followed by preliminary animal studies. (68)GaCl3 eluted from the generator was studied in terms of quality control factors including radiochemical purity (assessed by HPLC and RTLC), chemical purity (assessed by ICP-EOS), radionuclide purity (evaluated by HPGe), and breakthrough. (68)Ga-citrate was prepared from eluted (68)GaCl3 and sodium citrate under various reaction conditions. Stability of the complex was evaluated in human serum for 2 h at 370C, followed by biodistribution studies in rats for 120 min. (68)Ga-citrate was prepared with acceptable radiochemical purity (>97 ITLC and >98% HPLC), specific activity (4-6 GBq/mM), chemical purity (Sn, Fequality control of (68)Ga-citrate, using a commercially available (68)Ge/(68)Ga generator for PET imaging throughout the country.

  8. Spirocyclic character of ixazomib citrate revealed by comprehensive XRD, NMR and DFT study

    Science.gov (United States)

    Skorepova, Eliska; Čerňa, Igor; Vlasáková, Růžena; Zvoníček, Vít; Tkadlecová, Marcela; Dušek, Michal

    2017-11-01

    Ixazomib citrate is a very recently approved anti-cancer drug. Until now, to the best of our knowledge, no one has been able to solve any crystal structures of this compound. In this work, we present the crystal structures of two isostructural solvates of ixazomib citrate. In all currently available literature, the molecule is characterized as containing a single optically active carbon atom and a borate cycle formed when ixazomib is reacted with citric acid to form a stabilized ixazomib citrate that can be administered orally. However, the crystal structures revealed that none of the up-to-date presented structural formulas of ixazomib citrate are fully accurate. In addition to the citrate ring, another 5-membered ring is formed. These two rings are connected by the boron atom, making this compound a spirocyclic borate. By spirocyclization, the boron atom becomes tetrahedral and therefore optically active. In the crystal structures, ixazomib citrate was found to be in forms of two RR and RS stereoisomers. The results are supported by solid-state and solution NMR and DFT quantum mechanical calculations.

  9. Sildenafil citrate (Viagra) enhances vasodilatation by atrial natriuretic peptide in normal dogs.

    Science.gov (United States)

    Ishikura, Fuminobu; Beppu, Shintaro; Asanuma, Toshihiko; Seward, James B; Khandheria, Bijoy K

    2007-12-01

    Sildenafil citrate (Viagra) is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5, which might enhance the vasorelaxant and natriuretic actions of atrial natriuretic peptide (ANP) in patients with heart failure. The objective of this study was to examine the combined effect of Viagra on hemodynamic changes during infusion of exogenous ANP. Healthy male beagles were used to assess systemic blood pressure, pulmonary artery pressure (PAP), and plasma levels of cGMP. After hemodynamic variables were measured, 0.1 microg.kg(-1).min(-1) of ANP was given during this study. One hour after initiating infusion of ANP, 2 mg/kg of sildenafil citrate or vehicle was given orally via a nasogastric tube. Hemodynamic changes were measured before and 1 h after these administrations. Mean systemic and PAP decreased during infusion of ANP, and further decreased after sildenafil citrate administration, however, mean systemic blood pressure decreased within 10 mmHg. Plasma levels of cGMP also increased after sildenafil citrate administration. In normal dogs, sildenafil citrate enhances the vasodilator effect of ANP by increasing the cGMP level, however, the concomitant use of sildenafil citrate with ANP will not induce severe hypotension.

  10. Distinct neurological disorders with ATP1A3 mutations

    Science.gov (United States)

    Heinzen, Erin L.; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J.; Gurrieri, Fiorella; Goldstein, David B.; Jóhannesson, Sigurður H.; Mikati, Mohamad A.; Neville, Brian; Nicole, Sophie; Ozelius, Laurie J.; Poulsen, Hanne; Schyns, Tsveta; Sweadner, Kathleen J.; van den Maagdenberg, Arn; Vilsen, Bente

    2014-01-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na+/K+-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in vitro and animal model systems, and the role of Na+/K+-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  11. A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

    2017-11-20

    For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. FIBROBLAST CYTOSKELETAL REMODELING INDUCED BY TISSUE STRETCH INVOLVES ATP SIGNALING

    OpenAIRE

    Langevin, HM; Fujita, T.; Bouffard, NA; Takano, T; Koptiuch, C; Badger, GJ; Nedergaard, M

    2013-01-01

    Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 minutes, returning to baseline at 60 min...

  13. Journey in guidelines for lipid management: From adult treatment panel (ATP-I to ATP-III and what to expect in ATP-IV

    Directory of Open Access Journals (Sweden)

    P G Talwalkar

    2013-01-01

    Full Text Available Adult Treatment Panel (ATP, an expert panel to supervise cholesterol management was set up under the aegis of National Cholesterol Education Program (NCEP in 1985. Since then NCEP-ATP has been revising and framing guidelines to enable clinician to deliver better treatment to cardiovascular patients and to educate general people. As a result, considerable reduction in cardiovascular related deaths has been observed in recent times. All three ATP guidelines viz. ATP-I, ATP-II and ATP-III have targeted low density lipoprotein as their primary goal. The ATP-III guideline was updated in the light of evidences from 5-major clinical trials and was released in 2004. It added therapeutic lifestyle changes, concept of risk equivalents, Framingham CHD-risk score non-high density lipoprotein cholesterol (non-HDL-C as secondary target and gave strong emphasis on metabolic risk factors. The earlier treat-to-target paradigm faced fierce criticism from clinicians across the globe because of insufficient proof of safety and benefits of treating patients with respect to an individual′s low density lipoprotein (LDL level. Further, demonstration of non-HDL-C and total cholesterol/HDL-C ratio as strong predictors of overall cardiovascular risk foresees new guidelines. A tailored-treatment approach was suggested instead of LDL-C target based treatment approach which was soundly based on direct clinical trials evidences and proposes treatment based on individual′s overall 5- to 10-year cardiovascular risk irrespective of LDL-C level, leading to lower number of people on high dose/s of statins. Recent report of the Cholesterol Treatment Trialist′s Collaborators meta-analysis strongly supported primary prevention of LDL with statins in low risk individuals and showed that its benefits completely outweighed its known hazards. Markers other than LDL-C like apolipoprotein B, non-HDL-C and total cholesterol/HDL-C ratio would take precedence in the risk assessment and

  14. Journey in guidelines for lipid management: From adult treatment panel (ATP)-I to ATP-III and what to expect in ATP-IV.

    Science.gov (United States)

    Talwalkar, P G; Sreenivas, C G; Gulati, Ashish; Baxi, Hemang

    2013-07-01

    Adult Treatment Panel (ATP), an expert panel to supervise cholesterol management was set up under the aegis of National Cholesterol Education Program (NCEP) in 1985. Since then NCEP-ATP has been revising and framing guidelines to enable clinician to deliver better treatment to cardiovascular patients and to educate general people. As a result, considerable reduction in cardiovascular related deaths has been observed in recent times. All three ATP guidelines viz. ATP-I, ATP-II and ATP-III have targeted low density lipoprotein as their primary goal. The ATP-III guideline was updated in the light of evidences from 5-major clinical trials and was released in 2004. It added therapeutic lifestyle changes, concept of risk equivalents, Framingham CHD-risk score non-high density lipoprotein cholesterol (non-HDL-C) as secondary target and gave strong emphasis on metabolic risk factors. The earlier treat-to-target paradigm faced fierce criticism from clinicians across the globe because of insufficient proof of safety and benefits of treating patients with respect to an individual's low density lipoprotein (LDL) level. Further, demonstration of non-HDL-C and total cholesterol/HDL-C ratio as strong predictors of overall cardiovascular risk foresees new guidelines. A tailored-treatment approach was suggested instead of LDL-C target based treatment approach which was soundly based on direct clinical trials evidences and proposes treatment based on individual's overall 5- to 10-year cardiovascular risk irrespective of LDL-C level, leading to lower number of people on high dose/s of statins. Recent report of the Cholesterol Treatment Trialist's Collaborators meta-analysis strongly supported primary prevention of LDL with statins in low risk individuals and showed that its benefits completely outweighed its known hazards. Markers other than LDL-C like apolipoprotein B, non-HDL-C and total cholesterol/HDL-C ratio would take precedence in the risk assessment and strong emphasis would

  15. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    Science.gov (United States)

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  16. A COMPARISON OF CLOMIPHENE CITRATE AND SEQUENTIAL CLOMIPHENE CITRATE PLUS HUMAN MENOPAUSAL GONADOTROPIN FOR USE IN CONJUNCTION WITH INTRAUTERINE INSEMINATION

    Directory of Open Access Journals (Sweden)

    B. H. Rashidi

    2005-06-01

    Full Text Available There are currently ‎many different protocols in use for controlled ovarian hyperstimulation (COH, but the optimal method has ‎not yet been determined. To compare the outcome of COH using ‎clomiphene citrate (CC versus CC plus human menopausal gonadotropin (hMG in conjunction with intrauterine insemination (IUI, we studied 117 infertile couples‎. IUI with CC was used in 92 cycles ‎(group A and IUI with CC plus hMG was used in 66 cycles (group B. ‎Data analysis demonstrated no significant difference between the two ‎groups with respect to patients’ age, duration and type of infertility, prior COH and endometrial thickness and pattern. Group A had a little ‎longer follicular phase length than group B. ‎Pregnancy rate for group A and B were 6.52% and 12.12%, respectively (P= 0.22. ‎Endometrial pattern and thickness had no impact on pregnancy rate. ‎There were no multiple gestation and obvious hyperstimulation syndrome. ‎For patients undergoing controlled ovarian hyperstimulation with IUI, ‎CC plus hMG protocol yields higher pregnancy rate than one using CC, although this ‎difference was not statistically significant because of limitation of number of ‎cycles.

  17. An ATP synthase harboring an atypical γ-subunit is involved in ATP synthesis in tomato fruit chromoplasts

    DEFF Research Database (Denmark)

    Pateraki, Irini; Renato, Marta; Azcõn-Bieto, Joaquín

    2013-01-01

    Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active...... synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes...... the involvement of a plastidial ATP synthase harboring an atypical γ-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ-subunits. Silencing of this atypical γ-subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize...

  18. Transport of citrate-coated silver nanoparticles in unsaturated sand

    Energy Technology Data Exchange (ETDEWEB)

    Kumahor, Samuel K., E-mail: samuel.kumahor@ufz.de [Department of Soil Physics, Helmholtz Centre for Environmental Research–UFZ, Theodor-Lieser-Strasse 4, 06120 Halle-Saale (Germany); Hron, Pavel, E-mail: pavel.hron@iwr.uni-heidelberg.de [Interdisciplinary Center for Scientific Computing, University of Heidelberg, Im Neuenheimer Feld 368, Raum 422, 69120 Heidelberg (Germany); Metreveli, George, E-mail: metreveli@uni-landau.de [Universität Koblenz-Landau, Institute for Environmental Sciences, Group of Environmental and Soil Chemistry, Fortstr. 7, D-76829 Landau (Germany); Schaumann, Gabriele E., E-mail: schaumann@uni-landau.de [Universität Koblenz-Landau, Institute for Environmental Sciences, Group of Environmental and Soil Chemistry, Fortstr. 7, D-76829 Landau (Germany); Vogel, Hans-Jörg, E-mail: hans-joerg.vogel@ufz.de [Department of Soil Physics, Helmholtz Centre for Environmental Research–UFZ, Theodor-Lieser-Strasse 4, 06120 Halle-Saale (Germany); Institute of Soil Science and Plant Nutrition, Martin-Luther-University Halle-Wittenberg, Von-Seckendorff-Platz 3, 06120 Halle-Saale (Germany)

    2015-12-01

    Chemical factors and physical constraints lead to coupled effects during particle transport in unsaturated porous media. Studies on unsaturated transport as typical for soils are currently scarce. In unsaturated porous media, particle mobility is determined by the existence of an air–water interface in addition to a solid–water interface. To this end, we measured breakthrough curves and retention profiles of citrate-coated Ag nanoparticles in unsaturated sand at two pH values (5 and 9) and three different flow rates corresponding to different water contents with 1 mM KNO{sub 3} as background electrolyte. The classical DLVO theory suggests unfavorable deposition conditions at the air–water and solid–water interfaces. The breakthrough curves indicate modification in curve shapes and retardation of nanoparticles compared to inert solute. Retention profiles show sensitivity to flow rate and pH and this ranged from almost no retention for the highest flow rate at pH = 9 to almost complete retention for the lowest flow rate at pH = 5. Modeling of the breakthrough curves, thus, required coupling two parallel processes: a kinetically controlled attachment process far from equilibrium, responsible for the shape modification, and an equilibrium sorption, responsible for particle retardation. The non-equilibrium process and equilibrium sorption are suggested to relate to the solid–water and air–water interfaces, respectively. This is supported by the DLVO model extended for hydrophobic interactions which suggests reversible attachment, characterized by a secondary minimum (depth 3–5 kT) and a repulsive barrier at the air–water interface. In contrast, the solid–water interface is characterized by a significant repulsive barrier and the absence of a secondary minimum suggesting kinetically controlled and non-equilibrium interaction. This study provides new insights into particle transport in unsaturated porous media and offers a model concept representing the

  19. Assembly of citrate gold nanoparticles on hydrophilic monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Vikholm-Lundin, Inger, E-mail: inger.vikholm-lundin@uta.fi [University of Tampere, BioMediTech, Tampere (Finland); Fimlab Laboratories Ltd., Tampere (Finland); Rosqvist, Emil; Ihalainen, Petri [Abo Akademi University, Center for Functional Materials, Laboratory of Physical Chemistry (Finland); Munter, Tony [VTT Technical Research Centre of Finland, Process Chemistry end Environmental Engineering, Tampere (Finland); Honkimaa, Anni [University of Tampere, Department of Virology, School of Medicine, Tampere (Finland); Marjomäki, Varpu [University of Jyväskylä, Department of Biological and Environmental Science, Nanoscience Center, Jyväskylä (Finland); Albers, Willem M. [BioNavis Oy Ltd., Ylöjärvi, Tampere (Finland); Peltonen, Jouko [Abo Akademi University, Center for Functional Materials, Laboratory of Physical Chemistry (Finland)

    2016-08-15

    Highlights: • The self-assembled layers were all hydrophilic with Lipa-pTHMMAA exhibiting close to full wetting. • The polyacrylamide layers smoothen the gold surface to a higher extent than the polyethylene glycol and lipoic acid terminated with an amino group. • SPR resonance curves shift to higher angles and become increasingly damped when large nanoparticles assembled on the surface. • Topographical images confirmed that the highest number of particles were assembled on the polyethylene glycol monolayer. • By increasing the interaction time more particles could be assembled on the surface. - Abstract: Self-assembled monolayers (SAMs) as model surfaces were linked onto planar gold films thorough lipoic acid or disulfide groups. The molecules used were polyethylene glycol (EG-S-S), N-[tris-(hydroxymethyl)methyl]acrylamide polymers with and without lipoic acid (Lipa-pTHMMAA and pTHMMAA) and a lipoic acid triazine derivative (Lipa-MF). All the layers, but Lipa-MF with a primary amino group were hydroxyl terminated. The layers were characterized by contact angle measurements and atomic force microscopy, AFM. Citrate stabilized nanoparticles, AuNPs in water and phosphate buffer were allowed to assemble on the layers for 10 min and the binding was followed in real-time with surface plasmon resonance, SPR. The SPR resonance curves were observed to shift to higher angles and become increasingly damped, while also the peaks strongly broaden when large nanoparticles assembled on the surface. Both the angular shift and the damping of the curve was largest for nanoparticles assembling on the EG-S-S monolayer. High amounts of particles were also assembled on the pTHMMAA layer without the lipoic acid group, but the damping of the curve was considerably lower with a more even distribution of the particles. Topographical images confirmed that the highest number of particles were assembled on the polyethylene glycol monolayer. By increasing the interaction time more

  20. The effect of sildenafil citrate (Viagra) on visual sensitivity.

    Science.gov (United States)

    Stockman, Andrew; Sharpe, Lindsay T; Tufail, Adnan; Kell, Philip D; Ripamonti, Caterina; Jeffery, Glen

    2007-06-08

    The erectile dysfunction medicine sildenafil citrate (Viagra) inhibits phosphodiesterase type 6 (PDE6), an essential enzyme involved in the activation and modulation of the phototransduction cascade. Although Viagra might thus be expected to impair visual performance, reports of deficits following its ingestion have so far been largely inconclusive or anecdotal. Here, we adopt tests sensitive to the slowing of the visual response likely to result from the inhibition of PDE6. We measured temporal acuity (critical fusion frequency) and modulation sensitivity in four subjects before and after the ingestion of a 100-mg dose of Viagra under conditions chosen to isolate the responses of either their short-wavelength-sensitive (S-) cone photoreceptors or their long- and middle-wavelength-sensitive (L- and M-) cones. When vision was mediated by S-cones, all subjects exhibited some statistically significant losses in sensitivity, which varied from mild to moderate. The two individuals who showed the largest S-cone sensitivity losses also showed comparable losses when their vision was mediated by the L- and M-cones. Some of the losses appear to increase with frequency, which is broadly consistent with Viagra interfering with the ability of PDE6 to shorten the time over which the visual system integrates signals as the light level increases. However, others appear to represent a roughly frequency-independent attenuation of the visual signal, which might also be consistent with Viagra lengthening the integration time (because it has the effect of increasing the effectiveness of steady background lights), but such changes are also open to other interpretations. Even for the more affected observers, however, Viagra is unlikely to impair common visual tasks, except under conditions of reduced visibility when objects are already near visual threshold.

  1. Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis.

    Science.gov (United States)

    Marusich, W C; Jensen, R A; Zamir, L O

    1981-01-01

    Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than a complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-Phenylalanine will also induce lyase synthesis during exponential growth in minimal in which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared from cultures induced with doubly labeled (U-14C; ring-4-3H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate. PMID:7195398

  2. Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis

    Energy Technology Data Exchange (ETDEWEB)

    Marusich, W.C.; Jensen, R.A.; Zamir, L.O.

    1981-06-01

    Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than in complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-phenylalanine will also induce lyase synthesis during exponential growth in minimal medium in which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared fom cultures induced with doubly labeled (U-/sup 14/C; ring-4-/sup 3/H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate.

  3. Citrate-Permeable Channels in the Plasma Membrane of Cluster Roots from White Lupin1

    Science.gov (United States)

    Zhang, Wen-Hao; Ryan, Peter R.; Tyerman, Stephen D.

    2004-01-01

    White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (−P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3− than Cl− (PCit/PCl = 26.3), while ORAC was selective for Cl− over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of −P plants, as well as from normal (noncluster) roots of plants grown with 25 μm phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of −P plants. However, the IRAC current from +P plants occurred less frequently than in the −P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux. PMID:15516510

  4. Localization of the calcium-regulated citrate transport process in proximal tubule cells.

    Science.gov (United States)

    Hering-Smith, Kathleen S; Mao, Weibo; Schiro, Faith R; Coleman-Barnett, Joycelynn; Pajor, Ana M; Hamm, L Lee

    2014-06-01

    Urinary citrate is an important inhibitor of calcium-stone formation. Most of the citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport. The present studies address several fundamental properties of this novel process: the polarity of the transport process, the location of the calcium-sensitivity and whether NaDC1 is present in OK cells. OK cells grown on permeable supports exhibited apical >basolateral citrate transport. Apical transport of both citrate and succinate was sensitive to extracellular calcium whereas basolateral transport was not. Apical calcium, rather than basolateral, was the predominant determinant of changes in transport. Also 2,3-dimethylsuccinate, previously identified as an inhibitor of basolateral dicarboxylate transport, inhibited apical citrate uptake. Although the calcium-sensitive transport process in OK cells is functionally not typical NaDC1, NaDC1 is present in OK cells by Western blot and PCR. By immunolocalization studies, NaDC1 was predominantly located in discrete apical membrane or subapical areas. However, by biotinylation, apical NaDC1 decreases in the apical membrane with lowering calcium. In sum, OK cells express a calcium-sensitive/regulated dicarboxylate process at the apical membrane which responds to variations in apical calcium. Despite the functional differences of this process compared to NaDC1, NaDC1 is present in these cells, but predominantly in subapical vesicles.

  5. Secreted Citrate Serves as Iron Carrier for the Marine Pathogen Photobacterium damselae subsp damselae

    Directory of Open Access Journals (Sweden)

    Miguel Balado

    2017-08-01

    Full Text Available Photobacterium damselae subsp damselae (Pdd is a Vibrionaceae that has a wide pathogenic potential against many marine animals and also against humans. Some strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there are virulent strains that do not produce vibrioferrin, but they still give a strong positive reaction in the CAS test for siderophore production. In an in silico search on the genome sequences of this type of strains we could not find any ORF which could be related to a siderophore system. To identify genes that could encode a siderophore-mediated iron acquisition system we used a mini-Tn10 transposon random mutagenesis approach. From more than 1,400 mutants examined, we could isolate a mutant (BP53 that showed a strong CAS reaction independently of the iron levels of the medium. In this mutant the transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase that participates in the tricarboxylic acid cycle. The mutant did not show any growth impairment in rich or minimal media, but it accumulated a noticeable amount of citrate (around 7 mM in the culture medium, irrespective of the iron levels. The parental strain accumulated citrate, but in an iron-regulated fashion, being citrate levels 5–6 times higher under iron restricted conditions. In addition, a null mutant deficient in citrate synthase showed an impairment for growth at high concentrations of iron chelators, and showed almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the iron-restricted culture supernatants resulted in a CAS-positive fraction with biological activity as siderophore. HPLC purification of that fraction yielded a pure compound which was identified as citrate from its MS and NMR spectral data. Although the production of another citrate-based compound with siderophore activity cannot be ruled out, our results suggest that Pdd secretes endogenous citrate and use it for

  6. Effects and mechanisms of action of sildenafil citrate in human chorionic arteries

    Directory of Open Access Journals (Sweden)

    Lynch Tadhg

    2009-04-01

    Full Text Available Abstract Objectives Sildenafil citrate, a specific phosphodiesterase-5 inhibitor, is increasingly used for pulmonary hypertension in pregnancy. Sildenafil is also emerging as a potential candidate for the treatment of intra-uterine growth retardation and for premature labor. Its effects in the feto-placental circulation are not known. Our objectives were to determine whether phosphodiesterase-5 is present in the human feto-placental circulation, and to characterize the effects and mechanisms of action of sildenafil citrate in this circulation. Study Design Ex vivo human chorionic plate arterial rings were used in all experiments. The presence of phosphodiesterase-5 in the feto-placental circulation was determined by western blotting and immunohistochemical staining. In a subsequent series of pharmacologic studies, the effects of sildenafil citrate in pre-constricted chorionic plate arterial rings were determined. Additional studies examined the role of cGMP and nitric oxide in mediating the effects of sildenafil. Results Phosphodiesterase-5 mRNA and protein was demonstrated in human chorionic plate arteries. Immunohistochemistry demonstrated phosphodiesterase-5 within the arterial muscle layer. Sildenafil citrate produced dose dependent vasodilatation at concentrations at and greater than 10 nM. Both the direct cGMP inhibitor methylene blue and the cGMP-dependent protein kinase inhibitor Rp-8-Br-PET-cGMPS significantly attenuated the vasodilation produced by sildenafil citrate. Inhibition of NO production with L-NAME did not attenuate the vasodilator effects of sildenafil. In contrast, sildenafil citrate significantly enhanced the vasodilation produced by the NO donor sodium nitroprusside. Conclusion Phosphodiesterase-5 is present in the feto-placental circulation. Sildenafil citrate vasodilates the feto-placental circulation via a cGMP dependent mechanism involving increased responsiveness to NO.

  7. Effects and mechanisms of action of sildenafil citrate in human chorionic arteries.

    LENUS (Irish Health Repository)

    Maharaj, Chrisen H

    2009-01-01

    OBJECTIVES: Sildenafil citrate, a specific phosphodiesterase-5 inhibitor, is increasingly used for pulmonary hypertension in pregnancy. Sildenafil is also emerging as a potential candidate for the treatment of intra-uterine growth retardation and for premature labor. Its effects in the feto-placental circulation are not known. Our objectives were to determine whether phosphodiesterase-5 is present in the human feto-placental circulation, and to characterize the effects and mechanisms of action of sildenafil citrate in this circulation. STUDY DESIGN: Ex vivo human chorionic plate arterial rings were used in all experiments. The presence of phosphodiesterase-5 in the feto-placental circulation was determined by western blotting and immunohistochemical staining. In a subsequent series of pharmacologic studies, the effects of sildenafil citrate in pre-constricted chorionic plate arterial rings were determined. Additional studies examined the role of cGMP and nitric oxide in mediating the effects of sildenafil. RESULTS: Phosphodiesterase-5 mRNA and protein was demonstrated in human chorionic plate arteries. Immunohistochemistry demonstrated phosphodiesterase-5 within the arterial muscle layer. Sildenafil citrate produced dose dependent vasodilatation at concentrations at and greater than 10 nM. Both the direct cGMP inhibitor methylene blue and the cGMP-dependent protein kinase inhibitor Rp-8-Br-PET-cGMPS significantly attenuated the vasodilation produced by sildenafil citrate. Inhibition of NO production with L-NAME did not attenuate the vasodilator effects of sildenafil. In contrast, sildenafil citrate significantly enhanced the vasodilation produced by the NO donor sodium nitroprusside. CONCLUSION: Phosphodiesterase-5 is present in the feto-placental circulation. Sildenafil citrate vasodilates the feto-placental circulation via a cGMP dependent mechanism involving increased responsiveness to NO.

  8. Cervical anterior transpedicular screw fixation (ATPS)--Part II. Accuracy of manual insertion and pull-out strength of ATPS.

    Science.gov (United States)

    Koller, Heiko; Acosta, Frank; Tauber, Mark; Fox, Michael; Martin, Hudelmaier; Forstner, Rosmarie; Augat, Peter; Penzkofer, Rainer; Pirich, Christian; Kässmann, H; Resch, Herbert; Hitzl, Wolfgang

    2008-04-01

    Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3-T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of "critical" pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566 g/cm(2

  9. Michael addition of dehydroalanine-containing MAPK peptides to catalytic lysine inhibits the activity of phosphothreonine lyase.

    Science.gov (United States)

    Zhang, Yuan; Yang, Ru; Huang, Juan; Liang, Qiujin; Guo, Yanmin; Bian, Weixiang; Luo, Lingfei; Li, Hongtao

    2015-11-30

    The phosphothreonine lyases OspF and SpvC irreversibly inactivate host dual-phosphorylated mitogen-activated protein kinases (MAPKs) [pThr-X-pTyr motif] through β-elimination. We found that dual-phosphorylated (pSer-X-pTyr) MAPK substrate peptides and their resulting catalytic products cross-link to OspF and SpvC. Mass spectrometry results revealed that these linkages form between lysine, which acts as a general base, and dehydroalanine (Dha) on catalytic products. The nucleophilic addition efficiency is dependent on the K136 residue being in a deprotonated state. Peptide cross-linking inhibits the activity of SpvC and blocks the inactivation of MAPK signaling by SpvC. Small compounds mimicking these sequences may act as phosphothreonine lyase inhibitors. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. [Establishment of kinetics digital model for hyaluronate lyase production based on fermentation optimization of Arthrobacter globiformis A152].

    Science.gov (United States)

    Jiang, Yanhui; Zhang, Jingliang; Jiang, Xiaolu

    2017-11-25

    In order to produce hyaluronate lyase of high yield, we optimized the fermentation Arthrobacter globiformis A152 in quadruple fermentation of 5 L, and studied the kinetics of fermentation. Both the highest biomass and enzyme activity could be achieved when the rotation speed was 400 r/min and the ventilation volume was 3.5 L/min. In addition, digital models of cell growth, product synthesis and substrate consumption were built by equation of logistic, luedeking-piret, product synthesis and substrate consumption. Nonlinear fitting and estimation of optimal parameters were obtained by MATLAB. The model correlated well between prediction and experimental data, and reflected the change rules of cell growth, hyaluronidase synthesis and substrate consumption during the process of producing hyaluronate lyase. The establishment of fermentation kinetics digital models could provide basis for controlling and prediction of the production process.

  11. ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes

    NARCIS (Netherlands)

    de Waart, Dirk R.; Naik, Jyoti; Utsunomiya, Karina S.; Duijst, Suzanne; Ho-Mok, Kam; Bolier, A. Ruth; Hiralall, Johan; Bull, Laura N.; Bosma, Piter J.; Oude Elferink, Ronald P. J.; Paulusma, Coen C.

    2016-01-01

    ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a

  12. Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

    OpenAIRE

    Doberenz, Claudia; Zorn, Michael; Falke, Dörte; Nannemann, David; Hunger, Doreen; Beyer, Lydia; Christian H Ihling; Meiler, Jens; Sinz, Andrea; Sawers, R. Gary

    2014-01-01

    The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in ...

  13. Evidence for a link between histone deacetylation and Ca²+ homoeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts.

    Science.gov (United States)

    Ihlefeld, Katja; Claas, Ralf Frederik; Koch, Alexander; Pfeilschifter, Josef M; Meyer Zu Heringdorf, Dagmar

    2012-11-01

    Embryonic fibroblasts from S1P (sphingosine-1-phosphate) lyase-deficient mice [Sgpl1-/- MEFs (mouse embryonic fibroblasts)] are characterized by intracellular accumulation of S1P, elevated cytosolic [Ca2+]i and enhanced Ca2+ storage. Since S1P, produced by sphingosine kinase 2 in the nucleus of MCF-7 cells, inhibited HDACs (histone deacetylases) [Hait, Allegood, Maceyka, Strub, Harikumar, Singh, Luo, Marmorstein, Kordula, Milstein et al. (2009) Science 325, 1254-1257], in the present study we analysed whether S1P accumulated in the nuclei of S1P lyase-deficient MEFs and caused HDAC inhibition. Interestingly, nuclear concentrations of S1P were disproportionally elevated in Sgpl1-/- MEFs. HDAC activity was reduced, acetylation of histone 3-Lys9 was increased and the HDAC-regulated gene p21 cyclin-dependent kinase inhibitor was up-regulated in these cells. Furthermore, the expression of HDAC1 and HDAC3 was reduced in Sgpl1-/- MEFs. In wild-type MEFs, acetylation of histone 3-Lys9 was increased by the S1P lyase inhibitor 4-deoxypyridoxine. The non-specific HDAC inhibitor trichostatin A elevated basal [Ca2+]i and enhanced Ca2+ storage, whereas the HDAC1/2/3 inhibitor MGCD0103 elevated basal [Ca2+]i without influence on Ca2+ storage in wild-type MEFs. Overexpression of HDAC1 or HDAC2 reduced the elevated basal [Ca2+]i in Sgpl1-/- MEFs. Taken together, S1P lyase-deficiency was associated with elevated nuclear S1P levels, reduced HDAC activity and down-regulation of HDAC isoenzymes. The decreased HDAC activity in turn contributed to the dysregulation of Ca2+ homoeostasis, particularly to the elevated basal [Ca2+]i, in Sgpl1-/- MEFs.

  14. Hyaluronate lyase activity of Streptococcus suis serotype 2 and modulatory effects of hyaluronic acid on the bacterium's virulence properties.

    Science.gov (United States)

    Haas, Bruno; Vaillancourt, Katy; Bonifait, Laetitia; Gottschalk, Marcelo; Grenier, Daniel

    2015-11-26

    Streptococcus suis serotype 2 is a major swine pathogen and zoonotic agent worldwide causing mainly meningitis and septicemia. Hyaluronate lyases are enzymes that degrade hyaluronic acid, a major constituent of animal tissues, and have been reported as virulence factors in various bacterial species. Since the hyaluronate lyase of S. suis has been considered ambiguously as a virulence factor, we screened 50 isolates from the three major clonal complexes found in North America (sequence type [ST] 1, ST25, and ST28) known to differ in their degree of virulence in order to link the presence or absence of this activity with the degree of virulence. Moreover, the effect of exogenous hyaluronic acid on S. suis virulence factor gene expression and the pro-inflammatory response of brain macrovascular endothelial cells (BMEC) was also investigated. We found that all but one ST1 isolates (high virulence) were devoid of hyaluronate lyase activity whereas all ST25 (intermediate virulence) and ST28 (low virulence) isolates possessed the activity. A 2 bp insertion was responsible for the lack of activity in ST1 strains. Since the most virulent isolates did not degrade hyaluronic acid, this tissue component may be found during the infectious process. Therefore, we investigated its effect on S. suis and host cells. Hyaluronic acid was found to modulate S. suis adhesion to BMEC, to increase S. suis virulence factor expression, and to enhance pro-inflammatory cytokine secretion by BMEC. These findings suggest that S. suis hyaluronate lyase does not represent a critical virulence factor in its active form. However, exogenous hyaluronic acid that is likely to interact with S. suis and host cells during the course of infection appears to modulate several virulence determinants of the bacterium, in addition to promote inflammation.

  15. Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1).

    Science.gov (United States)

    Müller, Tina A; Tobar, Michael A; Perian, Madison N; Hausinger, Robert P

    2017-04-04

    Alkbh1 is one of nine mammalian homologues of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5'-product. The studies described here extend the biochemical characterization of two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to those of other AP lyases. The α,β-unsaturated aldehyde of the 5'-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examined with a newly developed assay using a methylation-sensitive restriction endonuclease, and the enzymatic rate was found to be very low. Indeed, the demethylase activity was less than half that of the AP lyase activity when ALKBH1 samples were assayed using identical buffer conditions. The two enzymatic activities were examined using a series of site-directed variant proteins, revealing the presence of distinct but partially overlapping active sites for the two reactions. We postulate that the very low 6-methyl adenine oxygenase activity associated with ALKBH1 is unlikely to represent the major function of the enzyme in the cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.

  16. Citrate compared to low molecular weight heparin anticoagulation in chronic hemodialysis patients.

    Science.gov (United States)

    Janssen, M J; Deegens, J K; Kapinga, T H; Beukhof, J R; Huijgens, P C; van Loenen, A C; van der Meulen, J

    1996-03-01

    Citrate and nadroparin calcium, a low molecular weight heparin (LMWH), were compared in a randomized cross-over trial in 21 chronic hemodialysis patients regarding anticoagulation, calcium and magnesium kinetics, biocompatibility, dialysis efficiency, and aluminum contamination. Citrate was infused into the arterial line at a minimum rate of 0.68 mmol/min, combined with a calcium and magnesium-free dialysate and intravenous supplementation of calcium and magnesium at rates of 0.22 and 0.10 mmol/min, respectively. Seven patients with a dialysis session of six hours, received 2/3 of the nadroparin dose predialysis, and 1/3 after 2.5 hours (divided dose (DD) group). A single predialysis bolus injection of nadroparin was administered to eight patients not on coumarins [single dose (SD) group] and to six patients on coumarins [single dose + coumarins (SD + C) group], all with a dialysis session of four hours. Nineteen patients received a nadroparin dose of 200 ICU/kg. Two patients with a single dose, one of them on coumarins, received a dose of 150 ICU/kg because of a hematocrit containing 1.5 mmol/liter calcium and 0.75 mmol/liter magnesium used in combination with nadroparin. Ionized calcium increments during citrate were significant after the end of dialysis, while the dialysate containing 1.5 mmol/liter calcium induced significant increments during and postdialysis. No differences were observed between citrate and nadroparin regarding biocompatibility), (expressed as dialysis-induced leukopenia and thrombocytopenia), and dialysis efficiency [measured as dialyzer urea and creatinine clearance, normalized weekly whole body urea clearance (Kt/Vurea) and time averaged urea concentration (TACurea)]. The citrate solution, if sterilized in glass bottles, contained 2 to 3 micrograms aluminum per mmol citrate, the nadroparin solution 0.009 microgram per 1,000 ICU. Aluminum contamination of the citrate solution was prevented by sterilizing the solution in polypropylene

  17. Inhibition of calcium oxalate monohydrate growth by citrate and the effect of the background electrolyte

    Science.gov (United States)

    Weaver, Matthew L.; Qiu, S. Roger; Hoyer, John R.; Casey, William H.; Nancollas, George H.; De Yoreo, James J.

    2007-08-01

    Pathological mineralization is a common phenomenon in broad range of plants and animals. In humans, kidney stone formation is a well-known example that afflicts approximately 10% of the population. Of the various calcium salt phases that comprise human kidney stones, the primary component is calcium oxalate monohydrate (COM). Citrate, a naturally occurring molecule in the urinary system and a common therapeutic agent for treating stone disease, is a known inhibitor of COM. Understanding the physical mechanisms of citrate inhibition requires quantification of the effects of both background electrolytes and citrate on COM step kinetics. Here we report the results of an in situ AFM study of these effects, in which we measure the effect of the electrolytes LiCl, NaCl, KCl, RbCl, and CsCl, and the dependence of step speed on citrate concentration for a range of COM supersaturations. We find that varying the background electrolyte results in significant differences in the measured step speeds and in step morphology, with KCl clearly producing the smallest impact and NaCl the largest. The kinetic coefficient for the former is nearly three times larger than for the latter, while the steps change from smooth to highly serrated when KCl is changed to NaCl. The results on the dependence of step speed on citrate concentration show that citrate produces a dead zone whose width increases with citrate concentration as well as a continual reduction in kinetic coefficient with increasing citrate level. We relate these results to a molecular-scale view of inhibition that invokes a combination of kink blocking and step pinning. Furthermore, we demonstrate that the classic step-pinning model of Cabrera and Vermilyea (C-V model) does an excellent job of predicting the effect of citrate on COM step kinetics provided the model is reformulated to more realistically account for impurity adsorption, include an expression for the Gibbs-Thomson effect that is correct for all supersaturations

  18. Nicotinic acid metabolism enzymic preparation and absolute configuration of the substrate for 2,3-dimethylmalate lyase.

    Science.gov (United States)

    Lill, U; Pirzer, P; Kukla, D; Huber, R; Eggerer, H

    1980-01-01

    1) A convenient method for the enzymatic preparation of a chemically and optically pure isomer of 2,3-dimethylmalic acid in g-amounts is described. Propionate, pyruvate and partially purified 2,3-dimethylmalate lyase (from Clostridium barkeri) were applied. 2) The enzymically formed product, m.p. 99--100 degrees C, [alpha]D20 = -16.4 (water), is related to the known stereochemistry of the Senecio alkaloid jacobine and to a laevorotatory 2,3-dimethylmalic acid derived from jaconecic acid, a degradation product of the alkaloid. From this relationship it appears likely that the substrate of the lyase is a component of the threo racemate and is of (2R,3S) configuration. 3) A three-dimensional X-ray structure analysis was performed and the structure refined to an R value of 0.049. The asymmetric unit contains three independent threo dimethylmalic acid molecules. The anomalous dispersion effects of carbon and oxygen were used to determine the absolute configuration. These measurements yielded a (2R,3S) configuration. 4) We conclude from these results that (2R,3S)-2,3-dimethylmalate is the substrate of the lyase. The results also establish that previously isolated racemic 2,3-dimethylmalic acids, m.p. 143 degrees C and m.p. 104--106 degrees C, represent the erythro and threo pair, respectively.

  19. A Systems Chemical Biology Study of Malate Synthase and Isocitrate Lyase Inhibition in Mycobacterium tuberculosis During Active and NRP Growth

    Science.gov (United States)

    May, Elebeoba E.; Leitão, Andrei; Tropsha, Alexander; Oprea, Tudor I.

    2013-01-01

    The ability of Mycobacterium tuberculosis (Mtb) to survive in low oxygen environments enables the bacterium to persist in a latent state within host tissues. In vitro studies of Mtb growth have identified changes in isocitrate lyase (ICL) and malate synthase (MS) that enable bacterial persistent under low oxygen and other environmentally limiting conditions. Systems chemical biology (SCB) enables us to evaluate the effects of small molecule inhibitors not only on the reaction catalyzed by malate synthase and isocitrate lyase, but the effect on the complete tricarboxylic acid cycle (TCA) by taking into account complex network relationships within that system. To study the kinetic consequences of inhibition on persistent bacilli, we implement a systems-chemical biology (SCB) platform and perform a chemistry-centric analysis of key metabolic pathways believed to impact Mtb latency. We explore consequences of disrupting the function of malate synthase (MS) and isocitrate lyase (ICL) during aerobic and hypoxic non-replicating persistence (NRP) growth by using the SCB method to identify small molecules that inhibit the function of MS and ICL, and simulating the metabolic consequence of the disruption. Results indicate variations in target and non-target reaction steps, clear differences in the normal and low oxygen models, as well as dosage dependent response. Simulation results from singular and combined enzyme inhibition strategies suggest ICL may be the more effective target for chemotherapeutic treatment against Mtb growing in a microenvironment where oxygen is slowly depleted, which may favor persistence. PMID:24121675

  20. On the Isolation and Characterization of a C-S-Lyase Preparation from Leek,Allium porrum.

    Science.gov (United States)

    Lohmüller, E M; Landshuter, J; Knobloch, K

    1994-08-01

    The C-S-lyase protein from leek, ALLIUM PORRUM L., has been purified and characterized. The molecular mass of the native protein was determined with M (r) = 100 000, including two similar subunits, M (r) = 50 000. The tendency of the native protein to form a trimer, M (r) = 300 000, could be supported. The isoelectric point of the enzyme turned out to be close to pH 7.5. The C-S-lyase reaction revealed a wide pH optimum, in the range of 6.1 to 6.9. The temperature optimum was found to be at 41 degrees C. Pure (+)- and (-)-isomers of S-alk(en)yl- L-cysteine sulfoxides were inserted as the substrates. The highest turnover rate was achieved with (+)- S-allyl- L-cysteine sulfoxide (alliin). (+)- S-Propyl- L-cysteine sulfoxide (PCSO) exhibited the lowest K (m) value. Activation energies for the cleavage of the substrates were determined to be 23 kJ/mol for (+)- S-methyl- L-cysteine sulfoxide (MCSO), 38 kJ/mol for (-)-MCSO, 28 kJ/mol for (+)-alliin, and 54 kJ/mol for (+)-PCSO. On the basis of studies with specific inhibitors, pyridoxal 5'-phosphate was found to be part of the A. PORRUM C-S-lyase protein as a cofactor. Competitive inhibitory effects were observed with L-cysteine and related compounds.

  1. New Ulvan-Degrading Polysaccharide Lyase Family: Structure and Catalytic Mechanism Suggests Convergent Evolution of Active Site Architecture.

    Science.gov (United States)

    Ulaganathan, ThirumalaiSelvi; Boniecki, Michal T; Foran, Elizabeth; Buravenkov, Vitaliy; Mizrachi, Naama; Banin, Ehud; Helbert, William; Cygler, Miroslaw

    2017-05-19

    Ulvan is a complex sulfated polysaccharide biosynthesized by green seaweed and contains predominantly rhamnose, xylose, and uronic acid sugars. Ulvan-degrading enzymes have only recently been identified and added to the CAZy ( www.cazy.org ) database as family PL24, but neither their structure nor catalytic mechanism(s) are yet known. Several homologous, new ulvan lyases, have been discovered in Pseudoalteromonas sp. strain PLSV, Alteromonas LOR, and Nonlabens ulvanivorans, defining a new family PL25, with the lyase encoded by the gene PLSV_3936 being one of them. This enzyme cleaves the glycosidic bond between 3-sulfated rhamnose (R3S) and glucuronic acid (GlcA) or iduronic acid (IdoA) via a β-elimination mechanism. We report the crystal structure of PLSV_3936 and its complex with a tetrasaccharide substrate. PLSV_3936 folds into a seven-bladed β-propeller, with each blade consisting of four antiparallel β-strands. Sequence conservation analysis identified a highly conserved region lining at one end of a deep crevice on the protein surface. The putative active site was identified by mutagenesis and activity measurements. Crystal structure of the enzyme with a bound tetrasaccharide substrate confirmed the identity of base and acid residues and allowed determination of the catalytic mechanism and also the identification of residues neutralizing the uronic acid carboxylic group. The PLSV_3936 structure provides an example of a convergent evolution among polysaccharide lyases toward a common active site architecture embedded in distinct folds.

  2. Purification and biochemical characterization of an alkaline pectin lyase from Fusarium decemcellulare MTCC 2079 suitable for Crotalaria juncea fiber retting.

    Science.gov (United States)

    Yadav, Sangeeta; Dubey, Amit Kumar; Anand, Gautam; Kumar, Reetesh; Yadav, Dinesh

    2014-07-01

    An extracellular pectin lyase secreted by Fusarium decemcellulare MTCC 2079 under solid state fermentation condition has been purified to electrophoretic homogeniety by using ammonium sulfate fractionation, carboxymethyl cellulose and gel filtration (Sephadex G-100) column chromatographies. The purified enzyme showed single protein band corresponding to molecular mass 45 ± 01 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 9.0 and showed maximum stability in the pH range of 9.0-12.0. The optimum temperature of the purified enzyme was 50 °C and it showed maximum stability upto 40 °C. The energy of activation for the thermal denaturation (Ea ) was 59.06 kJ mol(-1)  K(-1). The Km and kcat values using citrus pectin as the substrate were 0.125 mg ml(-1) and 72.9 s(-1) in 100 mM sodium carbonate buffer pH 9.0 at 50 °C. The biophysical studies on pectin lyase showed that its secondary structure belongs to α + β class of protein with comparatively less of β-sheets. Purified pectin lyase showed efficient retting of Crotolaria juncea fibers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Study on the Antimicrobial Properties of Citrate-Based Biodegradable Polymers.

    Science.gov (United States)

    Su, Lee-Chun; Xie, Zhiwei; Zhang, Yi; Nguyen, Kytai Truong; Yang, Jian

    2014-01-01

    Citrate-based polymers possess unique advantages for various biomedical applications since citric acid is a natural metabolism product, which is biocompatible and antimicrobial. In polymer synthesis, citric acid also provides multiple functional groups to control the crosslinking of polymers and active binding sites for further conjugation of biomolecules. Our group recently developed a number of citrate-based polymers for various biomedical applications by taking advantage of their controllable chemical, mechanical, and biological characteristics. In this study, various citric acid derived biodegradable polymers were synthesized and investigated for their physicochemical and antimicrobial properties. Results indicate that citric acid derived polymers reduced bacterial proliferation to different degrees based on their chemical composition. Among the studied polymers, poly(octamethylene citrate) showed ~70-80% suppression to microbe proliferation, owing to its relatively higher ratio of citric acid contents. Crosslinked urethane-doped polyester elastomers and biodegradable photoluminescent polymers also exhibited significant bacteria reduction of ~20 and ~50% for Staphylococcus aureus and Escherichia coli, respectively. Thus, the intrinsic antibacterial properties in citrate-based polymers enable them to inhibit bacteria growth without incorporation of antibiotics, silver nanoparticles, and other traditional bacteria-killing agents suggesting that the citrate-based polymers are unique beneficial materials for wound dressing, tissue engineering, and other potential medical applications where antimicrobial property is desired.

  4. Ferric Citrate Hydrate as a Phosphate Binder and Risk of Aluminum Toxicity

    Directory of Open Access Journals (Sweden)

    Ajay Gupta

    2014-09-01

    Full Text Available Ferric citrate hydrate was recently approved in Japan as an oral phosphate binder to be taken with food for the control of hyperphosphatemia in patients with chronic kidney disease (CKD. The daily therapeutic dose is about 3 to 6 g, which comprises about 2 to 4 g of citrate. Oral citrate solubilizes aluminum that is present in food and drinking water, and opens the tight junctions in the intestinal epithelium, thereby increasing aluminum absorption and urinary excretion. In healthy animals drinking tap water, oral citrate administration increased aluminum absorption and, over a 4-week period, increased aluminum deposition in brain and bone by about 2- and 20-fold, respectively. Renal excretion of aluminum is impaired in patients with chronic kidney disease, thereby increasing the risk of toxicity. Based on human and animal studies it can be surmised that patients with CKD who are treated with ferric citrate hydrate to control hyperphosphatemia are likely to experience enhanced absorption of aluminum from food and drinking water, thereby increasing the risk of aluminum overload and toxicity.

  5. Ferric citrate hydrate as a phosphate binder and risk of aluminum toxicity.

    Science.gov (United States)

    Gupta, Ajay

    2014-09-26

    Ferric citrate hydrate was recently approved in Japan as an oral phosphate binder to be taken with food for the control of hyperphosphatemia in patients with chronic kidney disease (CKD). The daily therapeutic dose is about 3 to 6 g, which comprises about 2 to 4 g of citrate. Oral citrate solubilizes aluminum that is present in food and drinking water, and opens the tight junctions in the intestinal epithelium, thereby increasing aluminum absorption and urinary excretion. In healthy animals drinking tap water, oral citrate administration increased aluminum absorption and, over a 4-week period, increased aluminum deposition in brain and bone by about 2- and 20-fold, respectively. Renal excretion of aluminum is impaired in patients with chronic kidney disease, thereby increasing the risk of toxicity. Based on human and animal studies it can be surmised that patients with CKD who are treated with ferric citrate hydrate to control hyperphosphatemia are likely to experience enhanced absorption of aluminum from food and drinking water, thereby increasing the risk of aluminum overload and toxicity.

  6. Comparison of 24-hour urinary citrate excretion in stone formers and healthy volunteers

    Directory of Open Access Journals (Sweden)

    Mohammad Taghi Goodarzi

    2012-01-01

    Full Text Available Low urinary citrate excretion is a risk factor in stone formers (SF. This study aimed to measure the urinary citrate excretion in SF and healthy volunteers at our center from 12 June 2008 to 20 August 2009. There were 28 SF patients (18 males and ten females and 27 (18 males and nine females age-matched healthy adult volunteers who participated in this study. Both groups had a similar living environment, extrinsic factors, diet and genetic descent. After collecting 24-h urine, citrate was measured using an enzymatic kit. Routine urinalysis and 24-h creatinine and uric acid were also performed. There was a significant difference in urinary citrate excretion level among SF (mean 310, SD 260 mg/L and normal volunteer subjects (mean 800, SD 300 mg/L. By applying the previously defined normal values (320 mg/24 h of urinary citrate in the local population, 43% of the SF in our study group was hypocitric, and none among the controls. We conclude that prevalence of hypocitraturia in stone formers was higher than that in healthy volunteers in our population.

  7. Sodium citrate blood contamination by K2 -ethylenediaminetetraacetic acid (EDTA): impact on routine coagulation testing.

    Science.gov (United States)

    Lima-Oliveira, G; Salvagno, G L; Danese, E; Favaloro, E J; Guidi, G C; Lippi, G

    2015-06-01

    The potential cross-contamination of additives between primary blood tubes is a well-known problem during sample collection. The aim of this study was to assess the impact of citrated blood contamination with different amounts of dipotassium ethylenediaminetetraacetic (K2 EDTA blood) on activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen. Blood was collected from 15 ostensibly healthy volunteers into four 0.109 m citrate blood tubes followed by one K2 EDTA blood tube. The citrate tubes of each subject were pooled and divided in five aliquots. The whole blood of the K2 EDTA tube was then added in scalar amounts to autologous citrated blood aliquots, to obtain K2 EDTA contamination ranging from 0% to 43%, and thus mimic potential pre-analytical contamination. A statistically and clinically significant prolongation was observed for both APTT and PT between 29% and 43% K2 EDTA contamination, whereas the decrease of fibrinogen values became statistically and clinically significant at 43% K2 EDTA contamination. The results of this investigation show that contamination of citrated blood with as much as 29% of K2 EDTA blood generates a significant bias in results of routine clotting assays. This has serious implications for patient safety and management. © 2014 John Wiley & Sons Ltd.

  8. Effect of topically applied sildenafil citrate on wound healing: experimental study

    Directory of Open Access Journals (Sweden)

    Koray Gürsoy

    2014-08-01

    Full Text Available Wound healing is a complex process that necessitates organization of different cell types and several signalling molecules. The aim of this study is to evaluate the effect of different concentrations of sildenafil citrate, which decreases cGMP degradation, on wound healing by secondary intention.This study was performed using 25 Sprague Dawley rats weighing 200-250 grams. 4 dorsal defects were created. Four different treatment modalities which were 1% and 5% sildenafil citrate gel prepared with carbopol, pure carbopol gel without any drug in it and 0,9% NaCl solution; were applied to each lesion of the same rat. Randomly selected five rats (25 rats in total were sacrificed on 3rd, 5th, 7th, 10th, and 14th days; and the effect of each modality was evaluated by means of defect area measurement, histopathological examination and measurement of tissue hydroxyproline levels.Sildenafil citrate gel application decreased the defect areas in a dose independent manner starting from 3rd day and dose dependent manner after 7th day. By means of vascularization, sildenafil citrate increased vascularity starting from 3rd day. The strength of acute inflammation was superior in sildenafil groups starting from 5th day; and the amount and maturation of granulation in the wound bed, as well as the strength of chronic inflammation were superior in defects treated with sildenafil citrate as early as 7th day.

  9. Effects of commercial citrate-containing juices on urolithiasis in a Drosophila model

    Directory of Open Access Journals (Sweden)

    Chien-Yi Ho

    2013-09-01

    Full Text Available Diet modification plays an important role in nephrolithiasis. Development of an easy, ready-to-use beverage such as a commercial juice drink to use as a preventive treatment for renal calculi formation would be widely welcomed. We previously developed a novel Drosophila model for the study of nephrolithiasis. It provides a new well-established drug discovery platform for this common disease. In our current study, we used the Drosophila model to investigate the preventive effects of various commercial juices as potential treatments for nephrolithiasis. Our results showed that apple, cranberry, orange, and pomegranate juices failed to reduce calcium oxalate (CaOx crystal formation, whereas our positive control—potassium citrate (K-citrate—significantly prevented CaOx crystal formation. Unlike the commercial fruit juices that were tested, the administration of K-citrate significantly ameliorated the ethylene glycol (EG-induced life-span reduction in treated flies. These results indicate that EG-induced CaOx nephrolithiasis in Drosophila can be prevented by K-citrate, but not by commercial citrate-containing juices. However, the inhibitory capability of citrate-containing juices to reduce renal stone formation in humans requires further elucidation.

  10. Concentration of citrate in the mammary secretion of sows during lactogenesis II and established lactation.

    Science.gov (United States)

    Holmes, M A; Hartmann, P E

    1993-08-01

    The functional significance of citrate in the mammary secretion of six sows was investigated during the second stage of lactogenesis (lactogenesis II) and established lactation. The changes in the concentrations of progesterone and lactose in the maternal blood, and lactose, Na and K in the mammary secretion, suggested that lactogenesis II began during the final day of pregnancy. The concentration of citrate in the mammary secretion of the sows during lactogenesis II was high and varied from 5.4 (SEM 0.5) mM at day 0.5 post partum to 6.8 (SEM 0.4) mM at day 1.5 post partum. There was a decline of approximately 30% in the concentration of citrate in the milk of sows during the first week of lactation. These findings suggest that, in contrast to all other species studied previously, milk citrate is not a harbinger of lactogenesis II in the sow. However, the changes in the concentration of citrate in the mammary secretions of sows may reflect changes in the rate of de novo synthesis of fatty acids that take place in the mammary glands of sows during lactogenesis II and established lactation.

  11. Study on the Antimicrobial Properties of Citrate-Based Biodegradable Polymers

    Science.gov (United States)

    Su, Lee-Chun; Xie, Zhiwei; Zhang, Yi; Nguyen, Kytai Truong; Yang, Jian

    2014-01-01

    Citrate-based polymers possess unique advantages for various biomedical applications since citric acid is a natural metabolism product, which is biocompatible and antimicrobial. In polymer synthesis, citric acid also provides multiple functional groups to control the crosslinking of polymers and active binding sites for further conjugation of biomolecules. Our group recently developed a number of citrate-based polymers for various biomedical applications by taking advantage of their controllable chemical, mechanical, and biological characteristics. In this study, various citric acid derived biodegradable polymers were synthesized and investigated for their physicochemical and antimicrobial properties. Results indicate that citric acid derived polymers reduced bacterial proliferation to different degrees based on their chemical composition. Among the studied polymers, poly(octamethylene citrate) showed ~70–80% suppression to microbe proliferation, owing to its relatively higher ratio of citric acid contents. Crosslinked urethane-doped polyester elastomers and biodegradable photoluminescent polymers also exhibited significant bacteria reduction of ~20 and ~50% for Staphylococcus aureus and Escherichia coli, respectively. Thus, the intrinsic antibacterial properties in citrate-based polymers enable them to inhibit bacteria growth without incorporation of antibiotics, silver nanoparticles, and other traditional bacteria-killing agents suggesting that the citrate-based polymers are unique beneficial materials for wound dressing, tissue engineering, and other potential medical applications where antimicrobial property is desired. PMID:25023605

  12. Effect of topically applied sildenafil citrate on wound healing: experimental study

    Science.gov (United States)

    Gürsoy, Koray; Oruç, Melike; Kankaya, Yüksel; Ulusoy, M. Gürhan; Koçer, Uğur; Kankaya, Duygu; Gürsoy, R. Neslihan; Çevik, Özge; Öğüş, Elmas; Fidanci, Vildan

    2014-01-01

    Wound healing is a complex process that necessitates organization of different cell types and several signalling molecules. The aim of this study is to evaluate the effect of different concentrations of sildenafil citrate, which decreases cGMP degradation, on wound healing by secondary intention. This study was performed using 25 Sprague Dawley rats weighing 200-250 grams. 4 dorsal defects were created. Four different treatment modalities which were 1% and 5% sildenafil citrate gel prepared with carbopol, pure carbopol gel without any drug in it and 0,9% NaCl solution; were applied to each lesion of the same rat. Randomly selected five rats (25 rats in total) were sacrificed on 3rd, 5th, 7th, 10th, and 14th days; and the effect of each modality was evaluated by means of defect area measurement, histopathological examination and measurement of tissue hydroxyproline levels. Sildenafil citrate gel application decreased the defect areas in a dose independent manner starting from 3rd day and dose dependent manner after 7th day. By means of vascularization, sildenafil citrate increased vascularity starting from 3rd day. The strength of acute inflammation was superior in sildenafil groups starting from 5th day; and the amount and maturation of granulation in the wound bed, as well as the strength of chronic inflammation were superior in defects treated with sildenafil citrate as early as 7th day. PMID:25172969

  13. Dose-dependent effects of mosapride citrate on duodenal and cecal motility in donkeys (Equus asinus

    Directory of Open Access Journals (Sweden)

    Naglaa A. Gomaa

    2013-12-01

    Full Text Available Prokinetic drugs are used for the management of gastrointestinal motility disorders in horses; however, little is known about their efficacy in donkeys. Therefore, the aim of the present study was to evaluate the effect of different doses of mosapride citrate on duodenal and cecal motility in normal donkeys. Six donkeys (n = 6 were used in a crossover study. Mosapride citrate was administered orally via a nasogastric tube at dose rate of 1, 2 and 3 mg kg−1. Duodenal and cecal motility were evaluated using ultrasonography before administration and at 15, 30, 60, 120 and 180 min post-administration. There was a significant increase of duodenal contractions (p < 0.05 after 30 min of mosapride citrate administration at 3 mg kg−1 with a prolonged (p < 0.05 prokinetic effect at 2 mg kg−1. Cecal contractions were significantly increased (p < 0.05 after 15 min at different doses of mosapride with a prolonged effect at 3 mg kg−1. The results of the present study indicate that mosapride citrate has a dose-dependent prokinetic effect on the duodenal and cecal contractions in healthy donkeys. Further studies need to determine whether mosapride citrate is effective in treatment of intestinal disorders in donkey.

  14. Enhancing radium solubilization in soils by citrate, EDTA, and EDDS chelating amendments.

    Science.gov (United States)

    Prieto, C; Lozano, J C; Blanco Rodríguez, P; Tomé, F Vera

    2013-04-15

    The effect of three chelating agents (citrate, EDTA, and EDDS) on the solubilization of radium from a granitic soil was studied systematically, considering different soil pH values, chelating agent concentrations, and leaching times. For all the chelating agents tested, the amount of radium leached proved to be strongly dependent on the pH of the substrate: only for acidic conditions did the amount of radium released increase significantly relative to the controls. Under the best conditions, the radium released from the amended soil was greater by factors of 20 in the case of citrate, 18 for EDTA, and 14 for EDDS. The greatest improvement in the release of radium was obtained for the citrate amendment at the highest concentration tested (50 mmol kg(-1)). A slightly lower amount of radium was leached with EDTA at 5 mmol kg(-1) soil, but the solubilization over time was very different from that observed with citrate or EDDS. With EDTA, a maximum in radium leaching was reached on the first day after amendment, while with citrate, the maximum was attained on the fourth day. With EDDS, radium leaching increased slightly but steadily with time (until the sixth day), but the net effect for the period tested was the lowest of the three reagents. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. The role of tannic acid and sodium citrate in the synthesis of silver nanoparticles

    Science.gov (United States)

    Ranoszek-Soliwoda, Katarzyna; Tomaszewska, Emilia; Socha, Ewelina; Krzyczmonik, Pawel; Ignaczak, Anna; Orlowski, Piotr; Krzyzowska, Małgorzata; Celichowski, Grzegorz; Grobelny, Jaroslaw

    2017-08-01

    We describe herein the significance of a sodium citrate and tannic acid mixture in the synthesis of spherical silver nanoparticles (AgNPs). Monodisperse AgNPs were synthesized via reduction of silver nitrate using a mixture of two chemical agents: sodium citrate and tannic acid. The shape, size and size distribution of silver particles were determined by UV-Vis spectroscopy, dynamic light scattering (DLS) and scanning transmission electron microscopy (STEM). Special attention is given to understanding and experimentally confirming the exact role of the reagents (sodium citrate and tannic acid present in the reaction mixture) in AgNP synthesis. The oxidation and reduction potentials of silver, tannic acid and sodium citrate in their mixtures were determined using cyclic voltammetry. Possible structures of tannic acid and its adducts with citric acid were investigated in aqueous solution by performing computer simulations in conjunction with the semi-empirical PM7 method. The lowest energy structures found from the preliminary conformational search are shown, and the strength of the interaction between the two molecules was calculated. The compounds present on the surface of the AgNPs were identified using FT-IR spectroscopy, and the results are compared with the IR spectrum of tannic acid theoretically calculated using PM6 and PM7 methods. The obtained results clearly indicate that the combined use of sodium citrate and tannic acid produces monodisperse spherical AgNPs, as it allows control of the nucleation, growth and stabilization of the synthesis process. [Figure not available: see fulltext.

  16. Hemodynamic effects of sildenafil citrate (Viagra) on segmental branches of bilateral renal arteries.

    Science.gov (United States)

    Ardicoglu, Arslan; Kocakoc, Ercan; Yuzgec, Veysel; Bozgeyik, Zulkif; Canatan, Halit; Kiris, Adem

    2005-01-01

    Erectile dysfunction (ED) is one of the most encountered male health problem and sildenafil citrate is used worldwide for treatment of ED. Aim of the present study was to determine and evaluate hemodynamic effects of sildenafil citrate on segmental branches of bilateral renal arteries of cases with ED. Total of 35 male patients aging between 34 and 56 years were recruited into study. Patients were evaluated with renal color Doppler sonography (CDS) before and after (45-75 min) peroral sildenafil citrate (50 mg) administration. Peak systolic flow rate (V(max)), end diastolic flow rate (V(min)), resistive index (RI) and pulsatility index (PI) of renal artery segmental branches were determined before and after sildenafil citrate administration. V(min) measured at right lower segment was significantly reduced after sildenafil administration (p = 0.030). Accordingly mean RI and PI of right lower segment of renal artery slightly increased after drug administration (p = 0.007 and p = 0.012, respectively). The measurements on other segments had no significant change after sildenafil treatment when compared to baseline values. On this single dose administration no side effect was recorded. Findings of the present study demonstrated that peroral sildenafil citrate usage had slight effects on hemodynamic parameters of lower segmental branch of right renal artery. This result could not be interpreted as robust evidence for hemodynamic changes of renal arteries associated with sildenafil usage nevertheless warrants more comprehensive research with larger number of patients.

  17. Effect Of Clomiphene Citrate On Hematology And Serum Biochemistry Of Nigerian Indigenous Chicken

    Directory of Open Access Journals (Sweden)

    Urom

    2015-08-01

    Full Text Available The effect of different doses of clomiphene citrate on haematology and serum biochemistry of Nigerian indigenous chicken were evaluated at the poultry Unit Department of Animal Science and Fisheries Abia State University Umuahia using 48 sexually matured 26 weeks old local cocks each group was divided into 4 treatment groups of 4 matured chickens in a completely randomized design with 3 replications. Four levels of Clomiphene citrate treatments represented as T1 0mg T2 10.00mg T3 20.00mg and T4 30.00mg were administered to the birds. Haematology and Serum biochemical indices of the treated birds were determined at the end of the study. The result showed that there were significant differences P0.05 among the treatment groups in haematology and serum biochemical parameters except for the following parameters hemoglobin red blood cell mean corpuscular volume of the birds. It was concluded that the administration of Clomiphene citrate Clomid led to changes in heamatology and serum biochemistry in Nigerian indigenous chicken which showed that clomiphene citrate can be considered safe for chicken. 10mg and 20mg levels of clomiphene citrate can bring about improved hematological indices of Nigeria indigenous chicken

  18. ATP hydrolyzing salivary enzymes of caterpillars suppress plant defenses.

    Directory of Open Access Journals (Sweden)

    Shuang Wu

    Full Text Available The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5'-triphosphate (ATP in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5'-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned.

  19. Fibroblast cytoskeletal remodeling induced by tissue stretch involves ATP signaling.

    Science.gov (United States)

    Langevin, Helene M; Fujita, Takumi; Bouffard, Nicole A; Takano, Takahiro; Koptiuch, Cathryn; Badger, Gary J; Nedergaard, Maiken

    2013-09-01

    Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 min, returning to baseline at 60 min. No increase in ATP was observed in tissue incubated without stretch or tissue stretched in the presence of the Rho kinase inhibitor Y27632. The increase in fibroblast cross sectional area in response to tissue stretch was blocked by both suramin (a purinergic receptor blocker) and apyrase (an enzyme that selectively degrades extracellular ATP). Furthermore, connexin channel blockers (octanol and carbenoxolone), but not VRAC (fluoxetine) or pannexin (probenecid) channel blockers, inhibited fibroblast expansion. Together, these results support a mechanism in which extracellular ATP signaling via connexin hemichannels mediate the active change in fibroblast shape that occurs in response to a static increase in tissue length. Copyright © 2013 Wiley Periodicals, Inc.

  20. Phenomenological analysis of ATP dependence of motor protein

    CERN Document Server

    Zhang, Yunxin

    2011-01-01

    In this study, through phenomenological comparison of the velocity-force data of processive motor proteins, including conventional kinesin, cytoplasmic dynein and myosin V, we found that, the ratio between motor velocities of two different ATP concentrations is almost invariant for any substall, superstall or negative external loads. Therefore, the velocity of motor can be well approximated by a Michaelis-Menten like formula $V=\\atp k(F)L/(\\atp +K_M)$, with $L$ the step size, and $k(F)$ the external load $F$ dependent rate of one mechanochemical cycle of motor motion in saturated ATP solution. The difference of Michaelis-Menten constant $K_M$ for substall, superstall and negative external load indicates, the ATP molecule affinity of motor head for these three cases are different, though the expression of $k(F)$ as a function of $F$ might be unchanged for any external load $F$. Verifications of this Michaelis-Menten like formula has also been done by fitting to the recent experimental data.

  1. Cloning and characterization of a pectin lyase gene from Colletotrichum lindemuthianum and comparative phylogenetic/structural analyses with genes from phytopathogenic and saprophytic/opportunistic microorganisms

    Science.gov (United States)

    2011-01-01

    Background Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides. The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively. Results Here we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides. Conclusions The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin

  2. [ATP content in cryopreserved sperm of Siberian white cranes Grus leucogeranus (Aves: Gruiformes)].

    Science.gov (United States)

    Maksudov, G Iu; Erokhin, A S; Nesterenko, O N; Panchenko, V G

    2002-01-01

    ATP contents were studied in the native and cryoconserved sperm of Siberian white cranes Grus leucogeranus using bioluminescence analysis. The ATP content in freshly obtained spermatozoa was 12.7 nmol/10(8) cells. No ATP was found in the seminal plasma. In the process of freezing-thawing, the ATP concentration in the spermatozoa decreased by 30%. The differences in the dynamics of ATP content during cryoconservation of sperm of white cranes and other birds and mammals are discussed.

  3. 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

    DEFF Research Database (Denmark)

    Ghosh, Amit; Ando, David; Gin, Jennifer

    2016-01-01

    of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation...... to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source...... of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux...

  4. ATP level and caffeine efficiency on cytokinesis inhibition in plants.

    Science.gov (United States)

    López-Sáez, J F; Mingo, R; González-Fernández, A

    1982-06-01

    Plant cytokinesis appears to be a topographically organized process of exocytosis. Golgi vesicles which contain cell wall precursors are translocated during telophase, by interzonal microtubules, to the equatorial region of the mitotic apparatus where they fuse with each other giving rise to the new cell wall. Caffeine inhibits cytokinesis by hindering Golgi vesicle coalescence. The present results demonstrate that treatments which increase the cellular ATP level (adenosine, cycloheximide and anisomycin) counteract caffein-induced cytokinesis inhibition in meristem cells of onion root tips (Allium cepa L.), while treatments which decrease ATP level potentiate this caffeine effect (dinitrophenol, fluoroacetate, low oxygen tensions, etc.). We postulate that caffeine, in competition with the cellular ATP level, blocks cell plate formation by inhibiting a certain ATPase activity required for membrane fusion of Golgi vesicles.

  5. ATP measurements for monitoring microbial drinking water quality

    DEFF Research Database (Denmark)

    Vang, Óluva Karin

    Current standard methods for surveillance of microbial drinking water quality are culture based, which are laborious and time-consuming, where results not are available before one to three days after sampling. This means that the water may have been consumed before results on deteriorated water....... The overall aim of this PhD study was to investigate various methodological features of the ATP assay for a potential implementation on a sensor platform as a real-time parameter for continuous on-line monitoring of microbial drinking water quality. Commercial reagents are commonly used to determine ATP......, microbial quality in distributed water, detection of aftergrowth, biofilm formation etc. This PhD project demonstrated that ATP levels are relatively low and fairly stable in drinking water without chlorine residual despite different sampling locations, different drinking water systems and time of year...

  6. ATP economy of force maintenance in human tibialis anterior muscle

    DEFF Research Database (Denmark)

    Nakagawa, Yoshinao; Ratkevicius, Aivaras; Mizuno, Masao

    2005-01-01

    PURPOSE: The aim of this study was investigate ATP economy of force maintenance in the human tibialis anterior muscle during 60 s of anaerobic voluntary contraction at 50% of maximum voluntary contraction (MVC). METHODS: ATP turnover rate was evaluated using P magnetic resonance spectroscopy (P......) of the total ankle dorsiflexor muscle volume, which was 267 +/- 10 cm. Relative cross-sectional areas occupied by Type I, IIA, and IIB fibers in the tibialis anterior were 69.3 +/- 2.2, 27.4 +/- 2.76, and 3.2 +/- 1.0%, respectively. ATP economy of force maintenance did not change significantly during the 60-s...... contraction. It averaged at 4.81 +/- 0.42 N.s.micromol-1, and correlated with the relative cross-sectional area of the muscle occupied by Type I fiber (r = 0.73, P economy compared with those maintaining the force (3...

  7. Tamoxifen citrate loaded ethosomes for transdermal drug delivery system: preparation and characterization.

    Science.gov (United States)

    Sarwa, Khomendra Kumar; Suresh, Preeti K; Debnath, Manabendra; Ahmad, Mohammad Zaki

    2013-08-01

    Long term tamoxifen citrate therapy is imperative to treat several dermatological and hormonal sensitive disorders. Successful oral and parenteral administration of tamoxifen citrate has been challenging since it undergoes enzymatic degradation and has poor aqueous solubility issues. In the present work, tamoxifen citrate loaded ethosomes were prepared and characterized for transdermal applications. The prepared formulations were characterized for morphological features, particle size distribution, calorimetric attributes, zeta potential and drug entrapment. Permeation profile of prepared ethosomes was compared with liposomes and hydroethonalic solution across cellophane membrane and human cadaver skin. Results of the permeation studies indicate that ethosomes were able to deliver >90% drug within 24 hours of application, while liposomes and hydroethanolic solution delivered only 39.04% and 36.55% respectively. Skin deposition and stability studies are also reported.

  8. Women taking the “blue pill” (sildenafil citrate: such a big deal?

    Directory of Open Access Journals (Sweden)

    Lo Monte G

    2014-11-01

    Full Text Available Giuseppe Lo Monte, Angela Graziano, Isabella Piva, Roberto Marci Department of Morphology, Surgery, and Experimental Medicine, University of Ferrara, Ferrara, Italy Abstract: For years, phosphodiesterase type 5 inhibitors have been used for the treatment of erectile dysfunctions. Due to the similarities between male and female sexual response, several studies have assessed the effects of sildenafil citrate (Viagra® in women affected by female sexual arousal disorder. The results are still conflicting and the drug is not devoid of adverse effects. Furthermore, female sexual arousal disorder is a heterogeneous condition whose underlying causes are difficult to diagnose and appropriate treatment requires a thorough sexual, psychological, and medical history along with specialist consultations. The clinician should pursue a global approach to the patient with sexual difficulties, while non-hormonal treatment such as phosphodiesterase type 5 inhibitors (ie, sildenafil citrate should be kept as the last option. Keywords: phosphodiesterase type 5 inhibitors, female sexual arousal disorder (FSAD, sildenafil citrate

  9. Arabidopsis fructokinase-like protein associations are regulated by ATP.

    Science.gov (United States)

    Riggs, John W; Callis, Judy

    2017-05-10

    The Arabidopsis thaliana fructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated. Homology modeling using pfkB X-ray structures, sequence comparisons, and mutational analyses suggests that FLN proteins may bind their substrates differently from other pfkB proteins. We provide evidence that purified recombinant FLN1 undergoes an ATP-mediated change in binding affinity with both itself and recombinant FLN2. The ATP-mediated change in the affinity of FLN1 for FLN2 is not affected by mutations in conserved active-site residues known to affect catalysis in active pfkB enzymes. In contrast, recombinant FLN2 hetero-oligomerizes independently of ATP concentration. At ATP concentrations that promote FLN1 homomeric interactions, the FLN1-FLN2 hetero-oligomer is the dominant form in vitro We further present evidence that FLN1 associates with a large protein complex in chloroplasts independently of ATP. Given that ATP levels fluctuate between light-dark cycles in the 1-5 mM range, we propose that changes in FLN1 and FLN2 interactions are biologically meaningful. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  10. Mechanism of ATP turnover inhibition in the EJC

    Science.gov (United States)

    Nielsen, Klaus H.; Chamieh, Hala; Andersen, Christian B.F.; Fredslund, Folmer; Hamborg, Kristiane; Le Hir, Hervé; Andersen, Gregers R.

    2009-01-01

    The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF3, the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC–ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5–RNA–ADP–AlF3, suggesting that the use of ADP–AlF3 may be a valuable tool for examining DEAD-box ATPases in general. PMID:19033377

  11. The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells.

    Science.gov (United States)

    Neumann, Christina; Fraiture, Malou; Hernàndez-Reyes, Casandra; Akum, Fidele N; Virlogeux-Payant, Isabelle; Chen, Ying; Pateyron, Stephanie; Colcombet, Jean; Kogel, Karl-Heinz; Hirt, Heribert; Brunner, Frédéric; Schikora, Adam

    2014-01-01

    Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

  12. Production of endo-pectate lyase by two stage cultivation of Erwinia carotovora

    Energy Technology Data Exchange (ETDEWEB)

    Fukuoka, Satoshi; Kobayashi, Yoshiaki

    1987-02-26

    The productivity of endo-pectate lyase from Erwinia carotovora GIR 1044 was found to be greatly improved by two stage cultivation: in the first stage the bacterium was grown with an inducing carbon source, e.g., pectin, and in the second stage it was cultivated with glycerol, xylose, or fructose with the addition of monosodium L-glutamate as nitrogen source. In the two stage cultivation using pectin or glycerol as the carbon source the enzyme activity reached 400 units/ml, almost 3 times as much as that of one stage cultivation in a 10 liter fermentor. Using two stage cultivation in the 200 liter fermentor improved enzyme productivity over that in the 10 liter fermentor, with 500 units/ml of activity. Compared with the cultivation in Erlenmeyer flasks, fermentor cultivation improved enzyme productivity. The optimum cultivating conditions were agitation of 480 rpm with aeration of 0.5 vvm at 28 /sup 0/C. (4 figs, 4 tabs, 14 refs)

  13. The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells

    KAUST Repository

    Neumann, Christina

    2014-10-17

    Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

  14. S1P Lyase Regulation of Thymic Egress and Oncogenic Inflammatory Signaling

    Directory of Open Access Journals (Sweden)

    Ashok Kumar

    2017-01-01

    Full Text Available Sphingosine-1-phosphate (S1P is a potent lipid signaling molecule that regulates pleiotropic biological functions including cell migration, survival, angiogenesis, immune cell trafficking, inflammation, and carcinogenesis. It acts as a ligand for a family of cell surface receptors. S1P concentrations are high in blood and lymph but low in tissues, especially the thymus and lymphoid organs. S1P chemotactic gradients are essential for lymphocyte egress and other aspects of physiological cell trafficking. S1P is irreversibly degraded by S1P lyase (SPL. SPL regulates lymphocyte trafficking, inflammation and other physiological and pathological processes. For example, SPL located in thymic dendritic cells acts as a metabolic gatekeeper that controls the normal egress of mature T lymphocytes from the thymus into the circulation, whereas SPL deficiency in gut epithelial cells promotes colitis and colitis-associated carcinogenesis (CAC. Recently, we identified a complex syndrome comprised of nephrosis, adrenal insufficiency, and immunological defects caused by inherited mutations in human SGPL1, the gene encoding SPL. In the present article, we review current evidence supporting the role of SPL in thymic egress, inflammation, and cancer. Lastly, we summarize recent progress in understanding other SPL functions, its role in inherited disease, and SPL targeting for therapeutic purposes.

  15. Phenylalanine ammonia-lyase2.1 contributes to the soybean response towards Phytophthora sojae infection.

    Science.gov (United States)

    Zhang, Chuanzhong; Wang, Xin; Zhang, Feng; Dong, Lidong; Wu, Junjiang; Cheng, Qun; Qi, Dongyue; Yan, Xiaofei; Jiang, Liangyu; Fan, Sujie; Li, Ninghui; Li, Dongmei; Xu, Pengfei; Zhang, Shuzhen

    2017-08-03

    Phytophthora root and stem rot of soybean [Glycine max (L.) Merr.] caused by Phytophthora sojae is a destructive disease worldwide. Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes related to plant responses to biotic and abiotic stresses. However, the molecular mechanism of PAL in soybean in response to P. sojae is largely unclear. Here, we characterize a novel member of the soybean PAL gene family, GmPAL2.1, which is significantly induced by P. sojae. Overexpression and RNA interference analysis demonstrates that GmPAL2.1 enhances resistance to P. sojae in transgenic soybean plants. In addition, the PAL activity in GmPAL2.1-OX transgenic soybean is significantly higher than that of non-transgenic plants after infection with P. sojae, while that in GmPAL2.1-RNAi soybean plants is lower. Further analyses show that the daidzein, genistein and salicylic acid (SA) levels and the relative content of glyceollins are markedly increased in GmPAL2.1-OX transgenic soybean. Taken together, these results suggest the important role of GmPAL2.1 functioning as a positive regulator in the soybean response to P. sojae infection, possibly by enhancing the content of glyceollins, daidzein, genistein and SA.

  16. Manganese (Mn2+)-dependent storage stabilization of Rhodotorula glutinis phenylalanine ammonia-lyase activity.

    Science.gov (United States)

    Wall, Mark J; Quinn, Andrew J; D'Cunha, Godwin B

    2008-02-13

    Phenylalanine ammonia-lyase (PAL; E C 4.3.1.5) reverse reaction has been exploited for the commercial production of optically pure l-phenylalanine from trans-cinnamic acid. Optimal conditions for the growth and PAL activity of Rhodotorula glutinis cells and an improved method for the synthesis of l-phenylalanine have been reported. A major problem encountered during these studies was rapid loss of PAL activity during storage of the yeast cells, which were therefore unsuitable for long-term and repeated use. Enhancement of enzyme stability in the presence of various additives including polyhydric compounds and metal ions is described. Whole cells retained nearly 85% of the original enzyme activity for at least 12 weeks when a low concentration of Mn2+ (0.01%) was included in the storage buffer medium (50 mM Tris-HCl, pH 8.8). In contrast, glutinis whole cells. The data suggest that Mn2+ ions may be responsible for the specific stabilization of a more active conformation of the enzyme. In addition, enzyme stability as a function of temperature was studied, and the optimal temperature for maximal activity retention was 0-2 degrees C. The effects of various additives on the induction of PAL have also been examined. These results could have direct implications in studies on activity, inhibition, and reaction mechanism of this biotechnologically important enzyme.

  17. Hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase from Rhodotorula glutinis.

    Science.gov (United States)

    Cui, Jian dong; Cui, Li li; Zhang, Song ping; Zhang, Yu fei; Su, Zhi guo; Ma, Guang hui

    2014-01-01

    Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

  18. Synthesis of specifically labelled L-phenylalanines using phenylalanine ammonia lyase activity

    Energy Technology Data Exchange (ETDEWEB)

    Haedener, A.; Tamm, Ch.

    1987-11-01

    Specifically labelled L-phenylalanines have been prepared using a variety of classical synthetic methods in combination with phenylalanine ammonia lyase (PAL) enzyme activity of the yeast Rhodosporidium toruloides ATCC 10788 or Rhodotorula glutinis IFO 0559, respectively. Thus, L-(2-/sup 2/H)phenyl-(2-/sup 2/H)alanine was formed from (E) -(2,2'-/sup 2/H/sub 2/)cinnamic acid and ammonia in 46% yield, whereas L-phenyl-(2-/sup 13/C, /sup 15/N)alanine was obtained from (E)-(2-/sup 13/C)cinnamic acid in 45% overall yield. Generally, labelled cinnamic acids were recovered in pure form from the reaction mixture, with a loss of 6-8%. Likewise, unchanged /sup 15/NH/sub 3/ was reisolated as /sup 15/NH/sub 4/Cl after steam distillation with overall losses of less than 4%. Labelled cinnamic acids were prepared by Knoevenagel condensations between appropriately labelled benzaldehydes and malonic acids. (2-/sup 2/H)Benzaldehyde was obtained from 2-bromotoluene by decomposition of the corresponding Grignard reagent with /sup 2/H/sub 2/O and subsequent oxidation. Since simple molecules, most of them commercially available in labelled form or otherwise easily accessible, may serve as starting material, and due to its defined stereochemistry, the reaction catalysed by PAL opens a short and attractive route to specifically labelled L-phenylalanines.

  19. Optimization of oligomeric enzyme activity in ionic liquids using Rhodotorula glutinis yeast phenylalanine ammonia lyase.

    Science.gov (United States)

    Barron, Christiaan C; Sponagle, Brandon J D; Arivalagan, Pugazhendhi; D'Cunha, Godwin B

    2017-01-01

    Phenylalanine ammonia lyase (E.C.4.3.1.24, PAL) activity of Rhodotorula glutinis yeast has been demonstrated in four commonly used ionic liquids. PAL forward reaction was carried out in 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO4]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium lactate ([BMIM][lactate]). Our experiments have revealed that PAL is catalytically active in ionic liquids and the enzyme activity in ([BMIM][PF6]) is comparable to that obtained in aqueous buffer medium. Different conditions were optimized for maximal PAL forward activity including time of incubation (30.0min)L-phenylalanine substrate concentration (30.0mM), nature of buffer (50.0mM Tris-HCl), pH (9.0), temperature (37°C), and speed of agitation (100 rev min-1). Under these optimized conditions, about 83% conversion of substrate to product was obtained for the PAL forward reaction that was determined using UV spectroscopy at 290nm. PAL reverse reaction in ([BMIM][PF6]) was determined spectrophotometrically at 520nm; and about 59% substrate conversion was obtained. This data provides further knowledge in enzyme biocatalysis in non-aqueous media, and may be of importance when studying the function of other oligomeric/multimeric proteins and enzymes in ionic liquids. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Immobilization of cross-linked phenylalanine ammonia lyase aggregates in microporous silica gel.

    Directory of Open Access Journals (Sweden)

    Jian Dong Cui

    Full Text Available A separable and highly-stable enzyme system was developed by adsorption of phenylalanine ammonia lyase (PAL from Rhodotorula glutinis in amino-functionalized macroporous silica gel and subsequent enzyme crosslinking. This resulted in the formation of cross-linked enzyme aggregates (PAL-CLEAs into macroporous silica gel (MSG-CLEAs. The effect of adsorptive conditions, type of aggregating agent, its concentration as well as that of cross-linking agent was studied. MSG-CLEAs production was most effective using ammonium sulfate (40%-saturation, followed by cross-linking for 1 h with 1.5% (v/v glutaraldehyde. The resulting MSG-CLEAs extended the optimal temperature and pH range compared to free PAL and PAL-CLEAs. Moreover, MSG-CLEAs exhibited the excellent stability of the enzyme against various deactivating conditions such as temperature and denaturants, and showed higher storage stability compared to the free PAL and the conventional PAL-CLEAs. Such as, after 6 h incubation at 60°C, the MSG-CLEAs still retained more than 47% of the initial activity whereas PAL-CLEAs only retained 7% of the initial activity. Especially, the MSG-CLEAs exhibited good reusability due to its suitable size and active properties. These results indicated that PAL-CLEAs on MSG might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

  1. Hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase from Rhodotorula glutinis.

    Directory of Open Access Journals (Sweden)

    Jian dong Cui

    Full Text Available Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM and confocal laser scanning microscopy (CLSM indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.

  2. Mutations underlying 3-Hydroxy-3-Methylglutaryl CoA Lyase deficiency in the Saudi population

    Directory of Open Access Journals (Sweden)

    Rashed Mohammed S

    2006-12-01

    Full Text Available Abstract Background 3-Hydroxy-3-Methylglutaric aciduria (3HMG, McKusick: 246450 is an autosomal recessive branched chain organic aciduria caused by deficiency of the enzyme 3-Hydroxy-3-Methylglutaryl CoA lyase (HL, HMGCL, EC 4.1.3.4. HL is encoded by HMGCL gene and many mutations have been reported. 3HMG is commonly observed in Saudi Arabia. Methods We utilized Whole Genome Amplification (WGA, PCR and direct sequencing to identify mutations underlying 3HMG in the Saudi population. Two patients from two unrelated families and thirty-four 3HMG positive dried blood spots (DBS were included. Results We detected the common missense mutation R41Q in 89% of the tested alleles (64 alleles. 2 alleles carried the frame shift mutation F305fs (-2 and the last two alleles had a novel splice site donor IVS6+1G>A mutation which was confirmed by its absence in more than 100 chromosomes from the normal population. All mutations were present in a homozygous state, reflecting extensive consanguinity. The high frequency of R41Q is consistent with a founder effect. Together the three mutations described account for >94% of the pathogenic mutations underlying 3HMG in Saudi Arabia. Conclusion Our study provides the most extensive genotype analysis on 3HMG patients from Saudi Arabia. Our findings have direct implications on rapid molecular diagnosis, prenatal and pre-implantation diagnosis and population based prevention programs directed towards 3HMG.

  3. Temporal and spatial expression of amygdalin hydrolase and (R)-(+)-mandelonitrile lyase in black cherry seeds.

    Science.gov (United States)

    Zheng, L; Poulton, J E

    1995-09-01

    In black cherry (Prunus serotina Ehrh.) macerates, the cyanogenic diglucoside (R)-amygdalin undergoes stepwise degradation to HCN catalyzed by amygdalin hydrolase (AH), prunasin hydrolase, and (R)-(+)-mandelonitrile lyase (MDL). A near full-length AH cDNA clone (pAH1), whose insert encodes the isozyme AH I, has been isolated and sequenced. AH I exhibits several features characteristic of beta-glucosidases of the BGA family, including their likely nucleophile center (isoleucine-threonine-glutamic acid-asparagine-glycine) and acid catalyst (asparagine-glutamic acid-proline/isoleucine) motifs. The temporal expression of AH and MDL in ripening fruit was analyzed by northern blotting. Neither mRNA was detectable until approximately 40 days after flowering (DAF), when embryos first became visible to the naked eye. Both mRNAs peaked at approximately 49 DAF before declining to negligible levels when the fruit matured (82 DAF). Taken together with enzyme activity data, these time courses suggest that AH and MDL expression may be under transcriptional control during fruit maturation. In situ hybridization analysis indicated that AH transcripts are restricted to the procambium, whereas MDL transcripts are localized within cotyledonary parenchyma cells. These tissue-specific distributions are consistent with the major locations of AH and MDL protein in mature seeds previously determined by immunocytochemistry (E. Swain, C.P. Li, and J.E. Poulton [1992] Plant Physiol 100:291-300).

  4. Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

    Directory of Open Access Journals (Sweden)

    Qing Jin

    Full Text Available In this study, a phenylalanine ammonia-lyase (PAL gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748 has 2,458 bps and contains a complete open reading frame (ORF of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

  5. Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

    Science.gov (United States)

    Jin, Qing; Yao, Yao; Cai, Yongping; Lin, Yi

    2013-01-01

    In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

  6. Purification and characterization of pectate lyase from banana (Musa acuminata) fruits.

    Science.gov (United States)

    Payasi, Anurag; Misra, Prakash C; Sanwal, Girdhar G

    2006-05-01

    Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.

  7. Effect of phytohormones on pectate lyase activity in ripening Musa acuminata.

    Science.gov (United States)

    Payasi, Anurag; Misra, P C; Sanwal, G G

    2004-12-01

    A differential activity peak of pectate lyase (PEL) was observed during ripening of banana fruits (Musa acuminata Harichhal) receiving different hormone treatments. Exposure of fruits to 25 ppm ethylene for 24 h, as well as dipping of M. acuminata fruits in 1 mM 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 h, hastened fruit ripening. Both PEL activity peak and climacteric peak were observed on the 4th and 10th days of treatment with ethylene and 2,4-D, respectively, compared to the 16th day in control fruits. Gibberellic acid (GA) treatment retarded fruit ripening and both PEL activity and climacteric peaks were observed on the 19th day. Treatment of fruits with ethylene or 2,4-D also advanced the appearance of a polygalacturonase (PG) peak and GA delayed its appearance, but the activity peaks always appeared in post-climacteric fruits, in contrast to PEL activity peaks coinciding with the respiratory peaks.

  8. Probing Reversible Chemistry in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase with Kinetic Isotope Effects

    Science.gov (United States)

    Jones, Alex R; Rentergent, Julius; Scrutton, Nigel S; Hay, Sam

    2015-01-01

    Coenzyme B12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5′-deoxyadenosyl moiety of the intrinsic coenzyme B12, it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5′-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5′-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small. PMID:25950663

  9. Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.

    Science.gov (United States)

    Jones, Alex R; Rentergent, Julius; Scrutton, Nigel S; Hay, Sam

    2015-06-08

    Coenzyme B12 -dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5'-deoxyadenosyl moiety of the intrinsic coenzyme B12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5'-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5'-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  10. Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta

    Science.gov (United States)

    Lauble, Hanspeter; Miehlich, Burkhard; Förster, Siegfried; Kobler, Christoph; Wajant, Harald; Effenberger, Franz

    2002-01-01

    Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and ∼450-fold, respectively, compared with the wild-type MeHNL. The crystal structure of the MeHNL-W128A substrate-free form at 2.1 Å resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL-W128A–4-hydroxybenzaldehyde complex structure at 2.1 Å resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center. PMID:11742123

  11. Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

    Science.gov (United States)

    Lovric, Svjetlana; Goncalves, Sara; Oskouian, Babak; Srinivas, Honnappa; Choi, Won-Il; Shril, Shirlee; Ashraf, Shazia; Tan, Weizhen; Rao, Jia; Airik, Merlin; Schapiro, David; Braun, Daniela A.; Sadowski, Carolin E.; Schmidt, Johanna Magdalena; Girik, Vladimir; Capitani, Guido; Suh, Jung H.; Lachaussée, Noëlle; Arrondel, Christelle; Patat, Julie; Furlano, Monica; Boyer, Olivia; Schmitt, Alain; Vuiblet, Vincent; Hashmi, Seema; Wilcken, Rainer; Bernier, Francois P.; Innes, A. Micheil; Parboosingh, Jillian S.; Lamont, Ryan E.; Midgley, Julian P.; Wright, Nicola; Majewski, Jacek; Zenker, Martin; Schaefer, Franz; Kuss, Navina; Giese, Thomas; Schwarz, Klaus; Catheline, Vilain; Franke, Ingolf; Sznajer, Yves; Truant, Anne S.; Adams, Brigitte; Désir, Julie; Biemann, Ronald; Pei, York; Lloberas, Nuria; Madrid, Alvaro; Dharnidharka, Vikas R.; Connolly, Anne M.; Willing, Marcia C.; Cooper, Megan A.; Lifton, Richard P.; Simons, Matias; Riezman, Howard; Antignac, Corinne; Saba, Julie D.

    2017-01-01

    Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS. PMID:28165339

  12. Over-expression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification.

    Science.gov (United States)

    Siritunga, Dimuth; Arias-Garzon, Diana; White, Wanda; Sayre, Richard T

    2004-01-01

    Cassava (Manihot esculenta, Crantz) roots are the primary source of calories for more than 500 million people, the majority of whom live in the developing countries of Africa. Cassava leaves and roots contain potentially toxic levels of cyanogenic glycosides. Consumption of residual cyanogens (linamarin or acetone cyanohydrin) in incompletely processed cassava roots can cause cyanide poisoning. Hydroxynitrile lyase (HNL), which catalyses the conversion of acetone cyanohydrin to cyanide, is expressed predominantly in the cell walls and laticifers of leaves. In contrast, roots have very low levels of HNL expression. We have over-expressed HNL in transgenic cassava plants under the control of a double 35S CaMV promoter. We show that HNL activity increased more than twofold in leaves and 13-fold in roots of transgenic plants relative to wild-type plants. Elevated HNL levels were correlated with substantially reduced acetone cyanohydrin levels and increased cyanide volatilization in processed or homogenized roots. Unlike acyanogenic cassava, transgenic plants over-expressing HNL in roots retain the herbivore deterrence of cyanogens while providing a safer food product.

  13. Diagnosis of adenylosuccinate lyase deficiency by metabolomic profiling in plasma reveals a phenotypic spectrum

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2016-09-01

    Full Text Available Adenylosuccinate lyase (ADSL deficiency is a rare autosomal recessive neurometabolic disorder that presents with a broad-spectrum of neurological and physiological symptoms. The ADSL gene produces an enzyme with binary molecular roles in de novo purine synthesis and purine nucleotide recycling. The biochemical phenotype of ADSL deficiency, accumulation of SAICAr and succinyladenosine (S-Ado in biofluids of affected individuals, serves as the traditional target for diagnosis with targeted quantitative urine purine analysis employed as the predominate method of detection. In this study, we report the diagnosis of ADSL deficiency using an alternative method, untargeted metabolomic profiling, an analytical scheme capable of generating semi-quantitative z-score values for over 1000 unique compounds in a single analysis of a specimen. Using this method to analyze plasma, we diagnosed ADSL deficiency in four patients and confirmed these findings with targeted quantitative biochemical analysis and molecular genetic testing. ADSL deficiency is part of a large a group of neurometabolic disorders, with a wide range of severity and sharing a broad differential diagnosis. This phenotypic similarity among these many inborn errors of metabolism (IEMs has classically stood as a hurdle in their initial diagnosis and subsequent treatment. The findings presented here demonstrate the clinical utility of metabolomic profiling in the diagnosis of ADSL deficiency and highlights the potential of this technology in the diagnostic evaluation of individuals with neurologic phenotypes.

  14. Attenuated adenylosuccinate lyase deficiency: a report of one case and a review of the literature.

    Science.gov (United States)

    Jurecka, Agnieszka; Zikanova, Marie; Jurkiewicz, Elżbieta; Tylki-Szymańska, Anna

    2014-02-01

    We present a 9-year follow-up of a patient with an attenuated (type II) adenylosuccinate lyase deficiency with no obvious signs of disease progression and degradation. We also review the literature, focusing on attenuated phenotype, and we report a positive effect of a ketogenic diet on seizure control. The patient presented at the age of 5 months with a history of global developmental delay. Screening of urinary purine metabolites revealed elevation of succinyladenosine and succinylaminoimidazolecarboxamide riboside (a ratio of 2:1). Mutation analysis revealed a compound heterozygosity for missense mutations: p.R426H and p.D268H. She began to walk independently at the age of 3 years. From the age of 4 years, her communication skills improved and she presented fewer autistic features. Due to poor results in seizure control, the ketogenic diet was introduced at the age of 7 years, resulting in reduction of seizure frequency. Currently, at the age of 9 years, the girl is attending a special kindergarten and is functioning very well in her preschool group. She began to make statements that form a logical continuity and make progress in simple manual operations. The patient participates in therapies such as pet therapy, hippotherapy, speech therapy, physiotherapy, hydrotherapy, and music therapy. Georg Thieme Verlag KG Stuttgart · New York.

  15. The Skin Bacterium Propionibacterium acnes Employs Two Variants of Hyaluronate Lyase with Distinct Properties.

    Science.gov (United States)

    Nazipi, Seven; Stødkilde-Jørgensen, Kristian; Scavenius, Carsten; Brüggemann, Holger

    2017-09-12

    Hyaluronic acid (HA) and other glycosaminoglycans are extracellular matrix components in the human epidermis and dermis. One of the most prevalent skin microorganisms, Propionibacterium acnes, possesses HA-degrading activity, possibly conferred by the enzyme hyaluronate lyase (HYL). In this study, we identified the HYL of P. acnes and investigated the genotypic and phenotypic characteristics. Investigations include the generation of a P. acneshyl knockout mutant and HYL activity assays to determine the substrate range and formed products. We found that P. acnes employs two distinct variants of HYL. One variant, HYL-IB/II, is highly active, resulting in complete HA degradation; it is present in strains of the phylotypes IB and II. The other variant, HYL-IA, has low activity, resulting in incomplete HA degradation; it is present in type IA strains. Our findings could explain some of the observed differences between P. acnes phylotype IA and IB/II strains. Whereas type IA strains are primarily found on the skin surface and associated with acne vulgaris, type IB/II strains are more often associated with soft and deep tissue infections, which would require elaborate tissue invasion strategies, possibly accomplished by a highly active HYL-IB/II.

  16. The effect of methyl-donated hydrogen bonding on active site conformations of hyaluronate lyase

    Science.gov (United States)

    Migues, Angela N.; Vergenz, Robert A.; Moore, Kevin B.

    2010-03-01

    Geometric evidence shows a val-A252 methyl-donated (MD) hydrogen bond (HB) in hyaluronate lyase (Streptococcus pneumoniae) interacts with nearby NH--O and OH--O HBs, distorting active-site helical structure. Results for model fragment A248-254 are based on experimental heavy atom positions with ab initio hydrogen atoms. The MDHB, with (H-O distance, donor-H-O angle) = (2.3å; 174^o), exhibits more favorable geometry than thr-A253 OH--O HB (1.8å; 170^o) to the same ala-249 C=O. Consequently, thr-253 N-H--O interaction is forced closer to lys-250 C=O than ala-249 C=O(2.6 versus 2.7å). A novel method has been developed to quantify the effects of atomic diplacements on motions of neighboring helices. A coordinate system was established to track the movement of specific residues and to ascertain the effect of such motions on active site conformations.

  17. In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

    Science.gov (United States)

    Stephen, Abish S; Millhouse, Emma; Sherry, Leighann; Aduse-Opoku, Joseph; Culshaw, Shauna; Ramage, Gordon; Bradshaw, David J; Burnett, Gary R; Allaker, Robert P

    2016-01-01

    Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (pgingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.

  18. PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris.

    Science.gov (United States)

    Jiang, Jingjing; Yao, Lina; Yu, Youjian; Lv, Meiling; Miao, Ying; Cao, Jiashu

    2014-11-01

    PECTATE LYASE-LIKE10 (PLL10) was previously identified as one of the differentially expressed genes both in microspores during the late pollen developmental stages and in pistils during the fertilization process in Chinese cabbage (Brassica campestris ssp. chinensis). Here, antisense-RNA was used to study the functions of BcPLL10 in Chinese cabbage. Abnormal pollen was identified in the transgenic lines (bcpll10-4, -5, and -6). In fertilization experiments, fewer seeds were harvested when the antisense-RNA lines were used as pollen donor. In vivo and in vitro pollen germination assays less germinated pollen tubes were observed in bcpll10 lines. Scanning electron microscopy observation verified that the tryphine materials were over accumulated around the pollen surface and sticked them together in bcpll10. Moreover, transmission electron microscopy observation revealed that the internal endintine was overdeveloped and predominantly occupied the intine, and disturbed the normal proportional distribution of the two layers in the non-germinal furrow region; and no obvious demarcation existed between them in the germinal furrow region in the bcpll10 pollen. Collectively, this study presented a novel PLL gene that played an important role during the pollen wall development in B. campestris, which may also possess potential importance for male sterility usage in agriculture. © 2014 Institute of Botany, Chinese Academy of Sciences.

  19. PECTATE LYASE-LIKE 9 from Brassica campestris is associated with intine formation.

    Science.gov (United States)

    Jiang, Jingjing; Yao, Lina; Yu, Youjian; Liang, Ying; Jiang, Jianxia; Ye, Nenghui; Miao, Ying; Cao, Jiashu

    2014-12-01

    Brassica campestris pectate lyase-like 9 (BcPLL9) was previously identified as a differentially expressed gene both in buds during late pollen developmental stage and in pistils during fertilization in Chinese cabbage. To characterize the gene's function, antisense-RNA lines of BcPLL9 (bcpll9) were constructed in Chinese cabbage. Self- and cross-fertilization experiments harvested half seed yields when bcpll9 lines were used as pollen donors. In vivo and in vitro pollen germination assays showed that nearly half of the pollen tubes in bcpll9 were irregular with shorter length and uneven surface. Aniline blue staining identified abnormal accumulation of a specific bright blue unknown material in the bcpll9 pollen portion. Scanning electron microscopy observation verified the abnormal outthrust material to be near the pollen germinal furrows. Transmission electron microscopy observation revealed the internal endintine layer was overdeveloped and predominantly occupied the intine. This abnormally formed intine likely induced the wavy structure and growth arrest of the pollen tube in half of the bcpll9 pollen grains, which resulted in less seed yields. Collectively, this study presented a novel PLL gene that has an important function in B. campestris intine formation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  20. An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification.

    Science.gov (United States)

    Xu, S X; Qin, X; Liu, B; Zhang, D Q; Zhang, W; Wu, K; Zhang, Y H

    2015-02-01

    The pectin lyase gene pnl-zj5a from Aspergillus niger ZJ5 was identified and expressed in Pichia pastoris. PNL-ZJ5A was purified by ultrafiltration, anion exchange and gel chromatography. The Km and Vmax values determined using citrus pectin were 0.66 mg ml(-1) and 32.6 μmol min(-1) mg(-1) , respectively. PNL-ZJ5A exhibited optimal activity at 43°C and retained activity over 25-50°C. PNL-ZJ5A was optimally active at pH 5 and effective in apple juice clarification. Compared with controls, PNL-ZJ5A increased the fruit juice yield significantly. Furthermore, PNL-ZJ5A reduced the viscosity of apple juice by 38.8% and increased its transmittance by 86.3%. PNL-ZJ5A combined with a commercial pectin esterase resulted in higher juice volume. © 2014 The Society for Applied Microbiology.

  1. Continuous synthesis of hexanal by immobilized hydroperoxide lyase in packed-bed reactor.

    Science.gov (United States)

    Liu, Qingqing; Hua, Yufei

    2015-12-01

    This study aimed to develop an optimal continuous procedure of immobilized hydroperoxide lyase (HPL)-catalyzed synthesis of hexanal. A central composite design was used to study the combined effect of substrate concentration and the residence time of the reactant on hexanal concentration. The optimum conditions for hexanal synthesis included a 13-HPOD concentration of 43.54 mM and a residence time of 60.99 min. The maximum hexanal concentration was 3560 ± 130 mg/L when 16 U of immobilized HPLwas used. Furthermore, the stability of immobilized HPL was significantly improved in the packed-bed reactor, as evidenced by the slowed enzyme inactivation and prolonged operation time. The immobilized HPL remained activity until 40 mL substrate solution flowed past the packed-bed reactor. The catalyst productivity of hexanal in the packed-bed reactor was 5.35 ± 0.34 mg/U, much higher than that in the batch stirred reactor. This study was greatly meaningful for providing a green method to the large-scale production of hexanal.

  2. Revised domain structure of ulvan lyase and characterization of the first ulvan binding domain.

    Science.gov (United States)

    Melcher, Rebecca L J; Neumann, Marten; Fuenzalida Werner, Juan Pablo; Gröhn, Franziska; Moerschbacher, Bruno M

    2017-03-22

    Biomass waste products from green algae have recently been given new life, as these polysaccharides have potential applications in industry, agriculture, and medicine. One such polysaccharide group called ulvans displays many different, potentially useful properties that arise from their structural versatility. Hence, performing structural analyses on ulvan is crucial for future applications. However, chemical reaction-based analysis methods cannot fully characterize ulvan and tend to alter its structure. Thus, better methods require well-characterized ulvan-degrading enzymes. Therefore, we analysed a previously sequenced ulvan lyase (Genebank TM reference number JN104480) and characterized its domains. We suggest that the enzyme consists of a shorter than previously described catalytic domain, a newly identified substrate binding domain, and a C-terminal type 9 secretion system signal peptide. By separately expressing the two domains in E. coli, we confirmed that the binding domain is ulvan specific, having higher affinity for ulvan than most lectins for their ligands (affinity constant: 10 5  M -1 ). To our knowledge, this is the first description of an ulvan-binding domain. Overall, identifying this new binding domain is one step towards engineering ulvan enzymes that can be used to characterize ulvan, e.g. through enzymatic/mass spectrometric fingerprinting analyses, and help unlock its full potential.

  3. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

    Directory of Open Access Journals (Sweden)

    Juliana Oliveira Lima

    Full Text Available Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120 h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

  4. Potential Inhibitors for Isocitrate Lyase of Mycobacterium tuberculosis and Non-M. tuberculosis: A Summary

    Directory of Open Access Journals (Sweden)

    Yie-Vern Lee

    2015-01-01

    Full Text Available Isocitrate lyase (ICL is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle, especially Mycobacterium tuberculosis (MTB. In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a MTB ICL with natural compounds; (b MTB ICL with synthetic compounds; (c non-MTB ICL with natural compounds; and (d non-MTB ICL with synthetic compounds. Each of the approaches is capable of overcoming different challenges of inhibitor discovery. We hope that this paper will benefit the discovery of better inhibitor for ICL.

  5. Molecular Cloning and Characterization of Hydroperoxide Lyase Gene in the Leaves of Tea Plant (Camellia sinensis).

    Science.gov (United States)

    Deng, Wei-Wei; Wu, Yi-Lin; Li, Ye-Yun; Tan, Zhen; Wei, Chao-Ling

    2016-03-02

    Hydroperoxide lyase (HPL, E.C. 4.1.2.) is the major enzyme in the biosynthesis of natural volatile aldehydes and alcohols in plants, however, little was known about HPL in tea plants (Camellia sinensis). A unique cDNA fragment was isolated by suppressive subtractive hybridization (SSH) from a tea plant subjected to herbivory by tea geometrid Ectropis obliqua. This full length cDNA acquired by RACE was 1476 bp and encoded 491 amino acids. DNA and protein BLAST searches showed high homology to HPL sequences from other plants. The His-tag expression vector pET-32a(+)/CsHPL was constructed and transferred into Escherichia coli Rosetta (DE3). The expression product of recombinant CsHPL in E. coli was about 60 kDa. The enzyme activity of CsHPL was 0.20 μmol·min(-1)·mg(-1). Quantitative RT-PCR analysis indicated CsHPL was strongly up-regulated in tea plants after Ectropis obliqua attack, suggesting that it may be an important candidate for defense against insects in tea plants.

  6. Six phenylalanine ammonia-lyases from Camellia sinensis: Evolution, expression, and kinetics.

    Science.gov (United States)

    Wu, Yingling; Wang, Wenzhao; Li, Yanzhi; Dai, Xinlong; Ma, Guoliang; Xing, Dawei; Zhu, Mengqing; Gao, Liping; Xia, Tao

    2017-09-01

    Phenylalanine ammonia-lyase (PAL), the branch point enzyme controlling the flow of primary metabolism into second metabolism, converts the L-phenylalanine (L-Phe) to yield cinnamic acid. Based on the sequencing data available from eight transcriptome projects, six PAL genes have been screened out, cloned, and designated as CsPALa-CsPALf. The phylogenetic tree showed that CsPALs were divided into three subgroups, PALa and PALb, PALc and PALd, and PALe and PALf. All six CsPALs exhibited indiscriminate cytosolic locations in epidermis cells and mesophyll cells. Then, the expression profiles of six PAL genes were qualitatively investigated and they displayed tissue-/induced-expression specificity in several tissues or under different exogenous treatments. Furthermore, in vitro enzymatic assays showed that all six recombinant proteins were characterized by the strict substrate specificity toward L-Phe, but no activity toward L-Tyr, and they displayed subtle differences in kinetics and enzymatic properties. These results indicate that CsPALs play both distinct and overlapping roles in plant growth and responses to environmental cues. Copyright © 2017. Published by Elsevier Masson SAS.

  7. Adenylosuccinate lyase (ADSL) and infantile autism: Absence of previously reported point mutation

    Energy Technology Data Exchange (ETDEWEB)

    Fon, E.A.; Sarrazin, J.; Rouleau, G.A. [Montreal General Hospital (Canada)] [and others

    1995-12-18

    Autism is a heterogeneous neuropsychiatric syndrome of unknown etiology. There is evidence that a deficiency in the enzyme adenylosuccinate lyase (ADSL), essential for de novo purine biosynthesis, could be involved in the pathogenesis of certain cases. A point mutation in the ADSL gene, resulting in a predicted serine-to-proline substitution and conferring structural instability to the mutant enzyme, has been reported previously in 3 affected siblings. In order to determine the prevalence of the mutation, we PCR-amplified the exon spanning the site of this mutation from the genomic DNA of patients fulfilling DSM-III-R criteria for autistic disorder. None of the 119 patients tested were found to have this mutation. Furthermore, on preliminary screening using single-strand conformation polymorphism (SSCP), no novel mutations were detected in the coding sequence of four ADSL exons, spanning approximately 50% of the cDNA. In light of these findings, it appears that mutations in the ADSL gene represent a distinctly uncommon cause of autism. 12 refs., 2 figs.

  8. Use of Potassium Citrate to Reduce the Risk of Renal Stone Formation During Spaceflight

    Science.gov (United States)

    Whitson, P. A.; Pietrzyk, R. A.; Sams, C. F.; Jones, J. A.; Nelman-Gonzalez, M.; Hudson, E. K.

    2008-01-01

    Introduction: NASA s Vision for Space Exploration centers on exploration class missions including the goals of returning to the moon and landing on Mars. One of NASA s objectives is to focus research on astronaut health and the development of countermeasures that will protect crewmembers during long duration voyages. Exposure to microgravity affects human physiology and results in changes in the urinary chemical composition favoring urinary supersaturation and an increased risk of stone formation. Nephrolithiasis is a multifactorial disease and development of a renal stone is significantly influenced by both dietary and environmental factors. Previous results from long duration Mir and short duration Shuttle missions have shown decreased urine volume, pH, and citrate levels and increased calcium. Citrate, an important inhibitor of calcium-containing stones, binds with urinary calcium reducing the amount of calcium available to form stones. Citrate inhibits renal stone recurrence by preventing crystal growth, aggregation, and nucleation and is one of the most common therapeutic agents used to prevent stone formation. Methods: Thirty long duration crewmembers (29 male, 1 female) participated in this study. 24-hour urines were collected and dietary monitoring was performed pre, in, and postflight. Crewmembers in the treatment group received two potassium citrate (KCIT) pills, 10 mEq/pill, ingested daily beginning 3 days before launch, all inflight days and through 14 days postflight. Urinary biochemical and dietary analyses were completed. Results: KCIT treated subjects exhibited decreased urinary calcium excretion and maintained the levels of calcium oxalate supersaturation risk at their preflight levels. The increased urinary pH levels in these subjects reduced the risk of uric acid stones. Discussion: The current study investigated the use of potassium citrate as a countermeasure to minimize the risk of stone formation during ISS missions. Results suggest that

  9. Synthesis and quality control of 68Ga citrate for routine clinical PET.

    Science.gov (United States)

    Rizzello, Anna; Di Pierro, Donato; Lodi, Filippo; Trespidi, Silvia; Cicoria, Gianfranco; Pancaldi, Davide; Nanni, Cristina; Marengo, Mario; Marzola, Maria Cristina; Al-Nahhas, Adil; Rubello, Domenico; Boschi, Stefano

    2009-07-01

    Scintigraphic imaging of infection and inflammation with 67Ga-citrate is an established and powerful diagnostic tool in the management of patients with infectious or inflammatory diseases. 68Ga is a short-lived positron-emitting radionuclide (half-life 67.6 min, positron energy 2.92 MeV), which allows better imaging qualities than 67Ga using the high spatial resolution and the quantitative features of PET. The aim of this study was to develop a method of synthesis for 68Ga citrate with high and reproducible radiochemical yield using a commercial 68Ga-labelling module. The resultant 68Ga citrate would be suitable for use in the detection of infectious or inflammatory diseases in routine clinical practice. A simplified method of producing 68Ga citrate is described. Radiochemical purity, pyrogen testing were performed as per the standard protocols. After performing 10 syntheses of 68Ga citrate, the radiochemical yield was 64.1+/-6.0% (mean+/-standard deviation) with an average activity of 971.2+/-103.4 MBq available for labelling. Radiochemical purity determined by instant thin-layer chromatography-silica gel was higher than 98%. All the synthesized products were found to be sterile and pyrogen-free. In this study, the quality control step provided good and reproducible results. This is worth noting, especially in view of the stringent new rules adopted in most European countries for the in-house good manufacturing practice (GMP) synthesis of radiopharmaceuticals. The high radiochemical yield and purity showed that this method is a reliable tool for the production of 68Ga citrate to be used in the detection of inflammatory and infectious diseases using high resolution and qualitative PET.

  10. Is magnesium citrate treatment effective on pain, clinical parameters and functional status in patients with fibromyalgia?

    Science.gov (United States)

    Bagis, Selda; Karabiber, Mehmet; As, Ismet; Tamer, Lülüfer; Erdogan, Canan; Atalay, Ayçe

    2013-01-01

    The aims of this study were to investigate the relationship between magnesium levels and fibromyalgia symptoms and to determine the effect of magnesium citrate treatment on these symptoms. Sixty premenopausal women diagnosed with fibromyalgia according to the ACR criteria and 20 healthy women whose age and weight matched the premenopausal women were evaluated. Pain intensity, pain threshold, the number of tender points, the tender point index, the fibromyalgia impact questionnaire (FIQ), the Beck depression and Beck anxiety scores and patient symptoms were evaluated in all the women. Serum and erythrocyte magnesium levels were also measured. The patients were divided into three groups. The magnesium citrate (300 mg/day) was given to the first group (n = 20), amitriptyline (10 mg/day) was given to the second group (n = 20), and magnesium citrate (300 mg/day) + amitriptyline (10 mg/day) treatment was given to the third group (n = 20). All parameters were reevaluated after the 8 weeks of treatment. The serum and erythrocyte magnesium levels were significantly lower in patients with fibromyalgia than in the controls. Also there was a negative correlation between the magnesium levels and fibromyalgia symptoms. The number of tender points, tender point index, FIQ and Beck depression scores decreased significantly with the magnesium citrate treatment. The combined amitriptyline + magnesium citrate treatment proved effective on all parameters except numbness. Low magnesium levels in the erythrocyte might be an etiologic factor on fibromyalgia symptoms. The magnesium citrate treatment was only effective tender points and the intensity of fibromyalgia. However, it was effective on all parameters when used in combination with amitriptyline.

  11. Modulation of calcium oxalate monohydrate crystallization by citrate through selective binding to atomic steps

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, S R; Wierzbicki, A; Salter, E A; Zepeda, S; Orme, C A; Hoyer, J R; Nancollas, G H; Cody, A M; De Yoreo, J J

    2004-10-19

    The majority of human kidney stones are composed primarily of calcium oxalate monohydrate (COM) crystals. Thus, determining the molecular mechanisms by which urinary constituents modulate calcium oxalate crystallization is crucial for understanding and controlling urolithiassis in humans. A comprehensive molecular-scale view of COM shape modification by citrate, a common urinary constituent, obtained through a combination of in situ atomic force microscopy (AFM) and molecular modeling is now presented. We show that citrate strongly influences the growth morphology and kinetics on the (-101) face but has much lower effect on the (010) face. Moreover, binding energy calculations show that the strength of the citrate-COM interaction is much greater at steps than on terraces and is highly step-specific. The maximum binding energy, -166.5 kJ {center_dot} mol{sup -1}, occurs for the [101] step on the (-101) face. In contrast, the value is only -56.9 kJ {center_dot} mol-1 for the [012] step on the (010) face. The binding energies on the (-101) and (010) terraces are also much smaller, -65.4 and -48.9 kJ {center_dot} mol{sup -1} respectively. All other binding energies lie between these extremes. This high selectivity leads to preferential binding of citrate to the acute [101] atomic steps on the (-101) face. The strong citrate-step interactions on this face leads to pinning of all steps, but the anisotropy in interaction strength results in anisotropic reductions in step kinetics. These anisotropic changes in step kinetics are, in turn, responsible for changes in the shape of macroscopic COM crystals. Thus, the molecular scale growth morphology and the bulk crystal habit in the presence of citrate are similar, and the predictions of molecular simulations are fully consistent with the experimental observations.

  12. Comparison of Elaeagnus angustifolia Extract and Sildenafil Citrate on Female Orgasmic Disorders: A Randomized Clinical Trial.

    Science.gov (United States)

    Akbarzadeh, Marzieh; Zeinalzadeh, Sanaz; Zolghadri, Jaleh; Mohagheghzadeh, Abdolali; Faridi, Pouya; Sayadi, Mehrab

    2014-10-01

    Orgasmic disorder can create a feeling of deprivation and failure and provide mental problems, incompatibility and marital discord. This study aimed to compare the effects of Elaeagnus angustifolia flower extract and sildenafil citrate on female orgasmic disorder in women in 2013. In this randomized clinical trial, 125 women between 18-40 years old who suffered from orgasmic disorder were divided into three E. angustifolia, sildenafil citrate and control groups. The data were gathered using Female Sexual Function Index and through measurement of TSH and prolactin. The first intervention group had to consume 4.5 gr E. angustifolia extract in two divided doses for 35 days and the second one had to use 50 mg sildenafil citrate tablets for 4 weeks one hour before their sexual relationship. However, the control group had to consume the placebo. The data were analyzed using paired t-test, one-way ANOVA, and Bonferroni posthoc test and porgasmic disorder before the intervention was 41.5%, 40.5%, and 57.1% in E. angustifolia, sildenafil citrate, and control groups, respectively (p=0.23). However, these measures were respectively 29.3%, 16.7%, and 50% after the intervention (p=0.004). A significant difference between the two groups regarding sexual satisfaction after the intervention (p=0.003) compared to the beginning of the study (p=0.356). Besides, the highest reduction of changes after the intervention (58.82%) was observed in the sildenafil citrate group. Both E. angustifolia extract and sildenafil citrate were effective in reduction of the frequency of orgasmic disorder in women.

  13. Aggregation Kinetics of Citrate and Polyvinylpyrrolidone Coated Silver Nanoparticles in Monovalent and Divalent Electrolyte Solutions

    Science.gov (United States)

    Huynh, Khanh An; Chen, Kai Loon

    2011-01-01

    The aggregation kinetics of silver nanoparticles (AgNPs) that were coated with two commonly used capping agents—citrate and polyvinylpyrrolidone (PVP)—were investigated. Time-resolved dynamic light scattering (DLS) was employed to measure the aggregation kinetics of the AgNPs over a range of monovalent and divalent electrolyte concentrations. The aggregation behavior of citrate-coated AgNPs in NaCl was in excellent agreement with the predictions based on Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, and the Hamaker constant of citrate-coated AgNPs in aqueous solutions was derived to be 3.7 × 10-20 J. Divalent electrolytes were more efficient in destabilizing the citrate-coated AgNPs, as indicated by the considerably lower critical coagulation concentrations (2.1 mM CaCl2 and 2.7 mM MgCl2 vs. 47.6 mM NaCl). The PVP-coated AgNPs were significantly more stable than citrate-coated AgNPs in both NaCl and CaCl2, which is likely due to steric repulsion imparted by the large, non-charged polymers. The addition of humic acid resulted in the adsorption of the macromolecules on both citrate- and PVP-coated AgNPs. The adsorption of humic acid induced additional electrosteric repulsion that elevated the stability of both nanoparticles in suspensions containing NaCl or low concentrations of CaCl2. Conversely, enhanced aggregation occurred for both nanoparticles at high CaCl2 concentrations due to interparticle bridging by humic acid clusters. PMID:21630686

  14. The effect of caffeine citrate on neural breathing pattern in preterm infants.

    Science.gov (United States)

    Parikka, Vilhelmiina; Beck, Jennifer; Zhai, Qian; Leppäsalo, Juha; Lehtonen, Liisa; Soukka, Hanna

    2015-10-01

    Caffeine citrate is widely used to prevent and treat prematurity-associated apnea. The aim of this study was to characterize the effect of caffeine citrate on the neural control of breathing, especially central apnea, in premature infants. Preterm infants were evaluated for 30min before and 30min after caffeine citrate loading (20mg/kg). A feeding tube including miniaturized sensors was used to measure the diaphragm electrical activity (Edi) waveform. Central apnea was defined as any period where the Edi waveform was flat for >5s. Seventeen preterm infants with a mean age of three days and mean birth weight of 900 grams were evaluated. In addition to central apnea, several parameters including neural inspiratory time, neural respiratory rate, peak Edi, delta inspiratory change in Edi (phasic Edi) and minimum Edi on exhalation were measured. The majority of the apnea were short (5 to 10s) and the number of apnea correlated with birth weight (p=0.039). Caffeine citrate reduced significantly the number of 5-to-10-second-long central apnea during the 30-minute periods (12±11 to 7±7; p=0.02). Caffeine citrate increased both peak and phasic Edi leading to a significant increase in the diaphragm energy expenditure. Edi signal can be reliably measured and processed to study changes in premature infants' neural breathing. The beneficial effect of caffeine citrate on the reduction of the number of apnea is mediated through stimulated neural breathing increasing the diaphragm energy expenditure. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Endometrial Receptivity Markers in Mice Stimulated With Raloxifene Versus Clomiphene Citrate and Natural Cycles.

    Science.gov (United States)

    Chen, Cairong; Yan, Qiuxia; Liu, Kunping; Zhou, Xiuqin; Xian, Yingjie; Liang, Dali; Zhao, Xiaoying; Guo, Xiaoyan; Quan, Song

    2016-06-01

    Ovulation induction therapy with clomiphene citrate can suppress endometrial receptivity. Raloxifene may be an alternative therapeutic for women with ovulatory disorders. This study aimed to compare the expression of endometrial receptivity markers, including homeobox gene 10 (HOXA10), integrin β3, and leukemia inhibitory factor (LIF), as well as pinopode production during the implantation window in mice stimulated with raloxifene and clomiphene citrate and natural cycles. Thirty-six 8-week-old female Kunming mice were randomly divided into 3 groups (n = 12) and administered daily raloxifene (22 mg/kg), clomiphene citrate (18 mg/kg), and normal saline (1 mL), respectively, by gavage. Two days later, mice were injected with 5 IU human chorionic gonadotropin and mated. Successfully mated female animals were identified with vaginal plugs designated gestation day 1. At day 4.5, pregnant donor mice were euthanized, and uterus samples were collected for immunohistochemistry, quantitative polymerase chain reaction, Western blot, and scanning electron microscopy analyses. Homeobox gene 10, integrin β3, and LIF messenger RNA (mRNA) and protein levels were significantly higher in the raloxifene-treated animals compared with the clomiphene citrate group (all P raloxifene and saline groups; however, in the clomiphene citrate-treated mice, fewer and poorly developed pinopodes were obtained. In mice, raloxifene had no effect on HOXA10, integrin β3, and LIF expression as well as pinopode production, suggesting it has no adverse effects on endometrial receptivity. Raloxifene may provide a viable alternative oral ovulation induction agent to clomiphene citrate. © The Author(s) 2015.

  16. Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

    Science.gov (United States)

    Rauf, Sakandar; Nawaz, Haq; Akhtar, Kalsoom; Ghauri, Muhammad A; Khalid, Ahmad M

    2007-05-15

    The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

  17. MicroRNA-126 Suppresses Mesothelioma Malignancy by Targeting IRS1 and Interfering with the Mitochondrial Function

    Czech Academy of Sciences Publication Activity Database

    Tomasetti, M.; Nocchi, L.; Staffolani, S.; Manzella, N.; Amati, M.; Goodwin, J.; Klučková, Katarína; Nguyen, M.; Strafella, E.; Bajziková, Martina; Peterka, Martin; Lettlová, Sandra; Truksa, Jaroslav; Lee, W.; Dong, L.-F.; Santarelli, L.; Neužil, Jiří

    2014-01-01

    Roč. 21, č. 15 (2014), s. 2109-2125 ISSN 1523-0864 R&D Projects: GA ČR(CZ) GAP301/10/1937; GA ČR GAP305/12/1708; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : ATP CITRATE LYASE * OXIDATIVE STRESS * PLEURAL MESOTHELIOMA * CANCER-CELLS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.407, year: 2014

  18. Preparation and Quality Control of 68Ga-Citrate for PET Applications

    OpenAIRE

    Ayuob Aghanejad; Amir Jalilian; Khosro Ardaneh; Fatemeh Bolourinovin; Hassan Yousefnia; Ali Bahrami-Samani

    2015-01-01

    Objective(s): In nuclear medicine studies, gallium-68 (8Ga) citrate has been recently known as a suitable infection agent in positron emission tomography (PET). In this study, by applying an in-house produced 68Ge/68Ga generator, a simple technique for the synthesis and quality control of 68Ga-citrate was introduced; followed by preliminary animal studies. Methods: 68GaCl3 eluted from the generator was studied in terms of quality control factors including radiochemical purity (assessed by HPL...

  19. The influence of metronidazole resistance on the efficacy of ranitidine bismuth citrate triple therapy regimens for Helicobacter pylori infection

    NARCIS (Netherlands)

    Van der Wouden, EJ; Thijs, JC; Van Zwet, AA; Kooy, A; Kleibeuker, JH

    Aim: To assess the influence of metronidazole resistance on the efficacy of ranitidine bismuth citrate-based triple therapy regimens in two consecutive studies. Methods: In the first study, patients with a culture-proven Helicobacter pylori infection were treated with ranitidine bismuth citrate 400

  20. The safety and efficacy of ranitidine bismuth citrate in combination with antibiotics for the eradication of Helicobacter pylori

    NARCIS (Netherlands)

    Wyeth, J. W.; Pounder, R. E.; Duggan, A. E.; O'Morain, C. A.; Schaufelberger, H. D.; de Koster, E. H.; Rauws, E. A.; Bardhan, K. D.; Gilvarry, J.; Buckley, M. J.; Gummett, P. A.; Logan, R. P.

    1996-01-01

    Ranitidine bismuth citrate is a novel salt of ranitidine and a bismuth citrate complex. It has intrinsic antisecretory and anti-Helicobacter pylori activity, but monotherapy rarely eradicates H. pylori infection in man. A pilot study to investigate rates of H. pylori eradication achieved by

  1. Comparative evaluation of the methods of continuous hemodiafiltration and hemofiltration with citrate anticoagulation in cardiac surgery patients

    Directory of Open Access Journals (Sweden)

    А. С. Борисов

    2015-10-01

    Full Text Available CRRT with citrate anticoagulation can be conducted in various modalities. Each of them has its own characteristics of. Comparative evaluation of hemofiltration and hemodiafiltration was the aim of this work. It was found that for the regime hemodiafiltration as less citrate dose.

  2. 76 FR 33219 - Citric Acid and Certain Citrate Salts from the People's Republic of China: Preliminary Results of...

    Science.gov (United States)

    2011-06-08

    ... production of a specialized medical grade citric acid, and that it expired at the end of 2008. See GOC... International Trade Administration Citric Acid and Certain Citrate Salts from the People's Republic of China... administrative review of the countervailing duty order on citric acid and certain citrate salts from the People's...

  3. 77 FR 1455 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Extension of Time...

    Science.gov (United States)

    2012-01-10

    ... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China... Department'') published the initiation of the administrative review of the antidumping duty order on citric acid and certain citrate salts (``citric acid'') from the People's Republic of China (``PRC''). See...

  4. 76 FR 2648 - Citric Acid and Certain Citrate Salts From People's Republic of China: Extension of Time Limit...

    Science.gov (United States)

    2011-01-14

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF COMMERCE International Trade Administration Citric Acid and Certain Citrate Salts From People's Republic of China... initiation of administrative review of the countervailing duty order on citric acid and certain citrate salts...

  5. 77 FR 22560 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Extension of Time...

    Science.gov (United States)

    2012-04-16

    ... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China... Department'') published the initiation of the administrative review of the antidumping duty order on citric acid and certain citrate salts (``citric acid'') from the People's Republic of China (``PRC'').\\1\\ On...

  6. 76 FR 82275 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Extension of Time...

    Science.gov (United States)

    2011-12-30

    ... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China... countervailing duty order on citric acid and certain citrate salts from the People's Republic of China (PRC). See Countervailing Duty Orders and Amendments of Final Affirmative Countervailing Duty Determinations: Citric Acid...

  7. 76 FR 4288 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Notice of Extension of...

    Science.gov (United States)

    2011-01-25

    ... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China... review of the antidumping duty order on citric acid and certain citrate salts (``citric acid'') from the... administrative review of citric acid from the PRC within this time limit. Among other things, additional time is...

  8. 76 FR 56158 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Extension of Time...

    Science.gov (United States)

    2011-09-12

    ... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China... administrative review of the countervailing duty order on citric acid and certain citrate sales from People's Republic of China, covering the period September 19, 2008, through December 31, 2009. See Citric Acid and...

  9. 77 FR 74171 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Final Results of...

    Science.gov (United States)

    2012-12-13

    ... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China...'') published its Preliminary Results of administrative review of the antidumping duty order on citric acid and... Citric Acid and Certain Citrate Salts from the People's Republic of China: Post- Preliminary Analysis...

  10. 78 FR 34642 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Preliminary Results of...

    Science.gov (United States)

    2013-06-10

    ... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China... Department'') is conducting an administrative review of the antidumping duty order on citric acid and certain citrate salts (``citric acid'') from the People's Republic of China (``PRC''). The period of review (``POR...

  11. 77 FR 47370 - Citric Acid and Certain Citrate Salts from the People's Republic of China: Intent To Rescind...

    Science.gov (United States)

    2012-08-08

    ... International Trade Administration Citric Acid and Certain Citrate Salts from the People's Republic of China... countervailing duty (CVD) order on citric acid and certain citrate salts from the People's Republic of China.\\1...). Scope of the Order The scope of the order includes all grades and granulation sizes of citric acid...

  12. 76 FR 47146 - Citric Acid and Certain Citrate Salts From the People's Republic of China: Extension of Time...

    Science.gov (United States)

    2011-08-04

    ... International Trade Administration Citric Acid and Certain Citrate Salts From the People's Republic of China...'') published the initiation of the administrative review of the antidumping duty order on citric acid and certain citrate salts (``citric acid'') from the People's Republic of China (``PRC''). See Initiation of...

  13. Complementary metal ion specificity of the metal-citrate transporters CitM and CitH of Bacillus subtilis

    NARCIS (Netherlands)

    Krom, BP; Warner, JB; Konings, WN; Lolkema, JS; Warner, Jessica B.

    2000-01-01

    Citrate uptake in Bacillus subtilis is stimulated by a wide range of divalent metal ions. The metal ions were separated into two groups based on the expression pattern of the uptake system. The two groups correlated with the metal ion specificity of two homologous B, subtilis secondary citrate

  14. Pulmonary scintigraphy by citrate of Ga67 for HIV(+) patients or AIDS. Scintigraphie pulmonaire au citrate de Ga67 chez des patients VIH(+) ou sida

    Energy Technology Data Exchange (ETDEWEB)

    Daumal, J.; Pena, C.; Mata, F.; Paternostro, C.; Penafiel, A. (Hopital Son Dureta, Palma de Mallorca (Spain))

    1993-04-01

    Pulmonary scintigraphy with citrate of gallium 67 is useful when combined with a thorax radiography to establish the presence of an infection. When the two examinations are negative we can conclude there is no infection, but if the radiography is positive with a scintiscanning negative the most possible diagnosis is tuberculosis. If the scintiscanning is positive with a diffuse model we can envisage a pneumonia by Pneumocystis Carinii and if the model is ganglionic we can think to tuberculosis. 4 tabs.

  15. Thermosensitive bioadhesive gels for the vaginal delivery of sildenafil citrate: in vitro characterization and clinical evaluation in women using clomiphene citrate for induction of ovulation.

    Science.gov (United States)

    Soliman, Ghareb M; Fetih, Gihan; Abbas, Ahmed M

    2017-03-01

    The objective of this study is to develop and characterize in situ thermosensitive gels for the vaginal administration of sildenafil as a potential treatment of endometrial thinning occurring as a result of using clomiphene citrate for ovulation induction in women with type II eugonadotrophic anovulation. While sildenafil has shown promising results in the treatment of infertility in women, the lack of vaginal pharmaceutical preparation and the side effects associated with oral sildenafil limit its clinical effectiveness. Sildenafil citrate in situ forming gels were prepared using different grades of Pluronic® (PF-68 and PF-127). Mucoadhesive polymers as sodium alginate and hydroxyethyl cellulose were added to the gels in different concentrations and the effect on gel properties was studied. The formulations were evaluated in terms of viscosity, gelation temperature (Tsol-gel), mucoadhesion properties, and in vitro drug release characteristics. Selected formulations were evaluated in women with clomiphene citrate failure due to thin endometrium (Clinicaltrial.gov identifier NCT02766725). The Tsol-gel decreased with increasing PF-127 concentration and it was modulated by addition of PF-68 to be within the acceptable range of 28-37 °C. Increasing Pluronic® concentration increased gel viscosity and mucoadhesive force but decreased drug release rate. Clinical results showed that the in situ sildenafil vaginal gel significantly increased endometrial thickness and uterine blood flow with no reported side effects. Further, these results were achieved at lower frequency and duration of drug administration. Sildenafil thermosensitive vaginal gels might result in improved potential of pregnancy in anovulatory patients with clomiphene citrate failure due to thin endometrium.

  16. Citrate-stabilized Q-CdSe seed-mediated synthesis of silver nanoparticles: The role of citrate moieties anchored to the Q-CdSe surface

    Science.gov (United States)

    Ingole, Pravin P.; Bhat, Mohsin A.

    2016-03-01

    Here, we try to explore a new dimension/role for citrate molecules in the bound state, i.e. anchored to the surface of cadmium selenide quantum dots (Q-CdSe), in the synthesis of metal nanoparticles (MNPs). Being labile, the citrate molecule is considered a good candidate for the stabilization of semiconductor quantum dots (QDs) such as Q-CdSe that can be used for further functionalization/modification of the surface properties of the QDs. In its free/ionic form (i.e. not bound to the surface), it is well known for its role as a reducing as well as a capping agent in the synthesis of silver and gold MNPs. A simple strategy for the preparation of silver MNPs following the chemical reduction of silver ions that is mediated by citrate-stabilized Q-CdSe seeds without addition of an external reducing agent is presented. The citrate moieties anchored to the surface of Q-CdSe are found to play an important role in the chemical reduction of silver ions. The obtained product was analysed by spectroscopic, microscopic and structural characterization techniques such as surface plasmon resonance (SPR), transmission electron microscopy (TEM) and cyclic voltammetry. The characteristic redox behaviour observed in cyclic voltammograms (CVs) also supports the formation of Ag MNPs in the samples. Further, the impact of the reaction solution pH on the feasibility of silver ion reduction by Q-CdSe seeds resulting into the formation of Ag MNPs is also briefly discussed.

  17. ATP-induced reactivation of ram testicular, cauda epididymal, and ejaculated spermatozoa extracted with Triton X-100.

    Science.gov (United States)

    White, I G; Voglmayr, J K

    1986-02-01

    It was possible to demembrante and reactivate not only freshly collected testicular, cauda epididymal, and ejaculated ram sperm but also sperm that had been stored for several days at 0 degrees C and for several months at -196 degrees C in rete testis fluid or egg yolk citrate media. Sperm were usually washed free of seminal plasma before demembranation, but this was not essential for reactivation. Bovine serum albumin (1.0%) in the wash medium increased the survival of sperm, but more than 0.25% in the extraction medium decreased reactivation. A macro-molecular component of cauda epididymal fluid also inhibited the reactivation of testicular sperm. Triton X-100 concentrations between 0.01% and 1.00% in the extraction medium were satisfactory for demembranating the sperm. Rapid cooling (i.e., cold shock) mimicked the effect of detergent in making the sperm responsive to added ATP and demonstrated that damage to ram sperm in cold shock does not involve the axoneme. Ejaculated and cauda sperm were reactivated immediately on addition of ATP and activity persisted for up to 10 min. Testicular sperm, on the other hand, required about 4 min to become fully reactivated. The optimal ATP concentration for activation of sperm was 0.1-1.0 mM. Magnesium ions (0.1-1.0 mM) were important for reactivation, and testicular sperm required a higher magnesium concentration than did cauda or ejaculated sperm. Manganese ions were almost as effective as magnesium for reactivating cauda epididymal and ejaculated sperm. Cobalt and cadmium ions were much less active for cauda and ejaculated sperm and none of these ions were effective for testicular sperm. Fluoride (25-50 mM) inhibited reactivation. The presence of 50 microM cAMP in the extraction medium or preincubation of testicular sperm with theophylline or caffeine increased low levels of activation, but this was not evident with ejaculated or cauda sperm. We conclude that the motor apparatus is already functionally assembled in

  18. Abnormal expression of ATP1A1 and ATP1A2 in breast cancer [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Alexey Bogdanov

    2017-01-01

    Full Text Available Breast cancer is the first in incidence and the second in death among all solid tumors occurring in women. The identification of molecular genetic abnormalities in breast cancer is important to improve the results of treatment. In the present study, we analyzed microarray data of breast cancer expression profiling (NCBI GEO database, accession GSE65194, focusing on Na+/K+-ATPase coding genes. We found overexpression of the ATP1A1 and down-regulation of the ATP1A2. We expect that our research could help to improve the understanding of predictive and prognostic features of breast cancer.

  19. Plasma Membrane Na+-Coupled Citrate Transporter (SLC13A5 and Neonatal Epileptic Encephalopathy

    Directory of Open Access Journals (Sweden)

    Yangzom D. Bhutia

    2017-02-01

    Full Text Available SLC13A5 is a Na+-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na+:citrate3− stoichiometry of 4:1. In humans, the Michaelis constant for SLC13A5 to transport citrate is ~600 μM, which is physiologically relevant given that the normal concentration of citrate in plasma is in the range of 150–200 μM. Li+ stimulates the transport function of human SLC13A5 at concentrations that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ~30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metabolism in the sperm mitochondria to generate biological energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metabolism. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to experimentally induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy

  20. Bright luminescence of Vibrio fischeri aconitase mutants reveals a connection between citrate and the Gac/Csr regulatory system.

    Science.gov (United States)

    Septer, Alecia N; Bose, Jeffrey L; Lipzen, Anna; Martin, Joel; Whistler, Cheryl; Stabb, Eric V

    2015-01-01

    The Gac/Csr regulatory system is conserved throughout the γ-proteobacteria and controls key pathways in central carbon metabolism, quorum sensing, biofilm formation and virulence in important plant and animal pathogens. Here we show that elevated intracellular citrate levels in a Vibrio fischeri aconitase mutant correlate with activation of the Gac/Csr cascade and induction of bright luminescence. Spontaneous or directed mutations in the gene that encodes citrate synthase reversed the bright luminescence of aconitase mutants, eliminated their citrate accumulation and reversed their elevated expression of CsrB. Our data elucidate a correlative link between central metabolic and regulatory pathways, and they suggest that the Gac system senses a blockage at the aconitase step of the tricarboxylic acid cycle, either through elevated citrate levels or a secondary metabolic effect of citrate accumulation, and responds by modulating carbon flow and various functions associated with host colonization, including bioluminescence. © 2014 John Wiley & Sons Ltd.