WorldWideScience

Sample records for reductase glutamine synthetase

  1. ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro

    International Nuclear Information System (INIS)

    Woehle, D.L.; Lueddecke, B.A.; Ludden, P.W.

    1990-01-01

    Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [α- 32 P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [α- 32 P]ATP- and [α- 32 P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [α- 32 P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes. A 32 P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H 3 32 PO 4 -grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification

  2. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  3. Glutamine Synthetase: Localization Dictates Outcome

    Directory of Open Access Journals (Sweden)

    Alessandra Castegna

    2018-02-01

    Full Text Available Glutamine synthetase (GS is the adenosine triphosphate (ATP-dependent enzyme that catalyses the synthesis of glutamine by condensing ammonium to glutamate. In the circulatory system, glutamine carries ammonia from muscle and brain to the kidney and liver. In brain reduction of GS activity has been suggested as a mechanism mediating neurotoxicity in neurodegenerative disorders. In cancer, the delicate balance between glutamine synthesis and catabolism is a critical event. In vitro evidence, confirmed in vivo in some cases, suggests that reduced GS activity in cancer cells associates with a more invasive and aggressive phenotype. However, GS is known to be highly expressed in cells of the tumor microenvironment, such as fibroblasts, adipocytes and immune cells, and their ability to synthesize glutamine is responsible for the acquisition of protumoral phenotypes. This has opened a new window into the complex scenario of the tumor microenvironment, in which the balance of glutamine consumption versus glutamine synthesis influences cellular function. Since GS expression responds to glutamine starvation, a lower glutamine synthesizing power due to the absence of GS in cancer cells might apply a metabolic pressure on stromal cells. This event might push stroma towards a GS-high/protumoral phenotype. When referred to stromal cells, GS expression might acquire a ‘bad’ significance to the point that GS inhibition might be considered a conceivable strategy against cancer metastasis.

  4. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

    Directory of Open Access Journals (Sweden)

    A Theron

    Full Text Available Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

  5. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

    Science.gov (United States)

    Theron, A; Roth, R L; Hoppe, H; Parkinson, C; van der Westhuyzen, C W; Stoychev, S; Wiid, I; Pietersen, R D; Baker, B; Kenyon, C P

    2017-01-01

    Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

  6. Growth factors regulate glutamine synthetase activity in ...

    African Journals Online (AJOL)

    Khaled

    2012-07-10

    Jul 10, 2012 ... glutamate and ammonia, which in turn, cells are supplied with ammonia ... out to determine the maximum growth time at which cells will be .... Western blot technique for detection the glutamine synthetase enzyme. Lane 1;.

  7. Glutamine synthetase gene evolution: A good molecular clock

    International Nuclear Information System (INIS)

    Pesole, G.; Lanvave, C.; Saccone, C.; Bozzetti, M.P.; Preparata, G.

    1991-01-01

    Glutamine synthetase gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. The calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. The data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves

  8. Glutamine Synthetase Deficiency in Murine Astrocytes Results in Neonatal Death

    NARCIS (Netherlands)

    He, Youji; Hakvoort, Theodorus B. M.; Vermeulen, Jacqueline L. M.; Labruyère, Wilhelmina T.; de Waart, D. Rudi; van der Hel, W. Saskia; Ruijter, Jan M.; Uylings, Harry B. M.; Lamers, Wouter H.

    2010-01-01

    Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/f)l mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in

  9. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

    CSIR Research Space (South Africa)

    Theron, Anjo

    2017-10-01

    Full Text Available mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does...

  10. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    International Nuclear Information System (INIS)

    Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V.; Carvalho, Helena G.; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

    2014-01-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants

  11. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    Energy Technology Data Exchange (ETDEWEB)

    Torreira, Eva [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Seabra, Ana Rita [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Marriott, Hazel; Zhou, Min [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Llorca, Óscar [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Robinson, Carol V. [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Carvalho, Helena G. [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Pereira, Pedro José Barbosa, E-mail: cftornero@cib.csic.es [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2014-04-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  12. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

    NARCIS (Netherlands)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Lamers, Wouter H.; Chaudhry, Farrukh A.; Verlander, Jill W.; Weiner, I. David

    2016-01-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of

  13. Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

    Science.gov (United States)

    Anderson, P M

    1989-01-01

    The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for

  14. Changes in Activities of Glutamine Synthetase during Grain Filling and Their Relation to Rice Quality

    Directory of Open Access Journals (Sweden)

    Zheng-xun JIN

    2007-09-01

    Full Text Available Four japonica rice varieties differed in cooking and eating qualities were used in a pot experiment to study the relationship between the activities of glutamine synthetase during grain filling and rice quality. The activities of glutamine synthetase gradually increased and then declined as a single peak curve in the course of grain filling. The 15th day after heading was a turning point, before which the enzymatic activities in the inferior rice varieties with high protein content were higher than those in the superior rice varietie with low protein content, and after which it was converse. The activity of glutamine synthetase in grain was correlated with the taste meter value, peak viscosity and breakdown negatively at the early stage of grain filling whereas positively at the middle and late stages. Moreover, it was correlated with the protein content of rice grain and setback positively at the early stage and negatively at the middle and late stages. The correlation degree varied with the course of grain filling. From 15 days to 20 days after heading was a critical stage, in which the direction of correlation between the activity of glutamine synthetase and taste meter value and RVA properties of rice changed.

  15. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

    Science.gov (United States)

    Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

    2016-06-27

    This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    remobilisation from ageing plant parts. Thus, GS is highly involved in determining crop yield and NUE. The major objective of this PhD project was to investigate the NUE properties of transgenic barley designed to constitutively overexpress a GS1 isogene (HvGS1.1). These transgenic lines exhibited an increased...... for N demand. Of the GS isogenes, only the transcript levels of root HvGS1.1 increased when plants were transferred from high to low N. This change coincided with an increase in total GS activity. Pronounced diurnal variation was observed for root nitrate transporter genes and GS isogenes in both root...... fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

  17. Influence of the temporal and spatial variation of nitrate reductase, glutamine synthetase and soil composition in the N species content in lettuce (Lactuca sativa).

    Science.gov (United States)

    Pinto, Edgar; Fidalgo, Fernanda; Teixeira, Jorge; Aguiar, Ana A; Ferreira, Isabel M P L V O

    2014-04-01

    The variation of nitrate reductase (NR), glutamine synthetase (GS) and N content in lettuce was evaluated at 5 stages of lettuce growth. Soil physicochemical properties and its N content were also assessed to elucidate the soil-to-plant transfer of inorganic N and potential leaching to groundwater. A decrease of NR activity and an increase of NO3(-) and N-Kjeldahl content in lettuces were observed during plant growth, whereas GS activity and NH4(+) increased during the first few weeks of lettuce growth and then decreased. Although the temporal variation was similar in lettuces grown in different soils, quantitative differences were observed, indicating that high NO3(-) content in soil caused a higher NO3(-) accumulation in lettuce despite the higher NR activity during the initial stage of plant growth. Higher levels of NO3(-) and NH4(+) were correlated with higher levels of N-Kjeldahl in lettuce suggesting a positive effect of these N species in the biosynthesis of organic forms of N. Soil physicochemical properties influenced the mobility of inorganic N within the groundwater-soil-plant system. Sandy soils with low OM content allowed NO3(-) leaching, which was confirmed by higher NO3(-) levels in groundwater. Therefore, lettuces grown in those soils presented lower N content and the inputs of N to the environment were higher. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus.

    Science.gov (United States)

    Montanini, Barbara; Betti, Marco; Márquez, Antonio J; Balestrini, Raffaella; Bonfante, Paola; Ottonello, Simone

    2003-01-01

    The nucleotide sequences reported in this paper have been submitted to the GenBank(R)/EBI Nucleotide Sequence Databases with accession numbers AF462037 (glutamine synthetase) and AF462032 (glutamate synthase). Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity ( n =1.7+/-0.29) and an unusually high S(0.5) value (54+/-16 mM; S(0.5) is the substrate concentration value at which v =(1/2) V (max)) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells. PMID:12683951

  19. Noncoding RNA of Glutamine Synthetase I Modulates Antibiotic Production in Streptomyces coelicolor A3(2)

    NARCIS (Netherlands)

    D'Alia, Davide; Nieselt, Kay; Steigele, Stephan; Mueller, Jonas; Verburg, Ilse; Takano, Eriko; Alia, Davide D’; Müller, Jonas

    Overexpression of antisense chromosomal cis-encoded noncoding RNAss (ncRNAs) in glutamine synthetase I resulted in a decrease in growth, protein synthesis, and antibiotic production in Streptomyces coelicolor. In addition, we predicted 3,597 cis-encoded ncRNAs and validated 13 of them

  20. Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme

    Directory of Open Access Journals (Sweden)

    Ana Rita Seabra

    2015-07-01

    Full Text Available Glutamine Synthetase (GS catalyses the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of glutamine synthetase isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

  1. Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats.

    Science.gov (United States)

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne; Bak, Lasse K; Schousboe, Arne; Waagepetersen, Helle S; Simonsen, Mette; Ott, Peter; Vilstrup, Hendrik; Sørensen, Michael

    2014-03-01

    Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of (15)NH(4)(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of (15)NH(4)(+) into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of (15)NH(4)(+) into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia.

  2. New perspectives on glutamine synthetase in grasses.

    Science.gov (United States)

    Swarbreck, Stéphanie M; Defoin-Platel, M; Hindle, M; Saqi, M; Habash, Dimah Z

    2011-02-01

    Members of the glutamine synthetase (GS) gene family have now been characterized in many crop species such as wheat, rice, and maize. Studies have shown that cytosolic GS isoforms are involved in nitrogen remobilization during leaf senescence and emphasized a role in seed production particularly in small grain crop species. Data from the sequencing of genomes for model crops and expressed sequence tag (EST) libraries from non-model species have strengthened the idea that the cytosolic GS genes are organized in three functionally and phylogenetically conserved subfamilies. Using a bioinformatic approach, the considerable publicly available information on high throughput gene expression was mined to search for genes having patterns of expression similar to GS. Interesting new hypotheses have emerged from searching for co-expressed genes across multiple unfiltered experimental data sets in rice. This approach should inform new experimental designs and studies to explore the regulation of the GS gene family further. It is expected that understanding the regulation of GS under varied climatic conditions will emerge as an important new area considering the results from recent studies that have shown nitrogen assimilation to be critical to plant acclimation to high CO(2) concentrations.

  3. The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad.

    Science.gov (United States)

    Bieganowski, Pawel; Brenner, Charles

    2003-08-29

    Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.

  4. Inhibition of Glutamine Synthetase: A Potential Drug Target in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Sherry L. Mowbray

    2014-08-01

    Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6–9 months and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

  5. Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design.

    Science.gov (United States)

    Krajewski, Wojciech W; Collins, Ruairi; Holmberg-Schiavone, Lovisa; Jones, T Alwyn; Karlberg, Tobias; Mowbray, Sherry L

    2008-01-04

    Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 A. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 A and 2.6 A, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.

  6. Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies.

    Directory of Open Access Journals (Sweden)

    Benedikt Frieg

    2016-02-01

    Full Text Available Glutamine synthetase (GS catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.

  7. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

    Science.gov (United States)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Lamers, Wouter H.; Chaudhry, Farrukh A.; Verlander, Jill W.

    2016-01-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4+ with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na+-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression. PMID:27009341

  8. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

    Science.gov (United States)

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

    2012-04-01

    Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

  9. Ammonium assimilation in rice based on the occurrence of 15N and inhibition of glutamine synthetase activity

    Energy Technology Data Exchange (ETDEWEB)

    Magalhaes, J. R.; Huber, D. M.; Lee, T. C.; Tsai, C. Y.

    1995-07-01

    Assimilation of ammonium (NH4) into free amino acids and total reduced nitrogen (N) was monitored in both roots and shoots of two-week old rice seedlings supplied with 5 mM 99% (15NH4)2SO4 in aerated hydroponic culture with or without a 2 h preincubation with 1 mM methionine sulfoximine (MSX) an inhibitor of glutamine synthetase (GS) activity. 15NH4 was not assimilated into amino acids when the GS/GOGAT (glutamate synthase) cycle was inhibited by MSX. Inhibition of glutamine synthetase (GS) activity in roots with MSX increased both the amount of NH4 and the abundance of 15N labeled NH4. In contrast, the amount of Gln and Glu, and their proportions as 15N, decreased in roots when GS activity was inhibited. This research confirms the importance of GS/GOGAT in NH4 assimilation in rice roots. 15N-labeled studies indicate that NH4 ions incorporated by roots of rice are transformed primarily into glutamine (Gin) and glutamic acid (Glu) before being converted to other amino acids through transamination. The formation of amino acids such as aspartic acid (Asp) and alanine (Ala) directly from free NH4 in roots also has been reported. Translocation of free NH4 to plant shoots, based on the concentration of free NH4 in xylem exudate, has been reported in tomato, although NH4 in shoots primarily originates from nitrate reduction in the shoot. Photorespiration also can contribute to the accumulation of NH4 in leaves. The GS/GOGAT cycle appears to be primarily responsible for the assimilation of exogenously supplied NH4 and NH4 derived from nitrate reduction in leaves, as well as NH4 derived from photorespiration. Genetic evidence cited to support this conclusion includes the lethal effect of photorespiratory conditions on plant mutants deficient in chloroplast-localized GS and GOGAT activities, and the rapid accumulation of free NH4 in GS-deficient mutants under photorespiratory conditions. The present study was initiated to quantify the in vivo amino acid synthesis in rice

  10. Ammonium assimilation in rice based on the occurrence of 15N and inhibition of glutamine synthetase activity

    International Nuclear Information System (INIS)

    Magalhaes, J.R.; Huber, D.M.; Lee, T.C.; Tsai, C.Y.

    1995-01-01

    Assimilation of ammonium (NH4) into free amino acids and total reduced nitrogen (N) was monitored in both roots and shoots of two-week old rice seedlings supplied with 5 mM 99% (15NH4)2SO4 in aerated hydroponic culture with or without a 2 h preincubation with 1 mM methionine sulfoximine (MSX) an inhibitor of glutamine synthetase (GS) activity. 15NH4 was not assimilated into amino acids when the GS/GOGAT (glutamate synthase) cycle was inhibited by MSX. Inhibition of glutamine synthetase (GS) activity in roots with MSX increased both the amount of NH4 and the abundance of 15N labeled NH4. In contrast, the amount of Gln and Glu, and their proportions as 15N, decreased in roots when GS activity was inhibited. This research confirms the importance of GS/GOGAT in NH4 assimilation in rice roots. 15N-labeled studies indicate that NH4 ions incorporated by roots of rice are transformed primarily into glutamine (Gin) and glutamic acid (Glu) before being converted to other amino acids through transamination. The formation of amino acids such as aspartic acid (Asp) and alanine (Ala) directly from free NH4 in roots also has been reported. Translocation of free NH4 to plant shoots, based on the concentration of free NH4 in xylem exudate, has been reported in tomato, although NH4 in shoots primarily originates from nitrate reduction in the shoot. Photorespiration also can contribute to the accumulation of NH4 in leaves. The GS/GOGAT cycle appears to be primarily responsible for the assimilation of exogenously supplied NH4 and NH4 derived from nitrate reduction in leaves, as well as NH4 derived from photorespiration. Genetic evidence cited to support this conclusion includes the lethal effect of photorespiratory conditions on plant mutants deficient in chloroplast-localized GS and GOGAT activities, and the rapid accumulation of free NH4 in GS-deficient mutants under photorespiratory conditions. The present study was initiated to quantify the in vivo amino acid synthesis in rice

  11. Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Rydzak, Thomas [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Garcia, David [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Stevenson, David M. [Univ. of Wisconsin, Madison, WI (United States). Dept. of Bacteriology; Sladek, Margaret [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Klingeman, Dawn M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Holwerda, Evert K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Dartmouth College, Hanover, NH (United States). Thayer School of Engineering; Amador-Noguez, Daniel [Univ. of Wisconsin, Madison, WI (United States). Dept. of Bacteriology; Brown, Steven D. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Guss, Adam M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center

    2017-05-01

    Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. And while recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H2), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine and α-ketoglutarate levels indicative of nitrogen-rich conditions. Here, we propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine/α-ketoglutarate levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.

  12. Nonnutritive effects of glutamine.

    Science.gov (United States)

    Roth, Erich

    2008-10-01

    Glutamine is the most abundant free amino acid of the human body. Besides its role as a constituent of proteins and its importance in amino acid transamination, glutamine has regulatory capacity in immune and cell modulation. Glutamine deprivation reduces proliferation of lymphocytes, influences expression of surface activation markers on lymphocytes and monocytes, affects the production of cytokines, and stimulates apoptosis. Moreover, glutamine administration seems to have a positive effect on glucose metabolism in the state of insulin resistance. Glutamine influences a variety of different molecular pathways. Glutamine stimulates the formation of heat shock protein 70 in monocytes by enhancing the stability of mRNA, influences the redox potential of the cell by enhancing the formation of glutathione, induces cellular anabolic effects by increasing the cell volume, activates mitogen-activated protein kinases, and interacts with particular aminoacyl-transfer RNA synthetases in specific glutamine-sensing metabolism. Glutamine is applied under clinical conditions as an oral, parenteral, or enteral supplement either as the single amino acid or in the form of glutamine-containing dipeptides for preventing mucositis/stomatitis and for preventing glutamine-deficiency in critically ill patients. Because of the high turnover rate of glutamine, even high amounts of glutamine up to a daily administration of 30 g can be given without any important side effects.

  13. Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum.

    Science.gov (United States)

    Rydzak, Thomas; Garcia, David; Stevenson, David M; Sladek, Margaret; Klingeman, Dawn M; Holwerda, Evert K; Amador-Noguez, Daniel; Brown, Steven D; Guss, Adam M

    2017-05-01

    Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H 2 ), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum. Copyright © 2017. Published by Elsevier Inc.

  14. Haplotype analysis of the genes encoding glutamine synthetase plastic isoforms and their association with nitrogen-use- and yield-related traits in bread wheat.

    Science.gov (United States)

    Li, Xin-Peng; Zhao, Xue-Qiang; He, Xue; Zhao, Guang-Yao; Li, Bin; Liu, Dong-Cheng; Zhang, Ai-Min; Zhang, Xue-Yong; Tong, Yi-Ping; Li, Zhen-Sheng

    2011-01-01

    Glutamine synthetase (GS) plays a key role in the growth, nitrogen (N) use and yield potential of cereal crops. Investigating the haplotype variation of GS genes and its association with agronomic traits may provide useful information for improving wheat N-use efficiency and yield. We isolated the promoter and coding region sequences of the plastic glutamine synthetase isoform (GS2) genes located on chromosomes 2A, 2B and 2D in bread wheat. By analyzing nucleotide sequence variations of the coding region, two, six and two haplotypes were distinguished for TaGS2-A1 (a and b), TaGS2-B1 (a-f) and TaGS2-D1 (a and b), respectively. By analyzing the frequency data of different haplotypes and their association with N use and agronomic traits, four major and favorable TaGS2 haplotypes (A1b, B1a, B1b, D1a) were revealed. These favorable haplotypes may confer better seedling growth, better agronomic performance, and improved N uptake during vegetative growth or grain N concentration. Our data suggest that certain TaGS2 haplotypes may be valuable in breeding wheat varieties with improved agronomic performance and N-use efficiency. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

  15. Age-dependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer's disease mouse model: Mechanism for deficient glutamatergic transmission?

    Czech Academy of Sciences Publication Activity Database

    Olabarria, M.; Noristani, H. N.; Verkhratsky, Alexei; Rodríguez Arellano, Jose Julio

    2011-01-01

    Roč. 6, č. 1 (2011), s. 55-63 ISSN 1750-1326 R&D Projects: GA ČR GA309/09/1696; GA ČR(CZ) GAP304/11/0184; GA ČR GA305/08/1384; GA ČR GA309/08/1381 Institutional research plan: CEZ:AV0Z50390703 Keywords : astroglia * glutamine synthetase * Alzheimer ?'?s disease Subject RIV: FH - Neurology Impact factor: 4.278, year: 2011

  16. The transcriptional activator NtrC controls the expression and activity of glutamine synthetase in Herbaspirillum seropedicae.

    Science.gov (United States)

    Persuhn, D C; Souza, E M; Steffens, M B; Pedrosa, F O; Yates, M G; Rigo, L U

    2000-11-15

    The role of the Ntr system in Herbaspirillum seropedicae was determined via ntrB and ntrC mutants. Three phenotypes were identified in these mutants: Nif(-), deficiency in growth using nitrate, and low glutamine synthetase (GS) activity. All phenotypes were restored by the plasmid pKRT1 containing the intact glnA, ntrB and ntrC genes of H. seropedicae. The promoter region of glnA was subcloned into a beta-galactosidase fusion vector and the results suggested that NtrC positively regulates the glnA promoter in response to low nitrogen. The H. seropedicae ntrC and ntrB mutant strains showed a deficiency of adenylylation/deadenylylation of GS, indicating that NtrC and NtrB are involved in both transcription and activity control of GS in this organism.

  17. Glutamine synthetase activity in solanaceous cell suspensions accumulating alkaloids or not. 13C NMR and enzymatic assay

    International Nuclear Information System (INIS)

    Mesnard, F.; Marty, D.; Monti, J.P.; Gillet-Manceau, F.; Fliniaux, M.A.

    1999-01-01

    The metabolism of labelled pyruvate followed by 13 C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia. This activity could derive glutamate from the alkaloid synthesizing pathways. However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio. The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio. Therefore, the hypothesis of GS dysfunction for the non-biosynthesis of alkaloids in N. plumbaginifolia suspension cells can be discarded. This conclusion is strengthened by the results obtained when using a GS inhibitor. (author)

  18. The role of adrenal hormones in the response of glutamine synthetase to fasting in adult and old rats.

    Science.gov (United States)

    Mezzarobba, V; Torrent, A; Leydier, I; Alles, S; Brajon, B; Mignon, M; Attaix, D; Meynial-Denis, D

    2003-12-01

    During fasting, skeletal muscle exports increased amounts of glutamine (Gln) while increasing the production of this amino acid by glutamine synthetase (GS) in order to maintain the intramuscular Gln pool. Glucocorticoid hormones are believed to be the principal mediators of GS induction during stress conditions. The aim of this study was to evaluate (1) the effect of fasting on GS activity and expression in skeletal muscle during aging and consequently, (2) the role of glucocorticoids in fasting-induced GS activity. Male Wistar rats (6-, 22-month old) were fasted for 5 days and both the activity and expression of GS were measured in tibialis anterior muscle. To better demonstrate the role of glucocorticoids in the response of GS to fasting, we suppressed their action by RU38486 administration (a potent glucocorticoid antagonist) and their production by adrenalectomy in fed and fasted rats. An increase in fasting-induced GS activity was observed in skeletal muscles from both adult and aged rats. Adrenalectomy, but surprisingly not RU38486, suppressed the fasting-induced increase in GS activity and expression. The data clearly show that the GS responsiveness to fasting was not modified by aging in skeletal muscle.

  19. Glutamine domain of the chimeric protein, CAD, that initiates pyrimidine biosynthesis in mammalian cells

    International Nuclear Information System (INIS)

    Kelly, R.E.; Kim, H.; Evans, D.R.

    1986-01-01

    Glutamine dependent carbamyl phosphate synthesis, the first step in mammalian de novo pyrimidine biosynthesis, is catalyzed by a 240 kDa chimeric protein, CAD, that also has the aspartate transcarbamylase and dihydroorotase activities. The complex was found to have a separate glutaminase activity of 0.04 μmol/min/mg, that increased five fold in the presence of bicarbonate and ATP. To determine whether the glutaminase activity, which provides ammonia for carbamyl phosphate synthesis, is associated with a separate structural domain (GLN), CAD was subjected to controlled proteolysis with elastase. The glutaminase, glutamine and ammonia dependent carbamyl phosphate synthetase activities, as well as the partial reactions; carbamyl phosphate dependent ATP synthesis and bicarbonate dependent ATPase, were correlated with the concentration of the various proteolytic fragments that accumulated in the digest. While the glutamine dependent carbamyl phosphate synthetase was rapidly inactivated, the glutaminase activity was found to be very resistant to proteolysis. The glutamine binding site of CAD was also specifically modified with 6-diazo-5-oxo-L-norleucine (DON). The modification was accompanied by a loss of both glutaminase and glutamine dependent carbamyl phosphate synthetase activities. Bicarbonate and ATP increased the rate of reaction of CAD with DON, while glutamine protected against inactivation. The stoichiometry of the reaction and the identity of the modified proteolytic fragments was determined using 14 C labelled DON

  20. Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

    Directory of Open Access Journals (Sweden)

    Seabra Ana R

    2010-08-01

    Full Text Available Abstract Background Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS, occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. Results This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2 in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. Conclusions This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate

  1. 15N studies on the in-vivo assay of nitrate reductase in leaves

    International Nuclear Information System (INIS)

    Yoneyama, Tadakatsu

    1981-01-01

    The reduction of nitrate and nitrite in the leaf disks of seven di- and two mono-cotyledonous species under the in-vivo assay conditions of nitrate reductase was studied using N-15 labeled substrates. The significant reduction of both nitrate and nitrite into ammonia and amino acids was detected in the atmosphere of air. In the atmosphere of N 2 gas, anaerobic incubation enhanced the accumulation of nitrite, but the subsequent reduction to the basic nitrogen compounds was from 40 to 180 % of the aerobic rate. The present examination indicated that the in-vivo assay of nitrate reductase under aerobic condition may give greatly underestimated results due to nitrite reduction, and that the exclusion of oxygen from the in-vivo assay mixture is desirable. The addition of n- propanol may be desirable for the assay under aerobic condition. Significant difference was not observed in the reduction of nitrate supplied as sodium and potassium salts on the nitrite formation and on the incorporation of nitrate-N into basic fractions. The N-15 experiment on the dark assimilation of nitrate, nitrite and ammonia into amino acids in wheat leaves showed that these three nitrogen sources were assimilated through the same route, and that the glutamine synthetase/glutamate synthetase pathway was the main route. By anaerobic treatment, the incorporation of nitrogen into alanine and serine was relatively high. (Kako, I.)

  2. Glutamine synthetase activity in solanaceous cell suspensions accumulating alkaloids or not. {sup 13}C NMR and enzymatic assay; Activite de la glutamine synthetase dans des suspensions cellulaires de solanacees productrices ou non d'alcaloides. RMN du {sup 13}C et dosage enzymatique

    Energy Technology Data Exchange (ETDEWEB)

    Mesnard, F.; Marty, D.; Monti, J.P. [Faculte de Pharmacie, 80 - Amiens (France). Laboratoire de Biophysique, Groupe de Recherche des Biomolecules: micro-environnement et Metabolisme; Gillet-Manceau, F.; Fliniaux, M.A. [Faculte de Pharmacie, 80 - Amiens (France). Laboratoire de Phytotechnologie

    1999-09-01

    The metabolism of labelled pyruvate followed by {sup 13}C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia. This activity could derive glutamate from the alkaloidsynthesizing pathways. However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio. The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio. Therefore, the hypothesis of GS dysfunction for the non-biosynthesis of alkaloids in N. plumbaginifolia suspension cells can be discarded. This conclusion is strengthened by the results obtained when using a GS inhibitor. (author)

  3. Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte–neuron co-cultures

    Science.gov (United States)

    Dadsetan, Sherry; Kukolj, Eva; Bak, Lasse K; Sørensen, Michael; Ott, Peter; Vilstrup, Hendrik; Schousboe, Arne; Keiding, Susanne; Waagepetersen, Helle S

    2013-01-01

    Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of 15NH4+ in alanine during acute hyperammonemia. We observed a fourfold increased amount of 15NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to 15NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of 15NH4 into alanine together with increased 15N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy. PMID:23673435

  4. Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte-neuron co-cultures.

    Science.gov (United States)

    Dadsetan, Sherry; Kukolj, Eva; Bak, Lasse K; Sørensen, Michael; Ott, Peter; Vilstrup, Hendrik; Schousboe, Arne; Keiding, Susanne; Waagepetersen, Helle S

    2013-08-01

    Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of (15)NH4(+) in alanine during acute hyperammonemia. We observed a fourfold increased amount of (15)NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to (15)NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of (15)NH4 into alanine together with increased (15)N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy.

  5. Mineral nitrogen sources differently affect root glutamine synthetase isoforms and amino acid balance among organs in maize.

    Science.gov (United States)

    Prinsi, Bhakti; Espen, Luca

    2015-04-03

    Glutamine synthetase (GS) catalyzes the first step of nitrogen assimilation in plant cell. The main GS are classified as cytosolic GS1 and plastidial GS2, of which the functionality is variable according to the nitrogen sources, organs and developmental stages. In maize (Zea mays L.) one gene for GS2 and five genes for GS1 subunits are known, but their roles in root metabolism are not yet well defined. In this work, proteomic and biochemical approaches have been used to study root GS enzymes and nitrogen assimilation in maize plants re-supplied with nitrate, ammonium or both. The plant metabolic status highlighted the relevance of root system in maize nitrogen assimilation during both nitrate and ammonium nutrition. The analysis of root proteomes allowed a study to be made of the accumulation and phosphorylation of six GS proteins. Three forms of GS2 were identified, among which only the phosphorylated one showed an accumulation trend consistent with plastidial GS activity. Nitrogen availabilities enabled increments in root total GS synthetase activity, associated with different GS1 isoforms according to the nitrogen sources. Nitrate nutrition induced the specific accumulation of GS1-5 while ammonium led to up-accumulation of both GS1-1 and GS1-5, highlighting co-participation. Moreover, the changes in thermal sensitivity of root GS transferase activity suggested differential rearrangements of the native enzyme. The amino acid accumulation and composition in roots, xylem sap and leaves deeply changed in response to mineral sources. Glutamine showed the prevalent changes in all nitrogen nutritions. Besides, the ammonium nutrition was associated with an accumulation of asparagine and reducing sugars and a drop in glutamic acid level, significantly alleviated by the co-provision with nitrate. This work provides new information about the multifaceted regulation of the GS enzyme in maize roots, indicating the involvement of specific isoenzymes/isoforms, post

  6. Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.)

    DEFF Research Database (Denmark)

    Bernard, Stéphanie M.; Møller, Anders Laurell Blom; Dionisio, Giuseppe

    2008-01-01

    ). Phylogenetic analysis showed that the wheat GS sub-families together with the GS genes from other monocotyledonous species form four distinct clades. Immunolocalisation studies in leaves, stems and rachis in plants at flowering showed GS protein to be present in parenchyma, phloem companion and perifascicular......We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2...... sheath cells. In situ localisation confirmed that GS1 transcripts were present in the perifascicular sheath cells whilst those for GSr were confined to the vascular cells. Studies of the expression and protein profiles showed that all GS sub-families were differentially expressed in the leaves, peduncle...

  7. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

    DEFF Research Database (Denmark)

    Noh, Soo Min; Shin, Seunghyeon; Min Lee, Gyun

    2018-01-01

    To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1...... and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated...... in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation...

  8. Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

    Science.gov (United States)

    Yu, Da Young; Lee, Sang Yoon; Lee, Gyun Min

    2018-05-01

    Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. © 2018 Wiley Periodicals, Inc.

  9. Conformational changes induced by Mg2+ on the multiple forms of glutamine synthetase from Bacillus brevis Bb G1

    Directory of Open Access Journals (Sweden)

    Suja Abraham

    2013-01-01

    Full Text Available Conformational changes play an important role in the function of proteins. Glutamine synthetase, an important enzyme of nitrogen metabolism, was purified under sporulating (GSala and non-sporulating (GSpyr conditions and the effect of Mg2+ on these multiple forms was studied by fluorescence spectroscopy to detect possible conformational changes that occur in the presenceof Mg2+. The substantial changes in the fluorescence emission maximum, fluorescence intensity and lifetime that occur in the presence of different concentrations of Mg2+, indicated major changes in molecular conformations in both forms of this enzyme. The fluorescent changes produced by the effect of Mg2+ in GSala was much more prominent than in GSpyr. These observations strongly support the possibility that GSala and GSpyr undergoes a conformational change on binding with Mg2+.

  10. Glutamine Synthetases GLN1;2 and GLN2 in Relation to Arabidopsis Growth Response to Elevated Atmospheric Carbon Dioxide and Varying Nitrogen Forms

    DEFF Research Database (Denmark)

    Vurrakula, Swathi

    content while diluting nitrogen concentrations. Such a reduction in nitrogen concentration will affect plant response to stress and seed/grain yield. Glutamine synthetase (GS) is the central nitrogen-assimilatory enzyme, performing primary and secondary nitrogen assimilation, in response to environmental....... Plants grown under elevated CO2 absorbed ammonia from the atmosphere, except with a high ammonium supply. GLN1;2 had a non-redundant role in determining vegetative growth and ammonium tolerance in response to elevated CO2. Under elevated CO2, GLN1;2 was compensable by GLN2 in assimilating nitrate...

  11. Diet- and hormone-induced reversal of the carbamoylphosphate synthetase mRNA gradient in the rat liver lobulus

    NARCIS (Netherlands)

    Moorman, A. F.; de Boer, P. A.; Charles, R.; Lamers, W. H.

    1990-01-01

    A hybridocytochemical analysis of adult liver from normal control and from hormonally and dietary-treated rats was carried out, using radioactively-labelled probes for the mRNAs of glutamine synthetase (GS), carbamoylphosphate synthetase (CPS) and phosphoenolpyruvate carboxykinase (PEPCK). In line

  12. Growth hormone increases and maturation decreases glutamine synthetase turnover rate in rat liver

    International Nuclear Information System (INIS)

    Lin, C.K.

    1985-01-01

    An investigation was made of the effect of hypophysectomy and growth hormone (GH) replacement regimen (1 mg/100 g twice daily for 30 days); and maturation (from 25 up to 90 days) on the liver and brain glutamine synthetase (GS) mass and turnover rates in rats. The first order decay rate of enzyme 14 C radioactivity was determined between 1 and 4 days to obtain the half-life (T/sub 1/2/) of GS. The hepatic GS mass was determined by immunoassay. GS turnover (GS/sub s/) was calculated from T/sub 1/2/ and the GS mass (i.e., K = 0.693/T/sub 1/2/; GS/sub s/ = K x GS mass). It was concluded that: (1) GS specific activity is not decreased by hypophysectomy or increased by GH. These results suggested that observed endocrine induced changes in GS are due to changes in GS mass. (2) The liver GS turnover rate is significantly reduced by hypophysectomy and increased by GH replacement. It was proposed that GH specifically enhances synthesis of GS in the liver. (3) Maturation (25, 40, 60, and 90 days) decreases GS turnover rate in both liver and brain of normal rats. This similar effect of maturation suggests that the observed age induced decline in GS turnover rate is not related to GH in all tissues

  13. Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

    Science.gov (United States)

    Acosta, Gabriela Beatriz; Fernández, María Alejandra; Roselló, Diego Martín; Tomaro, María Luján; Balestrasse, Karina; Lemberg, Abraham

    2009-01-01

    AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into sham-operated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modifies the normal function in some brain regions. PMID:19533812

  14. Astrocytes and Glutamate Homoeostasis in Alzheimer's Disease: A Decrease in Glutamine Synthetase, But Not in Glutamate Transporter-1, in the Prefrontal Cortex

    Directory of Open Access Journals (Sweden)

    Magdalena Kulijewicz-Nawrot

    2013-09-01

    Full Text Available Astrocytes control tissue equilibrium and hence define the homoeostasis and function of the CNS (central nervous system. Being principal homoeostatic cells, astroglia are fundamental for various forms of neuropathology, including AD (Alzheimer's disease. AD is a progressive neurodegenerative disorder characterized by the loss of cognitive functions due to specific lesions in mnesic-associated regions, including the mPFC (medial prefrontal cortex. Here, we analyzed the expression of GS (glutamine synthetase and GLT-1 (glutamate transporter-1 in astrocytes in the mPFC during the progression of AD in a triple-transgenic mouse model (3xTg-AD. GS is an astrocyte-specific enzyme, responsible for the intracellular conversion of glutamate into glutamine, whereas the removal of glutamate from the extracellular space is accomplished mainly by astroglia-specific GLT-1. We found a significant decrease in the numerical density (Nv, cells/mm3 of GS-positive astrocytes from early to middle ages (1–9 months; at the age of 1 month by 17%, 6 months by 27% and 9 months by 27% when compared with control animals in parallel with a reduced expression of GS (determined by Western blots, which started at the age of 6 months and was sustained up to 12 months of age. We did not, however, find any changes in the expression of GLT-1, which implies an intact glutamate uptake mechanism. Our results indicate that the decrease in GS expression may underlie a gradual decline in the vital astrocyte-dependent glutamate–glutamine conversion pathway, which in turn may compromise glutamate homoeostasis, leading towards failures in synaptic connectivity with deficient cognition and memory.

  15. [Imbalance of system of glutamin - glutamic acid in the placenta and amniotic fluid at placental insufficiency].

    Science.gov (United States)

    Pogorelova, T N; Gunko, V O; Linde, V A

    2014-01-01

    Metabolism of glutamine and glutamic acid has been investigated in the placenta and amniotic fluid under conditions of placental insufficiency. The development of placental insufficiency is characterized by the increased content of glutamic acid and a decrease of glutamine in both placenta and amniotic fluid. These changes changes were accompanied by changes in the activity of enzymes involved in the metabolism of these amino acids. There was a decrease in glutamate dehydrogenase activity and an increase in glutaminase activity with the simultaneous decrease of glutamine synthetase activity. The compensatory decrease in the activity of glutamine keto acid aminotransferase did not prevent a decrease in the glutamine level. The impairments in the system glutamic acid-glutamine were more pronounced during the development of premature labor.

  16. Origins of domestication and polyploidy in oca (Oxalis Tuberosa: Oxalidaceae). 2. Chloroplast-expressed glutamine synthetase data.

    Science.gov (United States)

    Emshwiller, Eve; Doyle, Jeff J

    2002-07-01

    In continuing study of the origins of the octoploid tuber crop oca, Oxalis tuberosa Molina, we used phylogenetic analysis of DNA sequences of the chloroplast-active (nuclear encoded) isozyme of glutamine synthetase (ncpGS) from cultivated oca, its allies in the "Oxalis tuberosa alliance," and other Andean Oxalis. Multiple ncpGS sequences found within individuals of both the cultigen and a yet unnamed wild tuber-bearing taxon of Bolivia were separated by molecular cloning, but some cloned sequences appeared to be artifacts of polymerase chain reaction (PCR) recombination and/or Taq error. Nonetheless, three classes of nonrecombinant sequences each joined a different part of the O. tuberosa alliance clade on the ncpGS gene tree. Octoploid oca shares two sequence classes with the Bolivian tuber-bearing taxon (of unknown ploidy level). Fixed heterozygosity of these two sequence classes in all ocas sampled suggests that they represent homeologous loci and that oca is allopolyploid. A third sequence class, found in eight of nine oca plants sampled, might represent a third homeologous locus, suggesting that oca may be autoallopolyploid, and is shared with another wild tuber-bearing species, tetraploid O. picchensis of southern Peru. Thus, ncpGS data identify these two taxa as the best candidates as progenitors of cultivated oca.

  17. Nitrogen metabolism in a grapevine in vitro system

    Directory of Open Access Journals (Sweden)

    Nuria Llorens

    2002-09-01

    Full Text Available Ammonium, nitrate, nitrite, protein and individual and total amino acid contents were determined in grapevine (cv Sauvignon cultured in vitro. The enzyme activities of nitrate and nitrite reductases, glutamine synthetase, glutamate synthetase and dehydrogenase were also determined. The nitrogen taken up by the plants was 70% of the total nitrogen in the medium after 75 days of in vitro culture. Most of the nitrogen taken up was recovered in the leaves, yet only ammonia and amino acid concentrations were significantly higher in leaves. In roots, glutamine was the most abundant amino acid. In leaves, the most abundant amino acids were aspartate, glutamate, glutamine, alanine, arginine and g-aminobutirate. All enzyme activities were higher in roots than in leaves. These results suggest that both roots and leaves incorporate inorganic nitrogen into organic forms.

  18. Metabolic Alterations Caused by KRAS Mutations in Colorectal Cancer Contribute to Cell Adaptation to Glutamine Depletion by Upregulation of Asparagine Synthetase

    Directory of Open Access Journals (Sweden)

    Kosuke Toda

    2016-11-01

    Full Text Available A number of clinical trials have shown that KRAS mutations of colorectal cancer (CRC can predict a lack of responses to anti-epidermal growth factor receptor–based therapy. Recently, there have been several studies to elucidate metabolism reprogramming in cancer. However, it remains to be investigated how mutated KRAS can coordinate the metabolic shift to sustain CRC tumor growth. In this study, we found that KRAS mutation in CRC caused alteration in amino acid metabolism. KRAS mutation causes a marked decrease in aspartate level and an increase in asparagine level in CRC. Using several human CRC cell lines and clinical specimens of primary CRC, we demonstrated that the expression of asparagine synthetase (ASNS, an enzyme that synthesizes asparagine from aspartate, was upregulated by mutated KRAS and that ASNS expression was induced by KRAS-activated signaling pathway, in particular PI3K-AKT-mTOR pathway. Importantly, we demonstrated that KRAS-mutant CRC cells could become adaptive to glutamine depletion through asparagine biosynthesis by ASNS and that asparagine addition could rescue the inhibited growth and viability of cells grown under the glutamine-free condition in vitro. Notably, a pronounced growth suppression of KRAS-mutant CRC was observed upon ASNS knockdown in vivo. Furthermore, combination of L-asparaginase plus rapamycin markedly suppressed the growth of KRAS-mutant CRC xenografts in vivo, whereas either L-asparaginase or rapamycin alone was not effective. These results indicate ASNS might be a novel therapeutic target against CRCs with mutated KRAS.

  19. The Populus superoxide dismutase gene family and its responses to drought stress in transgenic poplar overexpressing a pine cytosolic glutamine synthetase (GS1a.

    Directory of Open Access Journals (Sweden)

    Juan Jesús Molina-Rueda

    Full Text Available BACKGROUND: Glutamine synthetase (GS plays a central role in plant nitrogen assimilation, a process intimately linked to soil water availability. We previously showed that hybrid poplar (Populus tremula X alba, INRA 717-1B4 expressing ectopically a pine cytosolic glutamine synthetase gene (GS1a display enhanced tolerance to drought. Preliminary transcriptome profiling revealed that during drought, members of the superoxide dismutase (SOD family were reciprocally regulated in GS poplar when compared with the wild-type control, in all tissues examined. SOD was the only gene family found to exhibit such patterns. RESULTS: In silico analysis of the Populus genome identified 12 SOD genes and two genes encoding copper chaperones for SOD (CCSs. The poplar SODs form three phylogenetic clusters in accordance with their distinct metal co-factor requirements and gene structure. Nearly all poplar SODs and CCSs are present in duplicate derived from whole genome duplication, in sharp contrast to their predominantly single-copy Arabidopsis orthologs. Drought stress triggered plant-wide down-regulation of the plastidic copper SODs (CSDs, with concomitant up-regulation of plastidic iron SODs (FSDs in GS poplar relative to the wild type; this was confirmed at the activity level. We also found evidence for coordinated down-regulation of other copper proteins, including plastidic CCSs and polyphenol oxidases, in GS poplar under drought conditions. CONCLUSIONS: Both gene duplication and expression divergence have contributed to the expansion and transcriptional diversity of the Populus SOD/CCS families. Coordinated down-regulation of major copper proteins in drought-tolerant GS poplars supports the copper cofactor economy model where copper supply is preferentially allocated for plastocyanins to sustain photosynthesis during drought. Our results also extend previous findings on the compensatory regulation between chloroplastic CSDs and FSDs, and suggest that this

  20. l-glutamine and l-alanine supplementation increase glutamine-glutathione axis and muscle HSP-27 in rats trained using a progressive high-intensity resistance exercise.

    Science.gov (United States)

    Leite, Jaqueline Santos Moreira; Raizel, Raquel; Hypólito, Thaís Menezes; Rosa, Thiago Dos Santos; Cruzat, Vinicius Fernandes; Tirapegui, Julio

    2016-08-01

    In this study we investigated the chronic effects of oral l-glutamine and l-alanine supplementation, either in their free or dipeptide form, on glutamine-glutathione (GLN-GSH) axis and cytoprotection mediated by HSP-27 in rats submitted to resistance exercise (RE). Forty Wistar rats were distributed into 5 groups: sedentary; trained (CTRL); and trained supplemented with l-alanyl-l-glutamine, l-glutamine and l-alanine in their free form (GLN+ALA), or free l-alanine (ALA). All trained animals were submitted to a 6-week ladder-climbing protocol. Supplementations were offered in a 4% drinking water solution for 21 days prior to euthanasia. Plasma glutamine, creatine kinase (CK), myoglobin (MYO), and erythrocyte concentration of reduced GSH and glutathione disulfide (GSSG) were measured. In tibialis anterior skeletal muscle, GLN-GSH axis, thiobarbituric acid reactive substances (TBARS), and the expression of heat shock factor 1 (HSF-1), 27-kDa heat shock protein (HSP-27), and glutamine synthetase were determined. In CRTL animals, high-intensity RE reduced muscle glutamine levels and increased GSSG/GSH rate and TBARS, as well as augmented plasma CK and MYO levels. Conversely, l-glutamine-supplemented animals showed an increase in plasma and muscle levels of glutamine, with a reduction in GSSG/GSH rate, TBARS, and CK. Free l-alanine administration increased plasma glutamine concentration and lowered muscle TBARS. HSF-1 and HSP-27 were high in all supplemented groups when compared with CTRL (p alanine, in both a free or dipeptide form, improve the GLN-GSH axis and promote cytoprotective effects in rats submitted to high-intensity RE training.

  1. A Modified Bacillus Calmette-Guérin (BCG Vaccine with Reduced Activity of Antioxidants and Glutamine Synthetase Exhibits Enhanced Protection of Mice despite Diminished in Vivo Persistence

    Directory of Open Access Journals (Sweden)

    Douglas S. Kernodle

    2013-01-01

    Full Text Available Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

  2. Plant nutritional status modulates glutamine synthetase levels in ripe tomatoes (Solanum lycopersicum cv. Micro-Tom).

    Science.gov (United States)

    Scarpeci, Telma E; Marro, Martin L; Bortolotti, Santiago; Boggio, Silvana B; Valle, Estela M

    2007-02-01

    Tomato (Solanum lycopersicum) fruit ripening implies that chloroplastic proteins are degraded and new proteins are synthesized. Supplementary nutrition is frequently required when tomato plants begin to fruit and continues until the end of the plant's life cycle. Ammonium assimilation is crucial in these fruit maturation and ripening processes. Glutamine synthetase (GS; EC 6.3.1.2), the main ammonium-fixing enzyme in plants, could not be detected in red fruits of several tomato varieties when growing under standard nutrition. In this paper, we analyze the influence of the nutritional status on the ammonium assimilation capacity of ripe tomato (cv. Micro-Tom) fruit. For this purpose, GS expression and protein profiles were followed in mature green and red fruits harvested from plants grown under standard or supplemented nutrition. Under standard nutrient regime (weekly supplied with 0.5 x Hoagland solution) GS activity was found in chloroplasts (GS2) of mature green fruits, but it was not detected either in the chromoplasts or in the cytosol of red fruits. When plants were shifted to a supplemented nutritional regime (daily supplied with 0.5 x Hoagland solution), GS was found in red fruits. Also, cytosolic transcripts (gs1) preferentially accumulated in red fruits under high nutrition. These results indicate that mature green Micro-Tom fruits assimilate ammonia through GS2 under standard nutrition, while ripe red fruits accumulate GS1 under high nutrition, probably in order to assimilate the extra N-compounds made available through supplemented nutrition.

  3. Evidence for an operative glutamine translocator in chloroplasts from maritime pine (Pinus pinaster Ait.) cotyledons.

    Science.gov (United States)

    Claros, M G; Aguilar, M L; Cánovas, F M

    2010-09-01

    In higher plants, ammonium is assimilated into amino acids through the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. This metabolic cycle is distributed in different cellular compartments in conifer seedlings: glutamine synthesis occurs in the cytosol and glutamate synthesis within the chloroplast. A method for preparing intact chloroplasts of pine cotyledons is presented with the aim of identifying a glutamine-glutamate translocator. Glutamine-glutamate exchange has been studied using the double silicone layer system, suggesting the existence of a translocator that imports glutamine into the chloroplast and exports glutamate to the cytoplasm. The translocator identified is specific for glutamine and glutamate, and the kinetic constants for both substrates indicate that it is unsaturated at intracellular concentrations. Thus, the experimental evidence obtained supports the model of the GS/GOGAT cycle in developing pine seedlings that accounts for the stoichiometric balance of metabolites. As a result, the efficient assimilation of free ammonia produced by photorespiration, nitrate reduction, storage protein mobilisation, phenylpropanoid pathway or S-adenosylmethionine synthesis is guaranteed.

  4. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies.

    Science.gov (United States)

    Noh, Soo Min; Shin, Seunghyeon; Lee, Gyun Min

    2018-03-29

    To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R 2  ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

  5. Structure-Function Relationship of Transporters in the Glutamate–Glutamine Cycle of the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Mariko Kato Hayashi

    2018-04-01

    Full Text Available Many kinds of transporters contribute to glutamatergic excitatory synaptic transmission. Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters to be released from presynaptic terminals. After synaptic vesicle release, glutamate is taken up by neurons or astrocytes to terminate the signal and to prepare for the next signal. Glutamate transporters on the plasma membrane are responsible for transporting glutamate from extracellular fluid to cytoplasm. Glutamate taken up by astrocyte is converted to glutamine by glutamine synthetase and transported back to neurons through glutamine transporters on the plasma membranes of the astrocytes and then on neurons. Glutamine is converted back to glutamate by glutaminase in the neuronal cytoplasm and then loaded into synaptic vesicles again. Here, the structures of glutamate transporters and glutamine transporters, their conformational changes, and how they use electrochemical gradients of various ions for substrate transport are summarized. Pharmacological regulations of these transporters are also discussed.

  6. Effect of intermittent glutamine supplementation on skeletal muscle is not long-lasting in very old rats.

    Science.gov (United States)

    Meynial-Denis, D; Beaufrère, A-M; Mignon, M; Patureau Mirand, P

    2013-01-01

    Muscle is the major site for glutamine synthesis via glutamine synthetase (GS). This enzyme is increased 1.5-2 fold in 25-27-mo rats and may be a consequence of aging-induced stress. This stimulation is similar to the induction observed following a catabolic state such as glucocorticoid treatment (6 to 24 months). Although oral glutamine supply regulates the plasma glutamine level, nothing is known if this supplementation is interrupted before the experiment. Adult (8-mo) and very old (27-mo) female rats were exposed to intermittent glutamine supplementation for 50 % of their age lifetime. Treated rats received glutamine added to their drinking water and control rats water alone but the effect of glutamine supplementation was only studied 15 days after the last supplementation. Glutamine pretreatment discontinued 15 days before the experiment increased plasma glutamine to ~ 0.6 mM, a normal value in very old rats. However, it failed to decrease the up-regulated GS activity in skeletal muscle from very old rats. Our results suggest that long-term treatment with glutamine started before advanced age but discontinued 15 days before rat sacrifice is effective in increasing plasma glutamine to recover basal adult value and in maintaining plasma glutamine in very old rats, but has no long-lasting effect on the GS activity of skeletal muscle with advanced age.

  7. Cyclopiazonic acid biosynthesis in Aspergillus sp.: characterization of a reductase-like R* domain in cyclopiazonate synthetase that forms and releases cyclo-acetoacetyl-L-tryptophan.

    Science.gov (United States)

    Liu, Xinyu; Walsh, Christopher T

    2009-09-15

    The fungal neurotoxin alpha-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase, has a pentacyclic indole tetramic acid scaffold that arises from one molecule of tryptophan, acetyl-CoA, malonyl-CoA, and dimethylallyl pyrophosphate by consecutive action of three enzymes, CpaS, CpaD, and CpaO. CpaS is a hybrid, two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that makes and releases cyclo-acetoacetyl-L-tryptophan (cAATrp), the tetramic acid that serves as substrate for subsequent prenylation and oxidative cyclization to the five ring CPA scaffold. The NRPS module in CpaS has a predicted four-domain organization of condensation, adenylation, thiolation, and reductase* (C-A-T-R*), where R* lacks the critical Ser-Tyr-Lys catalytic triad of the short chain dehydrogenase/reductase (SDR) superfamily. By heterologous overproduction in Escherichia coli of the 56 kDa Aspergillus flavus CpaS TR* didomain and the single T and R* domains, we demonstrate that CpaS catalyzes a Dieckmann-type cyclization on the N-acetoacetyl-Trp intermediate bound in thioester linkage to the phosphopantetheinyl arm of the T domain to form and release cAATrp. This occurs without any participation of NAD(P)H, so R* does not function as a canonical SDR family member. Use of the T and R* domains in in trans assays enabled multiple turnovers and evaluation of specific mutants. Mutation of the D3803 residue in the R* domain, conserved in other fungal tetramate synthetases, abolished activity both in in trans and in cis (TR*) activity assays. It is likely that cyclization of beta-ketoacylaminoacyl-S-pantetheinyl intermediates to released tetramates represents a default cyclization/release route for redox-incompetent R* domains embedded in NRPS assembly lines.

  8. Regulation of glutamine synthetase isoforms in two differentially drought-tolerant rice (Oryza sativa L.) cultivars under water deficit conditions.

    Science.gov (United States)

    Singh, Kamal Krishna; Ghosh, Shilpi

    2013-02-01

    KEY MESSAGE : The regulation of GS isoforms by WD was organ specific. Two GS isoforms i.e. OsGS1;1 and OsGS2 were differentially regulated in IR-64 (drought-sensitive) and Khitish (drought-tolerant) cultivars of rice. Water deficit (WD) has adverse effect on rice (Oryza sativa L.) and acclimation requires essential reactions of primary metabolism to continue. Rice plants utilize ammonium as major nitrogen source, which is assimilated into glutamine by the reaction of Glutamine synthetase (GS, EC 6.3.1.2). Rice plants possess one gene (OsGS2) for chloroplastic GS2 and three genes (OsGS1;1, OsGS1;2 and OsGS1;3) for cytosolic GS1. Here, we report the effect of WD on regulation of GS isoforms in drought-sensitive (cv. IR-64) and drought-tolerant (cv. Khitish) rice cultivars. Under WD, total GS activity in root and leaf decreased significantly in IR-64 seedlings in comparison to Khitish seedlings. The reduced GS activity in IR-64 leaf was mainly due to decrease in GS2 activity, which correlated with decrease in corresponding transcript and polypeptide contents. GS1 transcript and polypeptide accumulated in leaf during WD, however, GS1 activity was maintained at a constant level. Total GS activity in stem of both the varieties was insensitive to WD. Among GS1 genes, OsGS1;1 expression was differently regulated by WD in the two rice varieties. Its transcript accumulated more abundantly in IR-64 leaf than in Khitish leaf. Following WD, OsGS1;1 mRNA level in stem and root tissues declined in IR-64 and enhanced in Khitish. A steady OsGS1;2 expression patterns were noted in leaf, stem and root of both the cultivars. Results suggest that OsGS2 and OsGS1;1 expression may contribute to drought tolerance of Khitish cultivar under WD conditions.

  9. Insecticidal activity of glufosinate through glutamine depletion in a caterpillar.

    Science.gov (United States)

    Kutlesa, N J; Caveney, S

    2001-01-01

    The herbicide glufosinate-ammonium (GLA) is a competitive inhibitor of glutamine synthetase (GS), an enzyme converting glutamate to glutamine in both plants and animals. Because GS is essential for ammonia detoxification in plants, GLA treatment disrupts photorespiration by causing a build-up of ammonia and a loss of glutamine in plant tissues. This study reports that GLA applied to leaf surfaces is also toxic to 5th-instar caterpillars of the skipper butterfly Calpodes ethlius (LD50 = 400 mg kg-1). After ingesting GLA, caterpillars stopped feeding and became dehydrated through a loss of rectal function. Caterpillars showed symptoms of neurotoxicity, such as proleg tremors, body convulsions and complete paralysis before death. Incubation of several tissues isolated from normal feeding-stage caterpillars with the GS substrates glutamate and ammonium showed that GLA inhibited GS activity in vitro. Within 24 h of ingesting GLA, caterpillars had a greatly reduced glutamine content and the ammonium ion levels had more than doubled. Injection of ammonium chloride into non-GLA-treated caterpillars had no deleterious effect, suggesting that glutamine depletion, and not a rise in body ammonium, was the primary cause of GLA toxicity following GS inhibition. This was supported by the observation that the onset of the symptoms of GLA poisoning could be postponed by giving GLA-fed caterpillars several subsequent daily injections of glutamine. The effective GLA dose fed to 5th-instar caterpillars in this study was comparable to the amount that might realistically by acquired from feeding on GLA-treated crops.

  10. Glutamine acts as a neuroprotectant against DNA damage, beta-amyloid and H2O2-induced stress.

    Directory of Open Access Journals (Sweden)

    Jianmin Chen

    Full Text Available Glutamine is the most abundant free amino acid in the human blood stream and is 'conditionally essential' to cells. Its intracellular levels are regulated both by the uptake of extracellular glutamine via specific transport systems and by its intracellular synthesis by glutamine synthetase (GS. Adding to the regulatory complexity, when extracellular glutamine is reduced GS protein levels rise. Unfortunately, this excess GS can be maladaptive. GS overexpression is neurotoxic especially if the cells are in a low-glutamine medium. Similarly, in low glutamine, the levels of multiple stress response proteins are reduced rendering cells hypersensitive to H(2O(2, zinc salts and DNA damage. These altered responses may have particular relevance to neurodegenerative diseases of aging. GS activity and glutamine levels are lower in the Alzheimer's disease (AD brain, and a fraction of AD hippocampal neurons have dramatically increased GS levels compared with control subjects. We validated the importance of these observations by showing that raising glutamine levels in the medium protects cultured neuronal cells against the amyloid peptide, Aβ. Further, a 10-day course of dietary glutamine supplementation reduced inflammation-induced neuronal cell cycle activation, tau phosphorylation and ATM-activation in two different mouse models of familial AD while raising the levels of two synaptic proteins, VAMP2 and synaptophysin. Together, our observations suggest that healthy neuronal cells require both intracellular and extracellular glutamine, and that the neuroprotective effects of glutamine supplementation may prove beneficial in the treatment of AD.

  11. Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

    Directory of Open Access Journals (Sweden)

    Tartar Aurélien

    2010-06-01

    Full Text Available Abstract Background Glutamine synthetase (GS is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria to the Chloroplastida. Results GSII sequences were isolated from four species of green algae (Trebouxiophyceae, and additional green algal (Chlorophyceae and Prasinophytae and streptophyte (Charales, Desmidiales, Bryophyta, Marchantiophyta, Lycopodiophyta and Tracheophyta sequences were obtained from public databases. In Bayesian and maximum likelihood analyses, eubacterial (GSIIB and eukaryotic (GSIIE GSII sequences formed distinct clades. Both GSIIB and GSIIE were found in chlorophytes and early-diverging streptophytes. The GSIIB enzymes from these groups formed a well-supported sister clade with the γ-Proteobacteria, providing evidence that GSIIB in the Chloroplastida arose by horizontal gene transfer (HGT. Bayesian relaxed molecular clock analyses suggest that GSIIB and GSIIE coexisted for an extended period of time but it is unclear whether the proposed HGT happened prior to or after the divergence of the primary endosymbiotic lineages (the Archaeplastida. However, GSIIB genes have not been identified in glaucophytes or red algae, favoring the hypothesis that GSIIB was gained after the divergence of the primary endosymbiotic lineages. Duplicate copies of the GSIIB gene were present in Chlamydomonas reinhardtii, Volvox carteri f. nagariensis, and Physcomitrella patens. Both GSIIB proteins in C. reinhardtii and V. carteri f. nagariensis had N-terminal transit sequences, indicating they are targeted to the chloroplast or mitochondrion. In contrast, GSIIB proteins of P. patens lacked transit sequences, suggesting

  12. Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons

    DEFF Research Database (Denmark)

    Dadsetan, Sherry; Bak, Lasse Kristoffer; Sørensen, Michael

    2011-01-01

    study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5h with [U-(13)C]glucose to monitor de novo......It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present...

  13. A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

    Science.gov (United States)

    Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

    2005-04-01

    Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

  14. Redox status affects the catalytic activity of glutamyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Katz, Assaf; Banerjee, Rajat; de Armas, Merly

    2010-01-01

    Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles in t...... inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme....

  15. Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.

    Science.gov (United States)

    Yu, Da Young; Noh, Soo Min; Lee, Gyun Min

    2016-08-10

    To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (PMSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats

    DEFF Research Database (Denmark)

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne

    2014-01-01

    , and aspartate and incorporation of (15)NH4(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation...... of (15)NH4(+) into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of (15)NH4(+) into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined...

  17. Chronic exposure to glufosinate-ammonium induces spatial memory impairments, hippocampal MRI modifications and glutamine synthetase activation in mice.

    Science.gov (United States)

    Calas, André-Guilhem; Richard, Olivier; Même, Sandra; Beloeil, Jean-Claude; Doan, Bich-Thuy; Gefflaut, Thierry; Même, William; Crusio, Wim E; Pichon, Jacques; Montécot, Céline

    2008-07-01

    Glufosinate-ammonium (GLA), the active compound of a worldwide-used herbicide, acts by inhibiting the plant glutamine synthetase (GS) leading to a lethal accumulation of ammonia. GS plays a pivotal role in the mammalian brain where it allows neurotransmitter glutamate recycling within astroglia. Clinical studies report that an acute GLA ingestion induces convulsions and memory impairment in humans. Toxicological studies performed at doses used for herbicidal activity showed that GLA is probably harmless at short or medium range periods. However, effects of low doses of GLA on chronically exposed subjects are not known. In our study, C57BL/6J mice were treated during 10 weeks three times a week with 2.5, 5 and 10mg/kg of GLA. Effects of this chronic treatment were assessed at behavioral, structural and metabolic levels by using tests of spatial memory, locomotor activity and anxiety, hippocampal magnetic resonance imaging (MRI) texture analysis, and hippocampal GS activity assay, respectively. Chronic GLA treatments have effects neither on anxiety nor on locomotor activity of mice but at 5 and 10mg/kg induce (1) mild memory impairments, (2) a modification of hippocampal texture and (3) a significant increase in hippocampal GS activity. It is suggested that these modifications may be causally linked one to another. Since glutamate is the main neurotransmitter in hippocampus where it plays a crucial role in spatial memory, hippocampal MRI texture and spatial memory alterations might be the consequences of hippocampal glutamate homeostasis modification revealed by increased GS activity in hippocampus. The present study provides the first data that show cerebral alterations after chronic exposure to GLA.

  18. Inhibition of nitrogen-fixing activity of the cyanobiont affects the localization of glutamine synthetase in hair cells of Azolla.

    Science.gov (United States)

    Uheda, Eiji; Maejima, Kazuhiro

    2009-10-15

    In the Azolla-Anabaena association, the host plant Azolla efficiently incorporates and assimilates ammonium ions that are released from the nitrogen-fixing cyanobiont, probably via glutamine synthetase (GS; EC 6.3.1.2) in hair cells, which are specialized cells protruding into the leaf cavity. In order to clarify the regulatory mechanism underlying ammonium assimilation in the Azolla-Anabaena association, Azolla plants were grown under an argon environment (Ar), in which the nitrogen-fixing activity of the cyanobiont was inhibited specifically and completely. The localization of GS in hair cells was determined by immunoelectron microscopy and quantitative analysis of immunogold labeling. Azolla plants grew healthily under Ar when nitrogen sources, such as NO(3)(-) and NH(4)(+), were provided in the growth medium. Both the number of cyanobacterial cells per leaf and the heterocyst frequency of the plants under Ar were similar to those of plants in a nitrogen environment (N(2)). In hair cells of plants grown under Ar, regardless of the type of nitrogen source provided, only weak labeling of GS was observed in the cytoplasm and in chloroplasts. In contrast, in hair cells of plants grown under N(2), abundant labeling of GS was observed in both sites. These findings indicate that specific inhibition of the nitrogen-fixing activity of the cyanobiont affects the localization of GS isoenzymes. Ammonium fixed and released by the cyanobiont could stimulate GS synthesis in hair cells. Simultaneously, the abundant GS, probably GS1, in these cells, could assimilate ammonium rapidly.

  19. Eco-physiological studies on Indian arid zone plants. VI. Effect of sodium chloride and abscisic acid on amino-acid and protein metabolism in leaves of Phaseolus aconitifolius

    Energy Technology Data Exchange (ETDEWEB)

    Huber, W.; Kreutmeier, F.; Sankhla, N.

    1977-01-01

    The effect of sodium chloride (NaCl) and abscisic acid (ABA) on protein synthesis, protein hydrolysis, activities of alanine and aspartate aminotransferases, glutamate dehydrogenase, glutamine synthetase, ..delta..-pyrroline-5-carboxylate-reductase and amino-acid composition was investigated in the leaves of four days old Phaseolus aconitifolius seedlings. Both NaCl and ABA inhibited protein synthesis, but promoted the activities of leucine arylamidase, alanine and aspartate aminotransferases, glutamate dehydrogenase, glutamine synthetase and ..delta..-pyrroline-5-carboxylate-reductase. The results of the amino-acid analysis indicated following treatment with NaCl the amounts of proline, arginine, serine and glutamic acid increased significantly in the leaves. An increase of the proline concentration could be observed only up to a salt concentration of 8.5 x 10/sup -3/ M. Increasing concentrations of ABA also brought a corresponding rise in proline, serine and glutamic acid content. Interestingly the decrease of proline concentration by a salt concentration of more than 8.5 x 10/sup -3/ M is correlated with a decrease in endogenous ABA-content. The possible significance of the similarites between the action of abscisic acid and salinity in influencing the amino-acid and protein metabolism in Phaseolus aconitifolius seedlings during stress is discussed. 31 references, 8 figures, 2 tables.

  20. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    Science.gov (United States)

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  1. Response of Pearl Millet to nitrogen as affected by water deficit

    OpenAIRE

    Diouf , O.; Brou , Yao Télesphore; Diouf , M.; Sarr , B.; Eyletters , M.; Roy-Macauley , H.; Delhaye , J.

    2004-01-01

    International audience; In the Sahelian zone, low soil N could be as limiting as drought in pearl millet production. Although growth and crop productivity depend on several biochemical reactions in which the nitrogen metabolism plays a great role, there is little information available on how N uptake and key enzymes, nitrate reductase and glutamine synthetase, are affected by nitrogen and water interaction in millet. For this purpose, the millet variety cv. Souna III was grown in the field du...

  2. Value and Limits of Routine Histology Alone or Combined with Glutamine Synthetase Immunostaining in the Diagnosis of Hepatocellular Adenoma Subtypes on Surgical Specimens

    Directory of Open Access Journals (Sweden)

    Paulette Bioulac-Sage

    2013-01-01

    Full Text Available Immunohistochemistry is a valid method to classify hepatocellular adenoma (HCA. The aim was to test the performance of routine histology combined to glutamine synthetase (GS staining to identify the 2 major HCA subtypes: HNF1α inactivated (H-HCA and inflammatory HCA (IHCA. 114 surgical cases, previously classified by immunohistochemistry, were analysed. Group A comprised 45 H-HCAs, 44 IHCAs, and 9 β-catenin-activated IHCAs (b-IHCA, and group B, 16 b-HCA and unclassified HCA (UHCA. Steatosis was the hallmark of H-HCA. IHCA and b-IHCA were mainly characterized by inflammation, thick arteries, and sinusoidal dilatation; b-IHCA could not be differentiated from IHCA by routine histology. Group B was identified by default. A control set (91 cases was analyzed using routine and GS stainings (without knowing immunohistochemical results. Among the 45 H-HCAs and 27 IHCAs, 40 and 24 were correctly classified, respectively. Among the 10 b-IHCAs, 4 were identified as such using additional GS. Eight of the 9 HCAs that were neither H-HCA nor IHCA were correctly classified. Conclusion. Routine histology allows to diagnose >85% of the 2 major HCA subtypes. GS is essential to identify b-HCA. This study demonstrates that a “palliative” diagnostic approach can be proposed, when the panel of specific antibodies is not available.

  3. Light induces changes in activities of Na+/K+(NH4+-ATPase, H+/K+(NH4+-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam Tridacna squamosa

    Directory of Open Access Journals (Sweden)

    Alex Y K Ip

    2015-03-01

    Full Text Available The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control, on enzymatic activities of transporters involved in the transport of NH4+ or H+, and activities of enzymes involved in converting NH4+ to glutamate/glutamine in inner mantle, outer mantle and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH4+ in substitution for K+ to activate Na+/K+-ATPase (NKA, manifested as a significant increase in the Na+/NH4+-activated-NKA activity in the inner mantle. However, similar phenomena were not observed in the extensible outer mantle, which contained abundant symbiotic zooxanthellae. Hence, during light-enhanced calcification, H+ released from CaCO3 deposition could react with NH3 to form NH4+ in the extrapallial fluid, and NH4+ could probably be transported into the shell-facing inner mantle epithelium through NKA. Light also induced an increase in the activity of glutamine synthetase, which converts NH4+ and glutamate to glutamine, in the inner mantle. Taken together, these results explained observations reported elsewhere that light induced a significant increase in pH and a significant decrease in ammonia concentration in the extrapallial fluid, as well as a significant increase in the glutamine concentration in the inner mantle, of T. squamosa. Exposure of T. squamosa to light also led to a significant decrease in the N-ethylmaleimide (NEM-sensitive-V-H+-ATPase (VATPase in the inner mantle, and significant increases in the Na+/K+-activated-NKA, H+/NH4+-activated-H+/K+-ATPase and NEM-sensitive-VATPase activities in ctenidia, indicating that light-enhanced calcification might perturb Na+ homeostasis and acid/base balance in the hemolymph, and might involve the active uptake of NH4+ from the environment. This is the first report on light having direct enhancing effects on activities of certain

  4. NCBI nr-aa BLAST: CBRC-TTRU-01-0296 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available Class I Glutamine Synthetases In Eukaryotes That Has Undergone Evolutionary Re-Engineering For A Tissue-Spe...ss I Glutamine Synthetases In Eukaryotes That Has Undergone Evolutionary Re-Engineering...n Ancient Family Of Class I Glutamine Synthetases In Eukaryotes That Has Undergone Evolutionary Re-Engineering...gsin Is A Survivor Of An Ancient Family Of Class I Glutamine Synthetases In Eukaryotes That Has Undergone Evolutionary Re-Engineering...s That Has Undergone Evolutionary Re-Engineering For A Tissue-Specific Role In The Vertebrate Eye Lens. pdb|

  5. A sub-threshold dose of pilocarpine increases glutamine synthetase in reactive astrocytes and enhances the progression of amygdaloid-kindling epilepsy in rats.

    Science.gov (United States)

    Sun, Hong-Liu; Deng, Da-Ping; Pan, Xiao-Hong; Wang, Chao-Yun; Zhang, Xiu-Li; Chen, Xiang-Ming; Wang, Chun-Hua; Liu, Yu-Xia; Li, Shu-Cui; Bai, Xian-Yong; Zhu, Wei

    2016-03-02

    The prognosis of patients exposed to a sub-threshold dose of a proconvulsant is difficult to establish. In this study, we investigated the effect of a single sub-threshold dose of the proconvulsant pilocarpine (PILO) on the progression of seizures that were subsequently induced by daily electrical stimulation (kindling) of the amygdaloid formation. Male Sprague–Dawley rats were each implanted with an electrode in the right basolateral amygdala and an indwelling cannula in the right ventricle. The animals were randomized into groups and were administered one of the following treatments: saline, PILO, saline+L-α-aminoadipic acid (L-AAA; one dosage tested), PILO+L-AAA, or PILO+L-methionine sulfoximine (three dosages tested). Amygdaloid stimulation and electroencephalography were performed once daily. We performed immunohistochemistry and western blot for glial fibrillary acidic protein and glutamine synthetase (GS). We also assayed the enzymic activity of GS in discrete brain regions. An intraperitoneal injection of a sub-threshold PILO dose enhanced the progression of amygdaloid-kindling seizures and was accompanied by an increase in reactive-astrocyte and GS (content and activity) in the hippocampus and piriform cortex. L-AAA and L-methionine sulfoximine, inhibitors of astrocytic and GS function, respectively, abolished the effect of PILO on amygdaloid-kindling seizures. We conclude that one sub-threshold dose of a proconvulsant may enhance the progression of subsequent epilepsy and astrocytic GS may play a role in this phenomenon. Thus, a future therapy for epilepsy could be inhibition of astrocytes and/or GS.

  6. De Novo Glutamine Synthesis

    Science.gov (United States)

    He, Qiao; Shi, Xinchong; Zhang, Linqi; Yi, Chang; Zhang, Xuezhen

    2016-01-01

    Purpose: The aim of this study was to investigate the role of de novo glutamine (Gln) synthesis in the proliferation of C6 glioma cells and its detection with 13N-ammonia. Methods: Chronic Gln-deprived C6 glioma (0.06C6) cells were established. The proliferation rates of C6 and 0.06C6 cells were measured under the conditions of Gln deprivation along with or without the addition of ammonia or glutamine synthetase (GS) inhibitor. 13N-ammonia uptake was assessed in C6 cells by gamma counting and in rats with C6 and 0.06C6 xenografts by micro–positron emission tomography (PET) scanning. The expression of GS in C6 cells and xenografts was assessed by Western blotting and immunohistochemistry, respectively. Results: The Gln-deprived C6 cells showed decreased proliferation ability but had a significant increase in GS expression. Furthermore, we found that low concentration of ammonia was sufficient to maintain the proliferation of Gln-deprived C6 cells, and 13N-ammonia uptake in C6 cells showed Gln-dependent decrease, whereas inhibition of GS markedly reduced the proliferation of C6 cells as well as the uptake of 13N-ammoina. Additionally, microPET/computed tomography exhibited that subcutaneous 0.06C6 xenografts had higher 13N-ammonia uptake and GS expression in contrast to C6 xenografts. Conclusion: De novo Gln synthesis through ammonia–glutamate reaction plays an important role in the proliferation of C6 cells. 13N-ammonia can be a potential metabolic PET tracer for Gln-dependent tumors. PMID:27118759

  7. Possible role of glutamine synthetase in the NO signaling response in root nodules by contributing to the antioxidant defenses

    Directory of Open Access Journals (Sweden)

    Liliana Santos Silva

    2013-09-01

    Full Text Available Nitric oxide (NO is emerging as an important regulatory player in the Rhizobium-legume symbiosis. The occurrence of NO during several steps of the symbiotic interaction suggests an important, but yet unknown, signaling role of this molecule for root nodule formation and functioning. The identification of the molecular targets of NO is key for the assembly of the signal transduction cascade that will ultimately help to unravel NO function. We have recently shown that the key nitrogen assimilatory enzyme Glutamine Synthetase (GS is a molecular target of NO in root nodules of Medicago truncatula, being post-translationally regulated by tyrosine nitration in relation to nitrogen fixation. In functional nodules of M. truncatula NO formation has been located in the bacteroid containing cells of the fixation zone, where the ammonium generated by bacterial nitrogenase is released to the plant cytosol and assimilated into the organic pools by plant GS. We propose that the NO-mediated GS post-translational inactivation is connected to nitrogenase inhibition induced by NO and is related to metabolite channeling to boost the nodule antioxidant defenses. Glutamate, a substrate for GS activity is also the precursor for the synthesis of glutathione (GSH, which is highly abundant in root nodules of several plant species and known to play a major role in the antioxidant defense participating in the ascorbate/GSH cycle. Existing evidence suggests that upon NO-mediated GS inhibition, glutamate could be channeled for the synthesis of GSH. According to this hypothesis, GS would be involved in the NO-signaling responses in root nodules and the NO-signaling events would meet the nodule metabolic pathways to provide an adaptive response to the inhibition of symbiotic nitrogen fixation by reactive nitrogen species (RNS.

  8. ORF Alignment: NC_002755 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available tallographic Structure Of A Relaxed ... Glutamine Synthetase From Mycobacterium Tuberculosis ... ...m Mycobacterium ... Tuberculosis pdb|1HTQ|V Chain V, Multicopy ... ...Crystallographic Structure Of A Relaxed Glutamine ... Synthetase From Mycobacterium Tuberculosis pdb|... From Mycobacterium ... Tuberculosis pdb|1HTQ|T Chain T, Multicopy ... ... ... Crystallographic Structure Of A Relaxed Glutamine ... Synthetase From Mycobacterium Tuberculosis

  9. ORF Alignment: NC_002945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available tallographic Structure Of A Relaxed ... Glutamine Synthetase From Mycobacterium Tuberculosis ... ...m Mycobacterium ... Tuberculosis pdb|1HTQ|V Chain V, Multicopy ... ...Crystallographic Structure Of A Relaxed Glutamine ... Synthetase From Mycobacterium Tuberculosis pdb|... From Mycobacterium ... Tuberculosis pdb|1HTQ|T Chain T, Multicopy ... ... ... Crystallographic Structure Of A Relaxed Glutamine ... Synthetase From Mycobacterium Tuberculosis

  10. ORF Alignment: NC_000962 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available tallographic Structure Of A Relaxed ... Glutamine Synthetase From Mycobacterium Tuberculosis ... ...m Mycobacterium ... Tuberculosis pdb|1HTQ|V Chain V, Multicopy ... ...Crystallographic Structure Of A Relaxed Glutamine ... Synthetase From Mycobacterium Tuberculosis pdb|... From Mycobacterium ... Tuberculosis pdb|1HTQ|T Chain T, Multicopy ... ... ... Crystallographic Structure Of A Relaxed Glutamine ... Synthetase From Mycobacterium Tuberculosis

  11. [Effect of NH4(+) -N/NO3(-)-N ratio in applied supplementary fertilizer on nitrogen metabolism and main chemical composition of Pinellia ternata].

    Science.gov (United States)

    Hu, Long-Jiao; Wang, Kang-Cai; Li, Can-Wen

    2013-07-01

    To study the effect of nitrogen forms on nitrogen metabolism and main chemical composition of Pinellia ternate. Through the soilless cultivation experiment and based at the same nitrogen level and different NH4(+) -N/NO3(-) -N ratios, nitrate reductase (NR) activity, glutamine synthetase (GS) activity, the content of nitrate nitrogen and ammonium nitrogen in different parts of P. ternate were determined. The contents of total alkaloid, free total organic acids and guanosine in the tuber were determined. The yield of bulbil and tuber was calculated. The test results showed that, with the NH4(+) -N/NO3(-) -N ratio increasing, the activity of nitrate reductase decreased, the content of nitrate nitrogen in the leaves, petioles and tuber increasing initially, then decreased, and the content of nitrate nitrogen in the root decreased. Meanwhile, with the NH4(+) -N/NO3(-) -N ratio increasing, the activity of glutamine synthetase in the leaves, petioles and root increased, the activity of glutamine synthetase in the tuber increasing initially, then decreased. The contents of ammonium nitrogen in the leaves, tuber and root increased initially, then decreased, and the contents of ammonium nitrogen in the petioles increased with the NH4(+)(-N/NO3(-)-N ratio increasing. The yield of bulbil and tuber were the highest at the NH4(+)-N/NO3(-) -N ratio of 75: 25. The content of total alkaloid and guanosine in the tuber were the highest at the NH4(+)-N/NO3(-) -N ratio of 0: 100, and the contents were 0.245% and 0.0197% respectively. With the NH4(+)-N/NO3(-) -N ratio of 50: 50, the content of free total organic acids was the highest, it reached 0.7%, however, the content of free total organic acids was the lowest at the NH4(+) -N/NO3(-) -N ratio of 0: 100. Nitrogen fertilization significant influences the nitrogen metabolism, the yield and main chemical composition of P. ternate.

  12. Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

    Science.gov (United States)

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

  13. Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

    Directory of Open Access Journals (Sweden)

    Joana Faria

    2016-01-01

    Full Text Available A growing interest in asparagine (Asn metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

  14. Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons.

    Science.gov (United States)

    Dadsetan, Sherry; Bak, Lasse K; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Leke, Renata; Schousboe, Arne; Waagepetersen, Helle S

    2011-09-01

    It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5h with [U-(13)C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH(4)Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Computational Insights into the High-Fidelity Catalysis of Aminoacyl-tRNA Synthetases

    Science.gov (United States)

    Aboelnga, Mohamed M.

    Obtaining insights into the catalytic function of enzymes is an important area of research due to their widespread applications in the biotechnology and pharmaceutical industries. Among these enzymes, the aminoacyl-tRNA synthetases (aaRSs) are known for their remarkable fidelity in catalyzing the aminoacylation reactions of tRNA in protein biosynthesis. Despite the exceptional execution of this critical function, mechanistic details of the reactions catalyzed by aminoacyl-tRNA synthetases remain elusive demonstrating the obvious need to explore their remarkable chemistry. During the PhD studies reported in this thesis the mechanism of aminoacylation, pre?transfer editing and post?transfer editing catalyzed by different aaRS have been established using multi-scale computational enzymology. In the first two chapters a detailed information about aaRS and the addressed questions was given in addition to an overview of the used computational methodology currently used to investigate the enzymatic mechanisms. The aminoacylation mechanism of threonine by Threonyl-tRNA synthetases, glutamine by Glutaminyl-tRNA synthetases and glutamate by Glutamyl-tRNA synthetases have been clearly unveiled in chapter 3 and 4. Also, valuable information regarding the role of cofactors and active site residues has been obtained. While investigating the post-transfer editing mechanisms, which proceed in a remote and distinct active site, two different scenarios were experimentally suggested for two types of threonyl-tRNA synthetase species to correct the misacylation of the structurally related serine. We explored these two mechanisms as in chapters 5 and 6. Moreover, the synthetic site in which the aminoacylation reaction is catalyzed, is also responsible for a second type of proofreading reaction called pre-transfer editing mechanism. In chapter 7, this latter mechanism has been elucidated for both Seryl-tRNA synthetases and Isoleucyl-tRNA synthetases against their non-cognate substrates

  16. Endogenous glutamine production in critically ill patients: the effect of exogenous glutamine supplementation.

    Science.gov (United States)

    Mori, Maiko; Rooyackers, Olav; Smedberg, Marie; Tjäder, Inga; Norberg, Ake; Wernerman, Jan

    2014-04-14

    Glutamine rate of appearance (Ra) may be used as an estimate of endogenous glutamine production. Recently a technique employing a bolus injection of isotopically labeled glutamine was introduced, with the potential to allow for multiple assessments of the glutamine Ra over time in critically ill patients, who may not be as metabolically stable as healthy individuals. Here the technique was used to evaluate the endogenous glutamine production in critically ill patients in the fed state with and without exogenous glutamine supplementation intravenously. Mechanically ventilated patients (n = 11) in the intensive care unit (ICU) were studied on two consecutive days during continuous parenteral feeding. To allow the patients to be used as their own controls, they were randomized for the reference measurement during basal feeding without supplementation, before or after the supplementation period. Glutamine Ra was determined by a bolus injection of 13C-glutamine followed by a period of frequent sampling to establish the decay-curve for the glutamine tracer. Exogenous glutamine supplementation was given by intravenous infusion of a glutamine containing dipeptide, L-alanyl-L-glutamine, 0.28 g/kg during 20 hours. A 14% increase of endogenous glutamine Ra was seen at the end of the intravenous supplementation period as compared to the basal measurements (P = 0.009). The bolus injection technique to measure glutamine Ra to estimate the endogenous production of glutamine in critically ill patients was demonstrated to be useful for repetitive measurements. The hypothesized attenuation of endogenous glutamine production during L-alanyl-L-glutamine infusion given as a part of full nutrition was not seen.

  17. Activation of the TOR Signalling Pathway by Glutamine Regulates Insect Fecundity.

    Science.gov (United States)

    Zhai, Yifan; Sun, Zhongxiang; Zhang, Jianqing; Kang, Kui; Chen, Jie; Zhang, Wenqing

    2015-05-29

    The target of rapamycin (TOR) positively controls cell growth in response to nutrients such as amino acids. However, research on the specific nutrients sensed by TOR is limited. Glutamine (Gln), a particularly important amino acid involved in metabolism in organisms, is synthesised and catalysed exclusively by glutamine synthetase (GS), and our previous studies have shown that Gln may regulate fecundity in vivo levels of the brown planthopper (BPH) Nilaparvata lugens. Until now, it has remained unclear whether Gln activates or inhibits the TOR signalling pathway. Here, we performed the combined analyses of iTRAQ (isobaric tags for relative and absolute quantification) and DGE (tag-based digital gene expression) data in N. lugens at the protein and transcript levels after GS RNAi, and we found that 52 pathways overlap, including the TOR pathway. We further experimentally demonstrate that Gln activates the TOR pathway by promoting the serine/threonine protein kinase AKT and inhibiting the 5'AMP-activated protein kinase AMPK phosphorylation activity in the pest. Furthermore, TOR regulates the fecundity of N. lugens probably by mediating vitellogenin (Vg) expression. This work is the first report that Gln activates the TOR pathway in vivo.

  18. Glutamine alimentation in catabolic state.

    Science.gov (United States)

    Boelens, P G; Nijveldt, R J; Houdijk, A P; Meijer, S; van Leeuwen, P A

    2001-09-01

    Glutamine should be reclassified as a conditionally essential amino acid in the catabolic state because the body's glutamine expenditures exceed synthesis and low glutamine levels in plasma are associated with poor clinical outcome. After severe stress, several amino acids are mobilized from muscle tissue to supply energy and substrate to the host. Glutamine is one of the most important amino acids that provide this function. Glutamine acts as the preferred respiratory fuel for lymphocytes, hepatocytes and intestinal mucosal cells and is metabolized in the gut to citrulline, ammonium and other amino acids. Low concentrations of glutamine in plasma reflect reduced stores in muscle and this reduced availability of glutamine in the catabolic state seems to correlate with increased morbidity and mortality. Adding glutamine to the nutrition of clinical patients, enterally or parenterally, may reduce morbidity. Several excellent clinical trials have been performed to prove efficacy and feasibility of the use of glutamine supplementation in parenteral and enteral nutrition. The increased intake of glutamine has resulted in lower septic morbidity in certain critically ill patient populations. This review will focus on the efficacy and the importance of glutamine supplementation in diverse catabolic states.

  19. Advances in animal cell recombinant protein production: GS-NS0 expression system.

    Science.gov (United States)

    Barnes, L M; Bentley, C M; Dickson, A J

    2000-02-01

    The production of recombinant proteins using mammalian cell expression systems is of growing importance within biotechnology, largely due to the ability of specific mammalian cells to carry out post-translational modifications of the correct fidelity. The Glutamine Synthetase-NS0 system is now one such industrially important expression system.Glutamine synthetase catalyses the formation ofglutamine from glutamate and ammonia. NS0 cellscontain extremely low levels of endogenous glutaminesynthetase activity, therefore exogenous glutaminesynthetase can be used efficiently as a selectablemarker to identify successful transfectants in theabsence of glutamine in the media. In addition, theinclusion of methionine sulphoximine, an inhibitor ofglutamine synthetase activity, enables furtherselection of those clones producing relatively highlevels of transfected glutamine synthetase and henceany heterologous gene which is coupled to it. Theglutamine synthetase system technology has been usedfor research and development purposes during thisdecade and its importance is clearly demonstrated nowthat two therapeutic products produced using thissystem have reached the market place.

  20. The Enzymes of the Ammonia Assimilation in Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Janssen, Dick B.; Camp, Huub J.M. op den; Leenen, Pieter J.M.; Drift, Chris van der

    1980-01-01

    Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen

  1. Digestion of a single meal affects gene expression of ion and ammonia transporters and glutamine synthetase activity in the gastrointestinal tract of freshwater rainbow trout.

    Science.gov (United States)

    Bucking, Carol; Wood, Chris M

    2012-04-01

    Experiments on freshwater rainbow trout, Oncorhynchus mykiss, demonstrated how digestion affected the transcriptional expression of gastrointestinal transporters following a single satiating meal (~3% body mass ration) after a 1-week fast. Quantitative real-time polymerase chain reaction was employed to measure the relative mRNA expression of three previously cloned and sequenced transporters [H(+)-K(+)-ATPase (HKA), Na(+)/HCO(3)(-) cotransporter (NBC), and the Rhesus glycoprotein (Rhbg1; an ammonia transporter)] over a 24-h time course following feeding. Plasma total ammonia increased about threefold from pre-feeding levels to 288 μmol l(-1), whereas total ammonia levels in chyme supernatant reached a sixfold higher value (1.8 mmol l(-1)) than plasma levels. Feeding did not appear to have a statistically significant effect on the relative mRNA expression of the gastric HKA or Rhbg1. However, the relative mRNA expression of gastric NBC was increased 24 h following the ingestion of a meal. Along the intestinal tract, feeding increased the relative mRNA expression of Rhbg1, but had no effect on the expression of NBC. Expression of the gastric HKA was undetectable in the intestinal tract of freshwater rainbow trout. Digestion increased the activity of glutamine synthetase in the posterior intestine at 12 and 24 h following feeding. This study is among the first to show that there are digestion-associated changes in gene expression and enzyme activity in the gastrointestinal tract of teleost fish illustrating the dynamic plasticity of this organ. These post-prandial changes occur over the relative short-term duration of digesting a single meal.

  2. Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs

    International Nuclear Information System (INIS)

    Jayakumar, A.; Hong, J.S.; Barnes, E.M. Jr.

    1987-01-01

    When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system. Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 μM L-glutamine in the presence of chloramphenicol (100 μg/ml). The inhibition of transport by L-glutamine was noncompetitive with respect to the [ 14 C]methylammonium substrate. D-Glutamine had no significant effect. The glutamine analogs γ-L-glutamyl hydroxamate and γ-L-glutamyl hydrazide were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required. The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter. The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition. Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells, was equipotent in blocking transport in glnP mutants. Although ammonium transport is also subject to repression by growth of E. coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition

  3. A Transient Upregulation of Glutamine Synthetase in the Dentate Gyrus Is Involved in Epileptogenesis Induced by Amygdala Kindling in the Rat.

    Directory of Open Access Journals (Sweden)

    Hong-Liu Sun

    Full Text Available Reduction of glutamine synthetase (GS function is closely related to established epilepsy, but little is known regarding its role in epileptogenesis. The present study aimed to elucidate the functional changes of GS in the brain and its involvement in epileptogenesis using the amygdala kindling model of epilepsy induced by daily electrical stimulation of basolateral amygdala in rats. Both expression and activity of GS in the ipsilateral dentate gyrus (DG were upregulated when kindled seizures progressed to stage 4. A single dose of L-methionine sulfoximine (MSO, in 2 µl, a selective GS inhibitor, was administered into the ipsilateral DG on the third day following the first stage 3 seizure (just before GS was upregulated. It was found that low doses of MSO (5 or 10 µg significantly and dose-dependently reduced the severity of and susceptibility to evoked seizures, whereas MSO at a high dose (20 µg aggravated kindled seizures. In animals that seizure acquisition had been successfully suppressed with 10 µg MSO, GS upregulation reoccurred when seizures re-progressed to stage 4 and re-administration of 10 µg MSO consistently reduced the seizures. GLN at a dose of 1.5 µg abolished the alleviative effect of 10 µg MSO and deleterious effect of 20 µg MSO on kindled seizures. Moreover, appropriate artificial microRNA interference (1 and 1.5×10(6 TU/2 µl of GS expression in the ipsilateral DG also inhibited seizure progression. In addition, a transient increase of GS expression and activity in the cortex was also observed during epileptogenesis evoked by pentylenetetrazole kindling. These results strongly suggest that a transient and region-specific upregulation of GS function occurs when epilepsy develops into a certain stage and eventually promotes the process of epileptogenesis. Inhibition of GS to an adequate degree and at an appropriate timing may be a potential therapeutic approach to interrupting epileptogenesis.

  4. The Glutamine Transporters and Their Role in the Glutamate/GABA-Glutamine Cycle

    DEFF Research Database (Denmark)

    Leke, Renata; Schousboe, Arne

    2016-01-01

    in this neural communication, i.e., the transporters responsible for glutamine efflux from astrocytes and influx into the neurons, such as the members of the SNAT, LAT, y(+)LAT, and ASC families of transporters. The SNAT family consists of the transporter isoforms SNAT3 and SNAT5 that are related to efflux from......Glutamine is a key amino acid in the CNS, playing an important role in the glutamate/GABA-glutamine cycle (GGC). In the GGC, glutamine is transferred from astrocytes to neurons, where it will replenish the inhibitory and excitatory neurotransmitter pools. Different transporters participate...... the astrocytic compartment, and SNAT1 and SNAT2 that are associated with glutamine uptake into the neuronal compartment. The isoforms SNAT7 and SNAT8 do not have their role completely understood, but they likely also participate in the GGC. The isoforms LAT2 and y(+)LAT2 facilitate the exchange of neutral amino...

  5. Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Pendergast, A.M.

    1986-01-01

    The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with ( 32 P)orthophosphate

  6. Glutamine supplementation maintains intramuscular glutamine concentrations and normalizes lymphocyte function in infected early weaned pigs.

    Science.gov (United States)

    Yoo, S S; Field, C J; McBurney, M I

    1997-11-01

    Numerous studies in humans and rats have shown that glutamine supplementation during stressful conditions has favorable outcomes. However, the requirements for glutamine during weaning are unknown. Thus, the effects of glutamine supplementation in healthy and infected weaned pigs were investigated. At 21 d of age, pigs were weaned to an elemental diet supplemented with glutamine (+Gln) or an isonitrogenous diet containing nonessential amino acids (-Gln). At 26 d of age, pigs were intraperitoneally injected with Escherichia coli (+Ecoli) or buffered saline (-Ecoli) and killed at 28 d of age. Infection decreased (P Ecoli+Gln pigs were greater (P Ecoli-Gln pigs and not different than those of noninfected pigs. Hence, glutamine supplementation maintained muscular glutamine concentrations and normalized lymphocyte function in infected pigs.

  7. Specificity of exogenous acetate and glutamate as astrocyte substrates examined in acute brain slices from female mice using methionine sulfoximine (MSO) to inhibit glutamine synthesis

    DEFF Research Database (Denmark)

    Andersen, Jens Velde; McNair, Laura Frendrup; Schousboe, Arne

    2017-01-01

    Removal of endogenously released glutamate is mediated primarily by astrocytes and exogenous (13) C-labeled glutamate has been applied to study glutamate metabolism in astrocytes. Likewise, studies have clearly established the relevance of (13) C-labeled acetate as an astrocyte specific metabolic...... cortical slices from female NMRI mice were incubated in media containing [1,2-(13) C]acetate or [U-(13) C]glutamate, with or without methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Tissue extracts were analyzed by gas chromatography-mass spectrometry. Blocking GS abolished the majority...... of glutamine (13) C-labeling from [1,2-(13) C]acetate as intended. However, (13) C-labeling of GABA was only 40-50% reduced by MSO, suggesting considerable neuronal uptake of acetate. Moreover, labeling of glutamate from [1,2-(13) C]acetate in the presence of MSO exceeded the level probable from exclusive...

  8. A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

    2014-05-01

    The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

  9. Lanthanum (III) regulates the nitrogen assimilation in soybean seedlings under ultraviolet-B radiation.

    Science.gov (United States)

    Huang, Guangrong; Wang, Lihong; Zhou, Qing

    2013-01-01

    Ultraviolet-B (UV-B, 280-320 nm) radiation has seriously affected the growth of plants. Finding the technology/method to alleviate the damage of UV-B radiation has become a frontal topic in the field of environmental science. The pretreatment with rare earth elements (REEs) is an effective method, but the regulation mechanism of REEs is unknown. Here, the regulation effects of lanthanum (La(III)) on nitrogen assimilation in soybean seedlings (Glycine max L.) under ultraviolet-B radiation were investigated to elucidate the regulation mechanism of REEs on plants under UV-B radiation. UV-B radiation led to the inhibition in the activities of the key enzymes (nitrate reductase, glutamine synthetase, glutamate synthase) in the nitrogen assimilation, the decrease in the contents of nitrate and soluble proteins, as well as the increase in the content of amino acid in soybean seedlings. The change degree of UV-B radiation at the high level (0.45 W m(-2)) was higher than that of UV-B radiation at the low level (0.15 W m(-2)). The pretreatment with 20 mg L(-1) La(III) could alleviate the effects of UV-B radiation on the activities of nitrate reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase, promoting amino acid conversion and protein synthesis in soybean seedlings. The regulation effect of La(III) under UV-B radiation at the low level was better than that of UV-B radiation at the high level. The results indicated that the pretreatment with 20 mg L(-1) La(III) could alleviate the inhibition of UV-B radiation on nitrogen assimilation in soybean seedlings.

  10. The glutamate/GABA-glutamine cycle

    DEFF Research Database (Denmark)

    Bak, Lasse K; Schousboe, Arne; Waagepetersen, Helle S

    2006-01-01

    Neurons are metabolically handicapped in the sense that they are not able to perform de novo synthesis of neurotransmitter glutamate and gamma-aminobutyric acid (GABA) from glucose. A metabolite shuttle known as the glutamate/GABA-glutamine cycle describes the release of neurotransmitter glutamate...... or GABA from neurons and subsequent uptake into astrocytes. In return, astrocytes release glutamine to be taken up into neurons for use as neurotransmitter precursor. In this review, the basic properties of the glutamate/GABA-glutamine cycle will be discussed, including aspects of transport and metabolism...... of intercellular transfer of ammonia produced in neurons (when glutamine is deamidated to glutamate) and utilized in astrocytes (for amidation of glutamate) when the glutamate/GABA-glutamine cycle is operating. A main objective of this review is to endorse the view that the glutamate/GABA-glutamine cycle must...

  11. The Glutamine Transporters and Their Role in the Glutamate/GABA-Glutamine Cycle

    DEFF Research Database (Denmark)

    Leke, Renata; Schousboe, Arne

    2016-01-01

    in this neural communication, i.e., the transporters responsible for glutamine efflux from astrocytes and influx into the neurons, such as the members of the SNAT, LAT, y(+)LAT, and ASC families of transporters. The SNAT family consists of the transporter isoforms SNAT3 and SNAT5 that are related to efflux from...... and translational mechanisms, which are induced by several determinants such as amino acid deprivation, hormones, pH, and the activity of different signaling pathways. Dysfunctional glutamine transporter activity has been associated with the pathophysiological mechanisms of certain neurologic diseases......, such as Hepatic Encephalopathy and Manganism. However, there might also be other neuropathological conditions associated with an altered GGC, in which glutamine transporters are dysfunctional. Hence, it appears to be of critical importance that the physiological and pathological aspects of glutamine transporters...

  12. The Glutamine-Glutamate/GABA Cycle

    DEFF Research Database (Denmark)

    Walls, Anne B; Waagepetersen, Helle S; Bak, Lasse Kristoffer

    2015-01-01

    information about glutamine transfer. The present review will give information about glutamine trafficking and the tools used to map it as exemplified by discussions of published work employing brain cell cultures as well as intact animals. It will be documented that considerably more glutamine is transferred...

  13. S-Nitrosomycothiol Reductase and Mycothiol Are Required for Survival Under Aldehyde Stress and Biofilm Formation in Mycobacterium smegmatis

    Science.gov (United States)

    Vargas, Derek; Hageman, Samantha; Gulati, Megha; Nobile, Clarissa J.; Rawat, Mamta

    2017-01-01

    We show that Mycobacterium smegmatis mutants disrupted in mscR, coding for a dual function S-nitrosomycothiol reductase and formaldehyde dehydrogenase, and mshC, coding for a mycothiol ligase and lacking mycothiol (MSH), are more susceptible to S-nitrosoglutathione (GSNO) and aldehydes than wild type. MSH is a cofactor for MscR, and both mshC and mscR are induced by GSNO and aldehydes. We also show that a mutant disrupted in egtA, coding for a γ-glutamyl cysteine synthetase and lacking in ergothioneine, is sensitive to nitrosative stress but not to aldehydes. In addition, we find that MSH and S-nitrosomycothiol reductase are required for normal biofilm formation in M. smegmatis, suggesting potential new therapeutic pathways to target to inhibit or disrupt biofilm formation. PMID:27321674

  14. Ammonia Assimilation in Zea mays L. Infected with a Vesicular-Arbuscular Mycorrhizal Fungus Glomus fasciculatum.

    Science.gov (United States)

    Cliquet, J. B.; Stewart, G. R.

    1993-03-01

    To investigate nitrogen assimilation and translocation in Zea mays L. colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thax. sensu Gerd.), we measured key enzyme activities, 15N incorporation into free amino acids, and 15N translocation from roots to shoots. Glutamine synthetase and nitrate reductase activities were increased in both roots and shoots compared with control plants, and glutamate dehydrogenase activity increased in roots only. In the presence of [15N]ammonium, glutamine amide was the most heavily labeled product. More label was incorporated into amino acids in VAM plants. The kinetics of 15N labeling and effects of methionine sulfoximine on distribution of 15N-labeled products were entirely consistent with the operation of the glutamate synthase cycle. No evidence was found for ammonium assimilation via glutamate dehydrogenase. 15N translocation from roots to shoots through the xylem was higher in VAM plants compared with control plants. These results establish that, in maize, VAM fungi increase ammonium assimilation, glutamine production, and xylem nitrogen translocation. Unlike some ectomycorrhizal fungi, VAM fungi do not appear to alter the pathway of ammonium assimilation in roots of their hosts.

  15. Plasma Glutamine Concentrations in Liver Failure.

    Directory of Open Access Journals (Sweden)

    Gunnel Helling

    Full Text Available Higher than normal plasma glutamine concentration at admission to an intensive care unit is associated with an unfavorable outcome. Very high plasma glutamine levels are sometimes seen in both acute and chronic liver failure. We aimed to systematically explore the relation between different types of liver failure and plasma glutamine concentrations.Four different groups of patients were studies; chronic liver failure (n = 40, acute on chronic liver failure (n = 20, acute fulminant liver failure (n = 20, and post-hepatectomy liver failure (n = 20. Child-Pugh and Model for End-stage Liver Disease (MELD scores were assessed as indices of liver function. All groups except the chronic liver failure group were followed longitudinally during hospitalisation. Outcomes were recorded up to 48 months after study inclusion.All groups had individuals with very high plasma glutamine concentrations. In the total group of patients (n = 100, severity of liver failure correlated significantly with plasma glutamine concentration, but the correlation was not strong.Liver failure, regardless of severity and course of illness, may be associated with a high plasma glutamine concentration. Further studies are needed to understand whether high glutamine levels should be regarded as a biomarker or as a contributor to symptomatology in liver failure.

  16. Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

    Science.gov (United States)

    Pyne, C; Bognar, A L

    1992-03-01

    The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.

  17. Radioimmune assay of human platelet prostaglandin synthetase

    International Nuclear Information System (INIS)

    Roth, G.J.; Machuga, E.T.

    1982-01-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

  18. Glutaminase enzyme biosensor for determination of glutamine in cerebrospinal fluid, human serum and l-glutamine capsule

    International Nuclear Information System (INIS)

    Bagriyanik, D.B.; Karakus, E

    2014-01-01

    Ammonium-selective glutamine biosensor was prepared by immobilizing glutaminase on poly(vinylchloride) (PVC) ammonium membrane electrode containing palmitic acid prepared by using nonactine. The response of glutamine biosensor was linear over the concentration range of 1.0x10-11.0x10-4M and slope was Nernstian. We determined optimum working conditions of the biosensor such as buffer concentration, buffer pH, lifetime, response time, linear working range and other response characteristics. The optimum buffer concentration and pH of proposed glutamine biosensor were determined as 20mM and pH 7.5, respectively. The interference effects of some ions and amino acids that may be present in body fluids were also investigated. The Km and Vmax values of glutaminase were determined. Additionally, glutamine assay in several biological samples such as healthy human serum, cerebrospinal fluid (CSF) and commercial glutamine capsule were also successfully carried out by using the standard addition method. The results were good agreement with previously reported values. (author)

  19. Glutamine supplementation suppresses herpes simplex virus reactivation.

    Science.gov (United States)

    Wang, Kening; Hoshino, Yo; Dowdell, Kennichi; Bosch-Marce, Marta; Myers, Timothy G; Sarmiento, Mayra; Pesnicak, Lesley; Krause, Philip R; Cohen, Jeffrey I

    2017-06-30

    Chronic viral infections are difficult to treat, and new approaches are needed, particularly those aimed at reducing reactivation by enhancing immune responses. Herpes simplex virus (HSV) establishes latency and reactivates frequently, and breakthrough reactivation can occur despite suppressive antiviral therapy. Virus-specific T cells are important to control HSV, and proliferation of activated T cells requires increased metabolism of glutamine. Here, we found that supplementation with oral glutamine reduced virus reactivation in latently HSV-1-infected mice and HSV-2-infected guinea pigs. Transcriptome analysis of trigeminal ganglia from latently HSV-1-infected, glutamine-treated WT mice showed upregulation of several IFN-γ-inducible genes. In contrast to WT mice, supplemental glutamine was ineffective in reducing the rate of HSV-1 reactivation in latently HSV-1-infected IFN-γ-KO mice. Mice treated with glutamine also had higher numbers of HSV-specific IFN-γ-producing CD8 T cells in latently infected ganglia. Thus, glutamine may enhance the IFN-γ-associated immune response and reduce the rate of reactivation of latent virus infection.

  20. Emiliania Huxleyi (Prymnesiophyceae): Nitrogen-metabolism genes and their expression in response to external nitrogen souces

    DEFF Research Database (Denmark)

    Bruhn, Annette; LaRoche, Julie; Richardson, Katherine

    2010-01-01

    The availability and composition of dissolved nitrogen in ocean waters are factors that influence species composition in natural phytoplankton communities. The same factors affect the ratio of organic to inorganic carbon incorporation in calcifying species, such as the coccolithophore Emiliania...... huxleyi (Lohman) W. W. Hay et H. Mohler. E. huxleyi has been shown to thrive on various nitrogen sources, including dissolved organic nitrogen. Nevertheless, assimilation of dissolved nitrogen under nitrogen-replete and -limited conditions is not well understood in this ecologically important species....... In this study, the complete amino acid sequences for three functional genes involved in nitrogen metabolism in E. huxleyi were identified: a putative formamidase, a glutamine synthetase (GSII family), and assimilatory nitrate reductase. Expression patterns of the three enzymes in cells grown on inorganic...

  1. Transgenic algae engineered for higher performance

    Science.gov (United States)

    Unkefer, Pat J; Anderson, Penelope S; Knight, Thomas J

    2014-10-21

    The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. In particular, the disclosure relates to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase.

  2. ORF Alignment: NC_000913 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... glutamine [Escherichia coli K12] pdb|1WXI|A Chain A, ... E.Coli Nad Synthetase, Amp.Pp pdb|1WXH|A Chain A, E.Coli... ... Nad Synthetase, Nad pdb|1WXG|A Chain A, E.Coli... Nad ... Synthetase, Dnd pdb|1WXF|A Chain A, E.Coli Nad ... Synthetase pdb|1WXE|A Chain A, E.Coli

  3. Introduction to the Glutamate-Glutamine Cycle

    DEFF Research Database (Denmark)

    Sonnewald, Ursula; Schousboe, Arne

    2016-01-01

    . This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate......The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain (14)C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor...... released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion...

  4. Introduction to the Glutamate-Glutamine Cycle

    DEFF Research Database (Denmark)

    Sonnewald, Ursula; Schousboe, Arne

    2016-01-01

    released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion......The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain (14)C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor....... This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate...

  5. Glutamine and glutamate: Nonessential or essential amino acids?

    Directory of Open Access Journals (Sweden)

    Malcolm Watford

    2015-09-01

    Full Text Available Glutamine and glutamate are not considered essential amino acids but they play important roles in maintaining growth and health in both neonates and adults. Although glutamine and glutamate are highly abundant in most feedstuffs there is increasing evidence that they may be limiting during pregnancy, lactation and neonatal growth, particularly when relatively low protein diets are fed. Supplementation of diets with glutamine, glutamate or both at 0.5 to 1.0% to both suckling and recently weaned piglets improves intestinal and immune function and results in better growth. In addition such supplementation to the sow prevents some of the loss of lean body mass during lactation, and increases milk glutamine content. However, a number of important questions related to physiological condition, species under study and the form and amount of the supplements need to be addressed before the full benefits of glutamine and glutamate supplementation in domestic animal production can be realized. Keywords: Amino acid, Glutamate, Glutamine, Lactation, Pregnancy, Growth

  6. Enzymology of asparagine metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Unnithan, S.

    1985-01-01

    A highly sensitive method for assaying asparagine synthetase and its glutaminase activity simultaneously was developed using reversed-phase high performance liquid chromatography. The amino acids L-aspartate, L-asparagine, L-glutamate, and L-glutamine were separated after derivatization with O-phthaldialdehyde. This assay can detect picomoles of asparagine and glutamate when a fluorescence detector is used. Chemical modification of asparagine synthetase was carried out. Beef pancreatic asparagine synthetase was inactivated by treatment with the sulfhydryl reagent 5,5'-dithiobis(2-nitro benzoic acid), DTNB. Asparagine protected the glutamine-dependent and ammonia-dependent asparagine synthetase activities as well as the glutaminase activity. Kinetic studies with partially inactivated asparagine synthetase showed that the K/sub m/ values for aspartic acid and glutamine did not change when compared with the K/sub m/ values of the control enzyme. The stoichiometry of DTNB inhibition was measured using (/sup 14/C)DTNB. Differences in the enzymology of L-asparaginase sensitive and resistant L5178Y leukemia cells were examined. Finally, the transport characteristics of L-asparaginase sensitive and resistant cells were observed. The L-asparaginase sensitive cells showed a higher rate of transport of the amino acids glycine, serine, asparagine and aspartate into the cytoplasmic pool.

  7. Endogenous glutamine decrease is associated with pancreatic cancer progression.

    Science.gov (United States)

    Roux, Cecilia; Riganti, Chiara; Borgogno, Sammy Ferri; Curto, Roberta; Curcio, Claudia; Catanzaro, Valeria; Digilio, Giuseppe; Padovan, Sergio; Puccinelli, Maria Paola; Isabello, Monica; Aime, Silvio; Cappello, Paola; Novelli, Francesco

    2017-11-10

    Pancreatic ductal adenocarcinoma (PDAC) is becoming the second leading cause of cancer-related death in the Western world. The mortality is very high, which emphasizes the need to identify biomarkers for early detection. As glutamine metabolism alteration is a feature of PDAC, its in vivo evaluation may provide a useful tool for biomarker identification. Our aim was to identify a handy method to evaluate blood glutamine consumption in mouse models of PDAC. We quantified the in vitro glutamine uptake by Mass Spectrometry (MS) in tumor cell supernatants and showed that it was higher in PDAC compared to non-PDAC tumor and pancreatic control human cells. The increased glutamine uptake was paralleled by higher activity of most glutamine pathway-related enzymes supporting nucleotide and ATP production. Free glutamine blood levels were evaluated in orthotopic and spontaneous mouse models of PDAC and other pancreatic-related disorders by High-Performance Liquid Chromatography (HPLC) and/or MS. Notably we observed a reduction of blood glutamine as much as the tumor progressed from pancreatic intraepithelial lesions to invasive PDAC, but was not related to chronic pancreatitis-associated inflammation or diabetes. In parallel the increased levels of branched-chain amino acids (BCAA) were observed. By contrast blood glutamine levels were stable in non-tumor bearing mice. These findings demonstrated that glutamine uptake is measurable both in vitro and in vivo . The higher in vitro avidity of PDAC cells corresponded to a lower blood glutamine level as soon as the tumor mass grew. The reduction in circulating glutamine represents a novel tool exploitable to implement other diagnostic or prognostic PDAC biomarkers.

  8. Regulation of the subunit composition of plastidic glutamine synthetase of the wild-type and of the phytochrome-deficient aurea mutant of tomato by blue/UV-A- or by UV-B-light

    International Nuclear Information System (INIS)

    Migge, A.; Carrayol, E.; Hirel, B.; Lohmann, M.; Meya, G.; Becker, T.W.

    1998-01-01

    The photomorphogenetic aurea mutant of tomato severely deficient in spectrophotometrically active phytochromes was used to study the light-regulation of the single-copy nuclear gene encoding plastidic glutamine synthetase (GS-2; EC 6.1.3.2). The de-etiolation of dark-grown aurea mutant seedling cotyledons showed an obligatory dependency on blue light. A limited red light-responsiveness of etiolated aurea cotyledons is, however, retained as seen by the stimulation of both the GS-2 transcript and protein level in the cotyledons of aurea seedlings during growth in red light. The subunits of the octameric GS-2 enzyme were represented by polypeptides with similar electrophoretic mobilities (polypeptides a) in etiolated wild-type or aurea mutant cotyledons. GS-2 proteins with similar apparent molecular masses were also seen in the cotyledons of red light-grown aurea mutant seedlings. In contrast, GS-2 polypeptides with different apparent molecular masses (polypeptides a and b) were detected in the cotyledons of wild-type seedlings grown in red light. This difference indicates that the (post-translational) modification of tomato GS-2 subunit composition is mediated by the photoreceptor phytochrome. The illumination of etiolated wild-type or aurea cotyledons with UV-A- or UV-B-light light resulted in an increase in both the GS-2 transcript and protein level. Following illumination of etiolated wild-type seedlings with UV-A-light, the relative proportion of the GS-2 polypeptides a and b was similar than upon irradiation with blue light but different than after exposure to UV-B- or red light. This result suggests the involvement of a blue/ UV-A-light-specific photoreceptor in the regulation of tomato GS-2 subunit composition. (author)

  9. Introduction to the Glutamate-Glutamine Cycle.

    Science.gov (United States)

    Sonnewald, Ursula; Schousboe, Arne

    2016-01-01

    The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain 14 C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor. This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion but more recent research has seriously questioned this.This volume of Advances in Neurobiology is intended to provide a detailed discussion of recent developments in research aimed at delineating the functional roles of the cycle taking into account that in order for this system to work there must be a tight coupling between metabolism of glutamate in astrocytes, transfer of glutamine to neurons and de novo synthesis of glutamine in astrocytes. To understand this, knowledge about the activity and regulation of the enzymes and transporters involved in these processes is required and as can be seen from the table of contents these issues will be dealt with in detail in the individual chapters of the book.

  10. Glufosinate ammonium--some aspects of its mode of action in mammals.

    Science.gov (United States)

    Hack, R; Ebert, E; Ehling, G; Leist, K H

    1994-05-01

    The broad-spectrum herbicide glufosinate ammonium is a structural analogue of glutamate and acts in plants by inhibition of glutamine synthetase leading to a complete breakdown of ammonia metabolism. Owing to the structural analogy of glufosinate ammonium to glutamate, its effect on various glutamate-utilizing systems needed to be investigated in mammals. Although in laboratory animals glufosinate ammonium causes an inhibition of glutamine synthetase activity in different tissues, this inhibition led to slight increases of glutamate and ammonia levels at high sublethal and lethal doses only. After oral administration for 28 days, glufosinate ammonium had no effect on glutathione and carbohydrate metabolism and no effect on biosynthesis of non-essential amino acids in rats and dogs. Glufosinate ammonium does not interfere with various neurotransmitter receptors in vitro and does not influence the catecholamine neurotransmitter tissue concentrations after iv application. The results of these studies show that--in contrast to the plant metabolism--in mammals the inhibition of glutamine synthetase activity in various tissues does not lead to a breakdown of ammonia metabolism. The mammalian metabolism obviously compensates for this inhibition of glutamine synthetase activity by various other metabolic pathways. It is concluded that under the conditions of recommended use of glufosinate ammonium as an active ingredient in herbicides, a detrimental effect on the health of both users and consumers is extremely unlikely.

  11. Effect of Chromium(VI Toxicity on Enzymes of Nitrogen Metabolism in Clusterbean (Cyamopsis tetragonoloba L.

    Directory of Open Access Journals (Sweden)

    Punesh Sangwan

    2014-01-01

    Full Text Available Heavy metals are the intrinsic component of the environment with both essential and nonessential types. Their excessive levels pose a threat to plant growth and yield. Also, some heavy metals are toxic to plants even at very low concentrations. The present investigation (a pot experiment was conducted to determine the affects of varying chromium(VI levels (0.0, 0.5, 1.0, 2.0, and 4.0 mg chromium(VI kg−1 soil in the form of potassium dichromate on the key enzymes of nitrogen metabolism in clusterbean. Chromium treatment adversely affect nitrogenase, nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate dehydrogenase in various plant organs at different growth stages as specific enzyme activity of these enzymes decreased with an increase in chromium(VI levels from 0 to 2.0 mg chromium(VI kg−1 soil and 4.0 mg chromium(VI kg−1 soil was found to be lethal to clusterbean plants. In general, the enzyme activity increased with advancement of growth to reach maximum at flowering stage and thereafter decreased at grain filling stage.

  12. GLUTAMINE AND HYPERAMMONEMIC CRISES IN PATIENTS WITH UREA CYCLE DISORDERS

    Science.gov (United States)

    Lee, B.; Diaz, G.A.; Rhead, W.; Lichter-Konecki, U.; Feigenbaum, A.; Berry, S.A.; Le Mons, C.; Bartley, J.; Longo, N.; Nagamani, S.C.; Berquist, W.; Gallagher, R.C.; Harding, C.O.; McCandless, S.E.; Smith, W.; Schulze, A.; Marino, M.; Rowell, R.; Coakley, D.F.; Mokhtarani, M.; Scharschmidt, B.F.

    2016-01-01

    Blood ammonia and glutamine levels are used as biomarkers of control in patients with urea cycle disorders (UCDs). This study was undertaken to evaluate glutamine variability and utility as a predictor of hyperammonemic crises (HACs) in UCD patients. Methods The relationships between glutamine and ammonia levels and the incidence and timing of HACs were evaluated in over 100 adult and pediatric UCD patients who participated in clinical trials of glycerol phenylbutyrate. Results The median (range) intra-subject 24-hour coefficient of variation for glutamine was 15% (8–29%) as compared with 56% (28%–154%) for ammonia, and the correlation coefficient between glutamine and concurrent ammonia levels varied from 0.17 to 0.29. Patients with baseline (fasting) glutamine values >900 µmol/L had higher baseline ammonia levels (mean [SD]: 39.6 [26.2] µmol/L) than patients with baseline glutamine ≤900 µmol/L (26.6 [18.0] µmol/L). Glutamine values >900 µmol/L during the study were associated with an approximately 2-fold higher HAC risk (odds ratio [OR]=1.98; p=0.173). However, glutamine lost predictive significance (OR=1.47; p=0.439) when concomitant ammonia was taken into account, whereas the predictive value of baseline ammonia ≥ 1.0 upper limit of normal (ULN) was highly statistically significant (OR=4.96; p=0.013). There was no significant effect of glutamine >900 µmol/L on time to first HAC crisis (hazard ratio [HR]=1.14; p=0.813), but there was a significant effect of baseline ammonia ≥ 1.0 ULN (HR=4.62; p=0.0011). Conclusions The findings in this UCD population suggest that glutamine is a weaker predictor of HACs than ammonia and that the utility of the predictive value of glutamine will need to take into account concurrent ammonia levels. PMID:26586473

  13. Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

    Science.gov (United States)

    Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

    2014-05-01

    To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and

  14. Glutamine and glutamate as vital metabolites

    Directory of Open Access Journals (Sweden)

    Newsholme P.

    2003-01-01

    Full Text Available Glucose is widely accepted as the primary nutrient for the maintenance and promotion of cell function. This metabolite leads to production of ATP, NADPH and precursors for the synthesis of macromolecules such as nucleic acids and phospholipids. We propose that, in addition to glucose, the 5-carbon amino acids glutamine and glutamate should be considered to be equally important for maintenance and promotion of cell function. The functions of glutamine/glutamate are many, i.e., they are substrates for protein synthesis, anabolic precursors for muscle growth, they regulate acid-base balance in the kidney, they are substrates for ureagenesis in the liver and for hepatic and renal gluconeogenesis, they act as an oxidative fuel for the intestine and cells of the immune system, provide inter-organ nitrogen transport, and act as precursors of neurotransmitter synthesis, of nucleotide and nucleic acid synthesis and of glutathione production. Many of these functions are interrelated with glucose metabolism. The specialized aspects of glutamine/glutamate metabolism of different glutamine-utilizing cells are discussed in the context of glucose requirements and cell function.

  15. Comparative proteomics analysis of the rice roots colonized by Herbaspirillum seropedicae strain SmR1 reveals induction of the methionine recycling in the plant host.

    Science.gov (United States)

    Alberton, Dayane; Müller-Santos, Marcelo; Brusamarello-Santos, Liziane Cristina Campos; Valdameri, Glaucio; Cordeiro, Fabio Aparecido; Yates, Marshall Geoffrey; de Oliveira Pedrosa, Fabio; de Souza, Emanuel Maltempi

    2013-11-01

    Although the use of plant growth-promoting bacteria in agriculture is a reality, the molecular basis of plant-bacterial interaction is still poorly understood. We used a proteomic approach to study the mechanisms of interaction of Herbaspirillum seropedicae SmR1 with rice. Root proteins of rice seedlings inoculated or noninoculated with H. seropedicae were separated by 2-D electrophoresis. Differentially expressed proteins were identified by MALDI-TOF/TOF and MASCOT program. Among the identified proteins of H. seropedicae, the dinitrogenase reductase NifH and glutamine synthetase GlnA, which participate in nitrogen fixation and ammonium assimilation, respectively, were the most abundant. The rice proteins up-regulated included the S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1, all of which are involved in the methionine recycling. S-Adenosylmethionine synthetase catalyzes the synthesis of S-adenosylmethionine, an intermediate used in transmethylation reactions and in ethylene, polyamine, and phytosiderophore biosynthesis. RT-qPCR analysis also confirmed that the methionine recycling and phytosiderophore biosynthesis genes were up-regulated, while ACC oxidase mRNA level was down-regulated in rice roots colonized by bacteria. In agreement with these results, ethylene production was reduced approximately three-fold in rice roots colonized by H. seropedicae. The results suggest that H. seropedicae stimulates methionine recycling and phytosiderophore synthesis and diminishes ethylene synthesis in rice roots.

  16. Ammonia stress on nitrogen metabolism in tolerant aquatic plant-Myriophyllum aquaticum.

    Science.gov (United States)

    Zhou, Qingyang; Gao, Jingqing; Zhang, Ruimin; Zhang, Ruiqin

    2017-09-01

    Ammonia has been a major reason of macrophyte decline in the water environment, and ammonium ion toxicity should be seen as universal, even in species frequently labeled as "NH 4 + specialists". To study the effects of high NH 4 + -N stress of ammonium ion nitrogen on tolerant submerged macrophytes and investigate the pathways of nitrogen assimilation in different organisms, Myriophyllum aquaticum was selected and treated with various concentrations of ammonium ions at different times. Increasing of ammonium concentration leads to an overall increase in incipient ammonia content in leaves and stems of plants. In middle and later stages, high concentrations of NH 4 + ion nitrogen taken up by M. aquaticum decreased, whereas the content of NO 3 - ion nitrogen increased. Moreover, in M. aquaticum, the activities of the enzymes nitrate reductase, glutamine synthetase and asparagine synthetase changed remarkably in the process of alleviating NH 4 + toxicity and deficiency. The results of the present study may support the studies on detoxification of high ammonium ion content in NH 4 + -tolerant submerged macrophytes and exploration of tissue-specific expression systems. Copyright © 2017. Published by Elsevier Inc.

  17. Prolonged continuous intravenous infusion of the dipeptide L-alanine- L-glutamine significantly increases plasma glutamine and alanine without elevating brain glutamate in patients with severe traumatic brain injury.

    Science.gov (United States)

    Nägeli, Mirjam; Fasshauer, Mario; Sommerfeld, Jutta; Fendel, Angela; Brandi, Giovanna; Stover, John F

    2014-07-02

    Low plasma glutamine levels are associated with worse clinical outcome. Intravenous glutamine infusion dose- dependently increases plasma glutamine levels, thereby correcting hypoglutaminemia. Glutamine may be transformed to glutamate which might limit its application at a higher dose in patients with severe traumatic brain injury (TBI). To date, the optimal glutamine dose required to normalize plasma glutamine levels without increasing plasma and cerebral glutamate has not yet been defined. Changes in plasma and cerebral glutamine, alanine, and glutamate as well as indirect signs of metabolic impairment reflected by increased intracranial pressure (ICP), lactate, lactate-to-pyruvate ratio, electroencephalogram (EEG) activity were determined before, during, and after continuous intravenous infusion of 0.75 g L-alanine-L-glutamine which was given either for 24 hours (group 1, n = 6) or 5 days (group 2, n = 6) in addition to regular enteral nutrition. Lab values including nitrogen balance, urea and ammonia were determined daily. Continuous L-alanine-L-glutamine infusion significantly increased plasma and cerebral glutamine as well as alanine levels, being mostly sustained during the 5 day infusion phase (plasma glutamine: from 295 ± 62 to 500 ± 145 μmol/ l; brain glutamine: from 183 ± 188 to 549 ± 120 μmol/ l; plasma alanine: from 327 ± 91 to 622 ± 182 μmol/ l; brain alanine: from 48 ± 55 to 89 ± 129 μmol/ l; p alanine-L-glutamine infusion (0.75 g/ kg/ d up to 5 days) increased plasma and brain glutamine and alanine levels. This was not associated with elevated glutamate or signs of potential glutamate-mediated cerebral injury. The increased nitrogen load should be considered in patients with renal and hepatic dysfunction. Clinicaltrials.gov NCT02130674. Registered 5 April 2014.

  18. Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

    International Nuclear Information System (INIS)

    Bandaralage, Sahan P.S.; Farnaghi, Soheil; Dulhunty, Joel M.; Kothari, Alka

    2016-01-01

    Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

  19. Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

    Energy Technology Data Exchange (ETDEWEB)

    Bandaralage, Sahan P.S. [Gold Coast Hospital and Health Service, Southport, Queensland (Australia); Griffith University, School of Medicine, Southport, Queensland (Australia); Farnaghi, Soheil [Caboolture Hospital, Caboolture, Queensland (Australia); Dulhunty, Joel M.; Kothari, Alka [Redcliffe Hospital, Redcliffe, Queensland (Australia); The University of Queensland, School of Medicine, Herston, Queensland (Australia)

    2016-03-15

    Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

  20. Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

    Science.gov (United States)

    Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

    2005-01-01

    CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

  1. Ketopantoyl lactone reductase is a conjugated polyketone reductase.

    Science.gov (United States)

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-03-01

    Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.

  2. Arginine Deprivation Inhibits the Warburg Effect and Upregulates Glutamine Anaplerosis and Serine Biosynthesis in ASS1-Deficient Cancers

    Directory of Open Access Journals (Sweden)

    Jeff Charles Kremer

    2017-01-01

    Full Text Available Targeting defects in metabolism is an underutilized strategy for the treatment of cancer. Arginine auxotrophy resulting from the silencing of argininosuccinate synthetase 1 (ASS1 is a common metabolic alteration reported in a broad range of aggressive cancers. To assess the metabolic effects that arise from acute and chronic arginine starvation in ASS1-deficient cell lines, we performed metabolite profiling. We found that pharmacologically induced arginine depletion causes increased serine biosynthesis, glutamine anaplerosis, oxidative phosphorylation, and decreased aerobic glycolysis, effectively inhibiting the Warburg effect. The reduction of glycolysis in cells otherwise dependent on aerobic glycolysis is correlated with reduced PKM2 expression and phosphorylation and upregulation of PHGDH. Concurrent arginine deprivation and glutaminase inhibition was found to be synthetic lethal across a spectrum of ASS1-deficient tumor cell lines and is sufficient to cause in vivo tumor regression in mice. These results identify two synthetic lethal therapeutic strategies exploiting metabolic vulnerabilities of ASS1-negative cancers.

  3. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells

    Science.gov (United States)

    Cheng, Tzuling; Sudderth, Jessica; Yang, Chendong; Mullen, Andrew R.; Jin, Eunsook S.; Matés, José M.; DeBerardinis, Ralph J.

    2011-01-01

    Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how “glutamine-addicted” cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis. PMID:21555572

  4. Nitrogen Starvation and TorC1 Inhibition Differentially Affect Nuclear Localization of the Gln3 and Gat1 Transcription Factors Through the Rare Glutamine tRNACUG in Saccharomyces cerevisiae

    Science.gov (United States)

    Tate, Jennifer J.; Rai, Rajendra; Cooper, Terrance G.

    2015-01-01

    A leucine, leucyl-tRNA synthetase–dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of Gln3, a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of Gln3 occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of Gln3 and Gat1 (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear Gln3 localization. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on nitrogen-responsive control of Gln3 and Gat1. We found that nuclear Gln3 localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or ure2Δ mutation is abolished in a sup70-65 mutant. However, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a ure2Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with Gln3, Gat1 localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3 and Gat1 localization and their downstream production of nitrogenous precursors

  5. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

    Science.gov (United States)

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

    2014-01-01

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

  6. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    Science.gov (United States)

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

    2014-11-25

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

  7. Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation.

    Science.gov (United States)

    Jiang, Yu; Tao, Rongsheng; Shen, Zhengquan; Sun, Liangdong; Zhu, Fuyun; Yang, Sheng

    2016-12-01

    Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l -1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol -1 added cysteine and a productivity of 6.1 ± 0.0 g l -1  h -1 . This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.

  8. Assay of glutamine phosphoribosylpyrophosphate amidotransferase using [1-14C]phosphoribosylpyrophosphate

    International Nuclear Information System (INIS)

    Ross, G.R.; Idriss, S.D.; Willis, R.C.; Seegmiller, J.E.

    1983-01-01

    Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-α-D-ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction, 5-β-phosphoribosyl-1-amine, using [1- 14 C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent K/sub m/ values of the human lymphoblast enzyme are 0.46 mM for glutamine and 0.71 mM for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo-L-norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity

  9. Response of nitrogen metabolism to boron toxicity in tomato plants.

    Science.gov (United States)

    Cervilla, L M; Blasco, B; Ríos, J J; Rosales, M A; Rubio-Wilhelmi, M M; Sánchez-Rodríguez, E; Romero, L; Ruiz, J M

    2009-09-01

    Boron (B) toxicity has become important in areas close to the Mediterranean Sea where intensive agriculture has been developed. The objective of this research was to study the effects of B toxicity (0.5 mM and 2.0 mM B) on nitrogen (N) assimilation of two tomato cultivars that are often used in these areas. Leaf biomass, relative leaf growth rate (RGR(L)), concentration of B, nitrate (NO(3) (-)), ammonium (NH(4) (+)), organic N, amino acids and soluble proteins, as well as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), glutamate synthetase (GOGAT) and glutamate dehydrogenase (GDH) activities were analysed in leaves. Boron toxicity significantly decreased leaf biomass, RGR(L), organic N, soluble proteins, and NR and NiR activities. The lowest NO(3) (-) and NH(4) (+) concentration in leaves was recorded when plants were supplied with 2.0 mM B in the root medium. Total B, amino acids, activities of GS, GOGAT and GDH increased under B toxicity. Data from the present study prove that B toxicity causes inhibition of NO(3) (-) reduction and increases NH(4) (+) assimilation in tomato plants.

  10. Comparative effect of Al, Se, and Mo toxicity on NO3(-) assimilation in sunflower (Helianthus annuus L.) plants.

    Science.gov (United States)

    Ruiz, Juan M; Rivero, Rosa M; Romero, Luis

    2007-04-01

    Here, we study the effect caused by three trace elements--Al, Se, and Mo--applied at the same concentration (100 microM) and in their oxyanionic forms--NaAl(OH)(4), Na(2)SeO(4), and Na(2)MoO(4)--on NO(3)(-) assimilation (NO(3)(-), nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) activities, and concentrations of amino acids and proteins) in sunflower (Helianthus annuus L. var. Kasol) plants. The most harmful element for sunflower plants proved to be selenate, followed by aluminate. On the contrary, the application of molybdate had no negative effect on the growth of this plant, suggesting the possibility of using sunflower for the phytoremediation of this metal, mainly in agricultural zones used for grazing where the excess of this element can provoke problems of molybdenosis in ruminants (particularly in cattle). In addition, we found that the alteration of NO(3)(-) assimilation by SeO(4)(2-) and Al(OH)(4)(-) directly influences the growth and development of plants, foliar inhibition of NR activity by SeO(4)(2-) being more harmful than the decrease in foliar availability of NO(3)(-) provoked by Al(OH)(4)(-).

  11. Photoperiodic regulation of glycogen metabolism, glycolysis, and glutamine synthesis in tanycytes of the Siberian hamster suggests novel roles of tanycytes in hypothalamic function.

    Science.gov (United States)

    Nilaweera, Kanishka; Herwig, Annika; Bolborea, Matei; Campbell, Gill; Mayer, Claus D; Morgan, Peter J; Ebling, Francis J P; Barrett, Perry

    2011-11-01

    The objective of this study is to investigate the impact of photoperiod on the temporal and spatial expression of genes involved in glucose metabolism in the brain of the seasonal mammal Phodopus sungorus (Siberian hamster). In situ hybridization was performed on brain sections obtained from male hamsters held in long photoperiod (high body weight and developed testes) or short photoperiod (reduced body weight with testicular regression). This analysis revealed upregulation in expression of genes involved in glycogen and glucose metabolism in short photoperiod and localized to the tanycyte layer of the third ventricle. On the basis of these data and a previously identified photoperiod-dependent increase in activity of neighboring hypothalamic neurons, we hypothesized that the observed expression changes may reflect alteration in either metabolic fuel or precursor neurotransmitter supply to surrounding neurons. Gene expression analysis was performed for genes involved in lactate and glutamate transport. This analysis showed that the gene for the lactate transporter MCT2 and glutamate transporter GLAST was decreased in the tanycyte layer in short photoperiod. Expression of mRNA for glutamine synthetase, the final enzyme in the synthesis of the neuronal neurotransmitter precursor, glutamine, was also decreased in short photoperiod. These data suggest a role for tanycytes in modulating glutamate concentrations and neurotransmitter supply in the hypothalamic environment. Copyright © 2011 Wiley-Liss, Inc.

  12. Selective inhibition of type 2 fatty acid synthetase by the antibiotic thiolactomycin

    International Nuclear Information System (INIS)

    Nishida, Ikuo; Kawaguchi, Akihiko; Yamada, Mitsuhiro

    1984-01-01

    The antibiotic thiolactomycin inhibits the fatty acid synthesis from both [1- 14 C]-acetate and [2 14 C] malonyl-CoA of spinach leaves, developing castor bean endosperms and avocado mesocarp. On the other hand, fatty acid synthetases of Brevibacterium ammoniagenes and Corynebacterium glutamicum are much less sensitive to this antibiotic. As Hayashi et al. have indicated in their paper that thiolactomycin inhibits fatty acid synthetase of Escherichia coli but has little effect on the synthetases of yeast and rat liver, thiolactomycin is suggested to be a selective inhibitor of type 2 fatty acid synthetases. (author)

  13. Selective inhibition of type 2 fatty acid synthetase by the antibiotic thiolactomycin

    Energy Technology Data Exchange (ETDEWEB)

    Nishida, Ikuo; Kawaguchi, Akihiko; Yamada, Mitsuhiro (Tokyo Univ. (Japan). Faculty of Science)

    1984-03-01

    The antibiotic thiolactomycin inhibits the fatty acid synthesis from both (1-/sup 14/C)-acetate and (2/sup 14/C) malonyl-CoA of spinach leaves, developing castor bean endosperms and avocado mesocarp. On the other hand, fatty acid synthetases of Brevibacterium ammoniagenes and Corynebacterium glutamicum are much less sensitive to this antibiotic. As Hayashi et al. have indicated in their paper that thiolactomycin inhibits fatty acid synthetase of Escherichia coli but has little effect on the synthetases of yeast and rat liver, thiolactomycin is suggested to be a selective inhibitor of type 2 fatty acid synthetases.

  14. The nitrate-reduction gene cluster components exert lineage-dependent contributions to optimization of Sinorhizobium symbiosis with soybeans.

    Science.gov (United States)

    Liu, Li Xue; Li, Qin Qin; Zhang, Yun Zeng; Hu, Yue; Jiao, Jian; Guo, Hui Juan; Zhang, Xing Xing; Zhang, Biliang; Chen, Wen Xin; Tian, Chang Fu

    2017-12-01

    Receiving nodulation and nitrogen fixation genes does not guarantee rhizobia an effective symbiosis with legumes. Here, variations in gene content were determined for three Sinorhizobium species showing contrasting symbiotic efficiency on soybeans. A nitrate-reduction gene cluster absent in S. sojae was found to be essential for symbiotic adaptations of S. fredii and S. sp. III. In S. fredii, the deletion mutation of the nap (nitrate reductase), instead of nir (nitrite reductase) and nor (nitric oxide reductase), led to defects in nitrogen-fixation (Fix - ). By contrast, none of these core nitrate-reduction genes were required for the symbiosis of S. sp. III. However, within the same gene cluster, the deletion of hemN1 (encoding oxygen-independent coproporphyrinogen III oxidase) in both S. fredii and S. sp. III led to the formation of nitrogen-fixing (Fix + ) but ineffective (Eff - ) nodules. These Fix + /Eff - nodules were characterized by significantly lower enzyme activity of glutamine synthetase indicating rhizobial modulation of nitrogen-assimilation by plants. A distant homologue of HemN1 from S. sojae can complement this defect in S. fredii and S. sp. III, but exhibited a more pleotropic role in symbiosis establishment. These findings highlighted the lineage-dependent optimization of symbiotic functions in different rhizobial species associated with the same host. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  16. Ion exclusion chromatography for the purification of L-glutamine; Ion haijo chromatography ni yoru L-glutamine no seiseiho no kenkyu

    Energy Technology Data Exchange (ETDEWEB)

    Ito, H.; Nishi, A.; Naruse, M. [Ajinomoto Co. Inc., Kawasaki (Japan)

    1998-09-05

    Ion exclusion chromatography for the purification of L-glutamine is studied. L-glutamine is usually produced by fermentation and used in pharmaceuticals. By using a model solution of L-glutamine and L-glutamic acid, the optimum cation exchange resin is examined. As a result of the experiments, it is found that a cation exchange resin which has smaller crosslinkage and smaller diameter is better. Ammonium sulfate, L-glutamic acid and pyrrolidonecarboxylic acid, which are usually contained in fermentation broth as impurities, are effectively separated by this method. Moreover, the experimental data of the chromatography is expressed fairly well by the differential equations which express the mass transfer in the fixed bed. 8 refs., 5 figs., 2 tabs.

  17. Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

    International Nuclear Information System (INIS)

    Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

    1987-01-01

    Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR - nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second 14 CO 2 pulse, the total 14 C incorporation of the mutant leaves was approximately 20 5 of that of the control. The 14 C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second 14 CO 2 pulse followed by a 60 second chase with normal CO 2 , 14 C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus

  18. Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

    Energy Technology Data Exchange (ETDEWEB)

    Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

    1987-05-01

    Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR/sup -/ nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second /sup 14/CO/sub 2/ pulse, the total /sup 14/C incorporation of the mutant leaves was approximately 20/sup 5/ of that of the control. The /sup 14/C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second /sup 14/CO/sub 2/ pulse followed by a 60 second chase with normal CO/sub 2/, /sup 14/C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus.

  19. A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Enosse, Sonia; Pearce, Richard

    2005-01-01

    Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine....... However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA...

  20. Frequencies distribution of dihydrofolate reductase and dihydropteroate synthetase mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum population from Hadhramout Governorate, Yemen.

    Science.gov (United States)

    Bamaga, Omar A A; Mahdy, Mohammed A K; Lim, Yvonne A L

    2015-12-22

    Malaria in Yemen is mainly caused by Plasmodium falciparum and 25% of the population is at high risk. Sulfadoxine-pyrimethamine (SP) had been used as monotherapy against P. falciparum. Emergence of chloroquine resistance led to the shift in anti-malarial treatment policy in Yemen to artemisinin-based combination therapy, that is artesunate (AS) plus SP as first-line therapy for uncomplicated malaria and artemether-lumefantrine as second-line treatment. This study aimed to screen mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes associated with SP resistance among P. falciparum population in Hadhramout governorate, Yemen. Genomic DNA was extracted from dried blood spots of 137 P. falciparum isolates collected from a community-based study. DNA was amplified using nested polymerase chain reaction (PCR) and subsequently sequenced for Pfdhfr and Pfdhps genes. Sequences were analysed for mutations in Pfdhfr gene codons 51, 59, 108, and 164 and in Pfdhps gene codons 436, 437, and 540. A total of 128 and 114 P. falciparum isolates were successfully sequenced for Pfdhfr and Pfdhps genes, respectively. Each Pfdhfr mutant allele (I51 and N108) in P. falciparum population had a frequency of 84%. Pfdhfr R59 mutant allele was detected in one isolate. Mutation at codon 437 (G437) in the Pfdhps gene was detected in 44.7% of falciparum malaria isolates. Frequencies of Pfdhfr double mutant genotype (I51C59N108I164) and Pfdhfr/Pfdhps triple mutant genotype (I51C59N108I164-S436G437K540) were 82.8 and 39.3%, respectively. One isolate harboured Pfdhfr triple mutant genotype (I51, R59, N108, I164) and Pfdhfr/Pfdhps quadruple mutant genotype (I51R59N108I164-S436G437K540). High frequencies of Pfdhfr and Pfdhps mutant alleles and genotypes in P. falciparum population in Hadhramout, Yemen, highlight the risk of developing resistance for SP, the partner drug of AS, which subsequently will expose the parasite to AS monotherapy increasing then the

  1. Valyl-tRNA synthetase gene of Escherichia coli K12: Molecular genetic characterization and homology within a family of aminoacyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Heck, J.D. III.

    1988-01-01

    This work reports the subcloning and characterization of the molecular elements necessary for the expression of the Escherichia coli valS gene encoding valyl-tRNA synthetase. The valS gene was subcloned from plasmid pLC26-22 by genetic complementation of a valS ts strain. The DNA region encoding the valS structural gene was determined by in vitro coupled transcription-translation assays. Cells transformed with a plasmid containing a full length copy of the valS gene enhanced in vivo valyl-tRNA synthetase specific activity twelve-fold. DNA sequences flanking the valS structural gene are presented. The transcription initiation sites of the valS gene were determined, in vivo and in vitro, by S1 nuclease protection studies, primer-extension analysis and both [α- 32 P]labeled and [γ- 32 P]end-labeled in vitro transcription assays. The DNA sequence of the valS gene of Escherichia coli has been determined. Significant similarity at the primary sequence level was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. An extended open reading frame (ORF) encoded on the DNA strand opposite the valS structural gene is described

  2. Effects of glutamine, taurine and their association on inflammatory pathway markers in macrophages.

    Science.gov (United States)

    Sartori, Talita; Galvão Dos Santos, Guilherme; Nogueira-Pedro, Amanda; Makiyama, Edson; Rogero, Marcelo Macedo; Borelli, Primavera; Fock, Ricardo Ambrósio

    2018-06-01

    The immune system is essential for the control and elimination of infections, and macrophages are cells that act as important players in orchestrating the various parts of the inflammatory/immune response. Amino acids play important role in mediating functionality of the inflammatory response, especially mediating macrophages functions and cytokines production. We investigated the influence of glutamine, taurine and their association on the modulation of inflammatory pathway markers in macrophages. The RAW 264.7 macrophage cell line was cultivated in the presence of glutamine and taurine and proliferation rates, cell viability, cell cycle phases, IL-1α, IL-6, IL-10 and TNF-α as well as H 2 O 2 production and the expression of the transcription factor, NFκB, and its inhibitor, IκBα, were evaluated. Our results showed an increase in viable cells and increased proliferation rates of cells treated with glutamine concentrations over 2 mM, as well as cells treated with both glutamine and taurine. The cell cycle showed a higher percentage of cells in the phases S, G2 and M when they were treated with 2 or 10 mM glutamine, or with glutamine and taurine in cells stimulated with lipopolysaccharide. The pNFκB/NFκB showed reduced ratio expression when cells were treated with 10 mM of glutamine or with glutamine in association with taurine. These conditions also resulted in reduced TNF-α, IL-1α and H 2 O 2 production, and higher production of IL-10. These findings demonstrate that glutamine and taurine are able to modulate macrophages inflammatory pathways, and that taurine can potentiate the effects of glutamine, illustrating their immunomodulatory properties.

  3. Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-01

    Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

  4. Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae.

    Science.gov (United States)

    Fam, Rachel R S; Hiong, Kum C; Choo, Celine Y L; Wong, Wai P; Chew, Shit F; Ip, Yuen K

    2018-05-20

    Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. The kidney of chicken adapts to chronic metabolic acidosis: in vivo and in vitro studies.

    Science.gov (United States)

    Craan, A G; Lemieux, G; Vinay, P; Gougoux, A

    1982-08-01

    Renal adaptation to chronic metabolic acidosis was studies in Arbor Acre hens receiving ammonium chloride by stomach tube 0.75 g/kg/day during 6 days. During a 14-day study, it was shown that the animals could excrete as much as 60% of the acid load during ammonium chloride administration. At the same time urate excretion fell markedly but the renal contribution to urate excretion (14%) did not change. During acidosis, blood glutamine increased twofold and the tissue concentration of glutamine rose in both liver and kidney. Infusion of L-glutamine led to increased ammonia excretion and more so in acidotic animals. Glutaminase I, glutamate dehydrogenase, alanine aminotransferase (GPT), and malic enzyme activities increased in the kidney during acidosis but phosphoenolpyruvate carboxykinase (PEPCK) activity did not change. Glutaminase I was not found in the liver, but hepatic glutamine synthetase rose markedly during acidosis. Glutamine synthetase was not found in the kidney. Renal tubules incubated with glutamine and alanine were ammoniagenic and gluconeogenic to the same degree as rat tubules with the same increments in acidosis. Lactate was gluconeogenic without increment during acidosis. The present study indicates that the avian kidney adapts to chronic metabolic acidosis with similarities and differences when compared to dog and rat. Glutamine originating from the liver appears to be the major ammoniagenic substrate. Our data also support the hypothesis that hepatic urate synthesis is decreased during acidosis.

  6. Effect of L-glutamine levels in piglets diets challenged with Escherichia coli lipopolysacharides

    Directory of Open Access Journals (Sweden)

    Arturo Pardo L.

    2014-09-01

    Full Text Available Objective. To evaluate the effect of different levels of L-glutamine on weaned and immunologically challenged piglets with Escherichia coli lipopolysaccharides (LPS on performance parameters, serum cortisol and defense cells. Materials and methods. Four levels of L –glutamine were evaluated (0, 1.0, 1.5, 2.0% as well as the addition, or no addition, of LPS (0.3μg. 96 piglets were used (48 castrated males and 48 females of Agroceres x PenArlan lineage, with an initial age of 21 days and 6.06±0.852 kg live weight. An experimental design was used on randomized blocks in a factorial setting 4 x 2 (levels of L- glutamine with or without challenge. Results. Cubic effect was shown for daily weight gain of unchallenged animals, and was better with the addition of 0.41% L- glutamine. Feed conversion improved with increased levels of L -glutamine for challenged animals. In the evaluation of defense cells, there was interaction of leukocytes with the levels of L- glutamine and the immune challenge. Eosinophils and lymphocytes showed a quadratic effect for the levels of L –glutamine, with a maximum value of 1.30% and 0.5%, respectively. Conclusions. L -glutamine supplementation of up to 2% in the diet improves feed conversion and favors the immune serum of weaned piglets challenged with LPS of E. coli.

  7. Effects of hyperthyroidism and hypothyroidism on glutamine metabolism by skeletal muscle of the rat.

    Science.gov (United States)

    Parry-Billings, M; Dimitriadis, G D; Leighton, B; Bond, J; Bevan, S J; Opara, E; Newsholme, E A

    1990-01-01

    1. The effects of hyperthyroidism and hypothyroidism on the concentrations of glutamine and other amino acids in the muscle and plasma and on the rates of glutamine and alanine release from incubated isolated stripped soleus muscle of the rat were investigated. 2. Hyperthyroidism decreased the concentration of glutamine in soleus muscle but was without effect on that in the gastrocnemius muscle or in the plasma. Hyperthyroidism also increased markedly the rate of release of glutamine from the incubated soleus muscle. 3. Hypothyroidism decreased the concentrations of glutamine in the gastrocnemius muscle and plasma but was without effect on that in soleus muscle. Hypothyroidism also decreased markedly the rate of glutamine release from the incubated soleus muscle. 4. Thyroid status was found to have marked effects on the rate of glutamine release by skeletal muscle per se, and may be important in the control of this process in both physiological and pathological conditions. PMID:2268261

  8. Regulation of the glutamine transporter SN1 by extracellular pH and intracellular sodium ions

    International Nuclear Information System (INIS)

    Broeer, A.; Broeer, S.; Setiawan, I.; Lang, F.

    2001-01-01

    Full text: SN1 has recently been identified as one of the major glutamine transporters in hepatocytes and brain astrocytes. It appears to be the molecular correlate of the system N amino acid transporter. Two different transport mechanisms have been proposed for this transporter. Either an electroneutral mechanism, in which glutamine uptake is coupled to an exchange of 1Na + and 1H + , or an electrogenic mechanism coupled to the exchange of 2Na + against 1H + . This study was performed to solve the discrepancies and to investigate the reversibility of the transporter. When expressed in Xenopus laevis oocytes glutamine uptake activity increased strongly with increasing pH. In agreement with the pH-dependence we found that uptake of glutamine was accompanied by an alkalization of the cytosol, indicating that SN1 mediates Glutamine/H + -Antiport. Uptake of glutamine into oocytes was Na + -dependent. Analysis of the Na + -dependence of glutamine transport and Flux studies using 22 Na + indicated that two or more sodium ions were cotransported together with glutamine. However, at the same time intracellular Na + was exchanged against extracellular Na + . Taken together with the results of the pH-dependence it is proposed that SN1 mediates a Na + /Na + -exchange and a Na + /H + -exchange, both being coupled to the transport of glutamine. In agreement with this mechanism we found that acidic pH caused a reversal of the transporter. To investigate the source of the glutamine-induced inward currents, we compared inward currents generated by the 1Na + /glutamine cotransporter ATA1 with those generated by SN1. Currents induced by glutamine uptake in SN1 expressing oocytes were only a fraction of the currents induced by glutamine in ATA1 expressing oocytes, indicating that they were not generated by a stoichiometric uptake of ions. It is concluded that SN1 is tightly regulated by pH and intracellular Na + -ions and is capable of mediating glutamine uptake and release

  9. Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

    NARCIS (Netherlands)

    Kremer, D.R.; Veenhuis, M.; Fauque, G.; Peck Jr., H.D.; LeGall, J.; Lampreia, J.; Moura, J.J.G.; Hansen, T.A.

    1988-01-01

    The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were

  10. Glutamine granule-supplemented enteral nutrition maintains immunological function in severely burned patients.

    Science.gov (United States)

    Peng, Xi; Yan, Hong; You, Zhongyi; Wang, Pei; Wang, Shiliang

    2006-08-01

    Glutamine is an important energy source for immune cells. It is a necessary nutrient for cell proliferation, and serves as specific fuel for lymphocytes, macrophages, and enterocytes when it is present in appropriate concentrations. The purpose of this clinical study was to observe the effects of enteral nutrition supplemented with glutamine granules on immunologic function in severely burned patients. Forty-eight severely burned patients (total burn surface area 30-75%, full thickness burn area 20-58%) who met the requirements of the protocol joined this double-blind randomized controlled clinical trail. Patients were randomly divided into two groups: burn control group (B group, 23 patients) and glutamine treated group (Gln group, 25 patients). There was isonitrogenous and isocaloric intake in both groups, Gln and B group patents were given glutamine granules or placebo (glycine) at 0.5 g/kgd for 14 days with oral feeding or tube feeding, respectively. The plasma level of glutamine and several indices of immunologic function including lymphocyte transformation ratio, neutrophil phagocytosis index (NPI), CD4/CD8 ratio, the content of immunoglobulin, complement C3, C4 and IL-2 levels were determined. Moreover, wound healing rate of burn area was observed and then hospital stay was recorded. The results showed significantly reduced plasma glutamine and damaged immunological function after severe burn Indices of cellular immunity function were remarkably decreased from normal controls. After taking glutamine granules for 14 days, plasma glutamine concentration was significantly higher in Gln group than that in B group (607.86+/-147.25 micromol/L versus 447.63+/-132.38 micromol/L, P0.05). In addition, wound healing was better and hospital stay days were reduced in Gln group (46.59+/-12.98 days versus 55.68+/-17.36 days, Pburn; supplemented glutamine granules with oral feeding or tube feeding abate the degree of immunosuppression, improve immunological function

  11. De Novo Glutamine Synthesis: Importance for the Proliferation of Glioma Cells and Potentials for Its Detection With 13N-Ammonia.

    Science.gov (United States)

    He, Qiao; Shi, Xinchong; Zhang, Linqi; Yi, Chang; Zhang, Xuezhen; Zhang, Xiangsong

    2016-01-01

    The aim of this study was to investigate the role of de novo glutamine (Gln) synthesis in the proliferation of C6 glioma cells and its detection with (13)N-ammonia. Chronic Gln-deprived C6 glioma (0.06C6) cells were established. The proliferation rates of C6 and 0.06C6 cells were measured under the conditions of Gln deprivation along with or without the addition of ammonia or glutamine synthetase (GS) inhibitor. (13)N-ammonia uptake was assessed in C6 cells by gamma counting and in rats with C6 and 0.06C6 xenografts by micro-positron emission tomography (PET) scanning. The expression of GS in C6 cells and xenografts was assessed by Western blotting and immunohistochemistry, respectively. The Gln-deprived C6 cells showed decreased proliferation ability but had a significant increase in GS expression. Furthermore, we found that low concentration of ammonia was sufficient to maintain the proliferation of Gln-deprived C6 cells, and (13)N-ammonia uptake in C6 cells showed Gln-dependent decrease, whereas inhibition of GS markedly reduced the proliferation of C6 cells as well as the uptake of (13)N-ammoina. Additionally, microPET/computed tomography exhibited that subcutaneous 0.06C6 xenografts had higher (13)N-ammonia uptake and GS expression in contrast to C6 xenografts. De novo Gln synthesis through ammonia-glutamate reaction plays an important role in the proliferation of C6 cells. (13)N-ammonia can be a potential metabolic PET tracer for Gln-dependent tumors. © The Author(s) 2016.

  12. The role of glutamine transport in metabolism in the brain cortical tissue slice

    International Nuclear Information System (INIS)

    Hare, N.; Bubb, W.A.; Rae, C.; Broeer, S.

    2001-01-01

    The widely accepted 'glutamate/glutamine cycle' holds that glutamate released as a neurotransmitter in the brain is taken up by surrounding astrocytes, converted to neuro-inactive glutamine and transported back to neurons for reconversion to glutamate. Little, however, is known about the role of glutamine transport in this process. The situation is complicated by the fact that glutamine is transported by a variety of general amino-acid transporters of low specificity. The role of these transporters in flux of glutamine through the glutamate/glutamine cycle was investigated by 13 C NMR monitoring of the flux of C from [3- 13 C]L-lactate in guinea pig cortical tissue slices in the presence of competitive inhibitors of the A-type(α-(methylamino)isobutyrate; MeAIB) and N-type (histidine) transporters. The presence of each inhibitor (10 mM) produced no significant decrease in total metabolite pool size but resulted in a significant decrease in flux of [ 13 C] into the neurotransmitters glutamate and GABA and also into glutamine and alanine. The factional enrichment of glutamate and GABA was also significantly lower. By contrast there was no effect on the amount of [ 13 C] incorporated into aspartate isotopomers which may represent a predominantly astrocyte-labelled pool. These results are consistent with involvement of glutamine transporters in the recycling of synaptic glutamate by demonstrating partial blockage of incorporation of [ 13 C] label into neuronal metabolites

  13. Glutamine prevents oxidative stress in a model of portal hypertension.

    Science.gov (United States)

    Zabot, Gilmara Pandolfo; Carvalhal, Gustavo Franco; Marroni, Norma Possa; Licks, Francielli; Hartmann, Renata Minuzzo; da Silva, Vinícius Duval; Fillmann, Henrique Sarubbi

    2017-07-07

    To evaluate the protective effects of glutamine in a model of portal hypertension (PH) induced by partial portal vein ligation (PPVL). Male Wistar rats were housed in a controlled environment and were allowed access to food and water ad libitum . Twenty-four male Wistar rats were divided into four experimental groups: (1) control group (SO) - rats underwent exploratory laparotomy; (2) control + glutamine group (SO + G) - rats were subjected to laparotomy and were treated intraperitoneally with glutamine; (3) portal hypertension group (PPVL) - rats were subjected to PPVL; and (4) PPVL + glutamine group (PPVL + G) - rats were treated intraperitoneally with glutamine for seven days. Local injuries were determined by evaluating intestinal segments for oxidative stress using lipid peroxidation and the activities of glutathione peroxidase (GPx), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) after PPVL. Lipid peroxidation of the membrane was increased in the animals subjected to PH ( P 0.05). The activity of the antioxidant enzyme GTx was decreased in the gut of animals subjected to PH compared with that in the control group of animals not subjected to PH ( P 0.05). At least 10 random, non-overlapping images of each histological slide with 200 × magnification (44 pixel = 1 μm) were captured. The sum means of all areas, of each group were calculated. The mean areas of eNOS staining for both of the control groups were similar. The PPVL group showed the largest area of staining for eNOS. The PPVL + G group had the second highest amount of staining, but the mean value was much lower than that of the PPVL group ( P < 0.01). For iNOS, the control (SO) and control + G (SO + G) groups showed similar areas of staining. The PPVL group contained the largest area of iNOS staining, followed by the PPVL + G group; however, this area was significantly smaller than that of the group that underwent PH without glutamine ( P < 0.01). Treatment with

  14. Response to nitrate/ammonium nutrition of tomato (Solanum lycopersicum L.) plants overexpressing a prokaryotic NH4(+)-dependent asparagine synthetase.

    Science.gov (United States)

    Martínez-Andújar, Cristina; Ghanem, Michel Edmond; Albacete, Alfonso; Pérez-Alfocea, Francisco

    2013-05-01

    Nitrogen availability is an important limiting factor for plant growth. Although NH4(+) assimilation is energetically more favorable than NO3(-), it is usually toxic for plants. In order to study if an improved ammonium assimilatory metabolism could increase the plant tolerance to ammonium nutrition, tomato (Solanum lycopersicum L. cv P-73) plants were transformed with an NH4(+)-dependent asparagine synthetase (AS-A) gene from Escherichia coli (asnA) under the control of a PCpea promoter (pea isolated constitutive promotor). Homozygous (Hom), azygous (Az) asnA and wild type (WT) plants were grown hydroponically for 6 weeks with normal Hoagland nutrition (NO3(-)/NH4(+)=6/0.5) and high ammonium nutrition (NO3(-)/NH4(+)=3.5/3). Under Hoagland's conditions, Hom plants produced 40-50% less biomass than WT and Az plants. However, under NO3(-)/NH4(+)=3.5/3 the biomass of Hom was not affected while it was reduced by 40-70% in WT and Az plants compared to Hoagland, respectively. The Hom plants accumulated 1.5-4 times more asparagine, glycine, serine and soluble proteins and registered higher glutamine synthetase (GS) and glutamate synthase (GOGAT) activities in the light-adapted leaves than the other genotypes, but had similar NH4(+) and NO3(-) levels in all conditions. In the dark-adapted leaves, a protein catabolism occurred in the Hom plants with a concomitant 25-40% increase in organic acid concentration, while asparagine accumulation registered the highest values. The aforementioned processes might be responsible for a positive energetic balance as regards the futile cycle of the transgenic protein synthesis and catabolism. This explains growth penalty under standard nutrition and growth stability under NO3(-)/NH4(+)=3.5/3, respectively. Copyright © 2013 Elsevier GmbH. All rights reserved.

  15. Expressed sequence tags related to nitrogen metabolism in maize inoculated with Azospirillum brasilense.

    Science.gov (United States)

    Pereira-Defilippi, L; Pereira, E M; Silva, F M; Moro, G V

    2017-05-31

    The relative quantitative real-time expression of two expressed sequence tags (ESTs) codifying for key enzymes in nitrogen metabolism in maize, nitrate reductase (ZmNR), and glutamine synthetase (ZmGln1-3) was performed for genotypes inoculated with Azospirillum brasilense. Two commercial single-cross hybrids (AG7098 and 2B707) and two experimental synthetic varieties (V2 and V4) were raised under controlled greenhouse conditions, in six treatment groups corresponding to different forms of inoculation and different levels of nitrogen application by top-dressing. The genotypes presented distinct responses to inoculation with A. brasilense. Increases in the expression of ZmNR were observed for the hybrids, while V4 only displayed a greater level of expression when the plants received nitrogenous fertilization by top-dressing and there was no inoculation. The expression of the ZmGln1-3EST was induced by A. brasilense in the hybrids and the variety V4. In contrast, the variety V2 did not respond to inoculation.

  16. Equilibria and partitioning of complexes in the S-adenosylmethionine synthetase reaction

    International Nuclear Information System (INIS)

    Markham, G.D.

    1987-01-01

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a reaction in which the [enzyme-ATP-methionine] complex reacts to form an intermediate [enzyme-AdoMet-PPPi] complex: hydrolysis of PPPi yields an [enzyme-AdoMet-PPi-Pi] complex from which AdoMet is the last product to dissociate. Analysis of reaction mixtures which were quenched with acid during turnover of E. coli AdoMet synthetase with saturating substrates containing [α - 32 P]ATP showed that PPPi is present in an amount corresponding to 45% of the total enzyme active sites, reflecting the portion of enzyme present in an [enzyme-AdoMet-PPPi] complex. Similar experiments in which excess pyrophosphatase was included (to hydrolyze PPi as it was released from AdoMet synthetase), showed that enzyme-bound PPi is present in an amount corresponding to 22% of the total AdoMet synthetase. The enzyme not present in complexes with PPPi or PPi is probably distributed between the [enzyme-ATP-methionine] and the [enzyme-AdoMet] complexes. AdoMet synthetase forms enzyme-bound 32 PPPi from added 32 PPi and Pi; the equilibrium constant [enzyme-AdoMet-PPi-Pi]/[enzyme-AdoMet-PPPi] is 2.0, greatly displaced from the equilibrium for hydrolysis of free PPPi. Since the ratio of enzyme-bound PPi to PPPi is 0.5 during the steady state, the PPPi hydrolysis step is not at equilibrium during turnover. Formation of [ 32 P]ATP from the [enzyme-AdoMet- 32 PPPi] complex was not detected

  17. Early Administration of Glutamine Protects Cardiomyocytes from Post-Cardiac Arrest Acidosis

    Directory of Open Access Journals (Sweden)

    Yan-Ren Lin

    2016-01-01

    Full Text Available Postcardiac arrest acidosis can decrease survival. Effective medications without adverse side effects are still not well characterized. We aimed to analyze whether early administration of glutamine could improve survival and protect cardiomyocytes from postcardiac arrest acidosis using animal and cell models. Forty Wistar rats with postcardiac arrest acidosis (blood pH < 7.2 were included. They were divided into study (500 mg/kg L-alanyl-L-glutamine, n=20 and control (normal saline, n=20 groups. Each of the rats received resuscitation. The outcomes were compared between the two groups. In addition, cardiomyocytes derived from human induced pluripotent stem cells were exposed to HBSS with different pH levels (7.3 or 6.5 or to culture medium (control. Apoptosis-related markers and beating function were analyzed. We found that the duration of survival was significantly longer in the study group (p<0.05. In addition, in pH 6.5 or pH 7.3 HBSS buffer, the expression levels of cell stress (p53 and apoptosis (caspase-3, Bcl-xL markers were significantly lower in cardiomyocytes treated with 50 mM L-glutamine than those without L-glutamine (RT-PCR. L-glutamine also increased the beating function of cardiomyocytes, especially at the lower pH level (6.5. More importantly, glutamine decreased cardiomyocyte apoptosis and increased these cells’ beating function at a low pH level.

  18. Importance of glutamine metabolism in leukemia cells by energy production through TCA cycle and by redox homeostasis.

    Science.gov (United States)

    Goto, Mineaki; Miwa, Hiroshi; Shikami, Masato; Tsunekawa-Imai, Norikazu; Suganuma, Kazuto; Mizuno, Shohei; Takahashi, Miyuki; Mizutani, Motonori; Hanamura, Ichiro; Nitta, Masakazu

    2014-07-01

    Some cancer cells depend on glutamine despite of pronounced glycolysis. We examined the glutamine metabolism in leukemia cells, and found that HL-60 cells most depended on glutamine in the 4 acute myelogenous leukemia (AML) cell lines examined: growth of HL-60 cells was most suppressed by glutamine deprivation and by inhibition of glutaminolysis, which was rescued by tricarboxylic acid (TCA) cycle intermediate, oxaloacetic acid. Glutamine is also involved in antioxidant defense function by increasing glutathione. Glutamine deprivation suppressed the glutathione content and elevated reactive oxygen species most evidently in HL-60 cells. Glutamine metabolism might be a therapeutic target in some leukemia.

  19. Effects of hyperthyroidism and hypothyroidism on glutamine metabolism by skeletal muscle of the rat.

    OpenAIRE

    Parry-Billings, M; Dimitriadis, G D; Leighton, B; Bond, J; Bevan, S J; Opara, E; Newsholme, E A

    1990-01-01

    1. The effects of hyperthyroidism and hypothyroidism on the concentrations of glutamine and other amino acids in the muscle and plasma and on the rates of glutamine and alanine release from incubated isolated stripped soleus muscle of the rat were investigated. 2. Hyperthyroidism decreased the concentration of glutamine in soleus muscle but was without effect on that in the gastrocnemius muscle or in the plasma. Hyperthyroidism also increased markedly the rate of release of glutamine from the...

  20. Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Taylor, G.R.; Barclay, B.J.; Storms, R.K.; Friesen, J.D.; Haynes, R.H.

    1982-01-01

    The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp/sup +/ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy/sup +/ transformants directly, it was found that all pTL1 transformants were phenotypically Thy/sup +/ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-/sup 3/H]dUMP to [6-/sup 3/H]dTMP. In protein extracts from the thymidylate auxotroph (tmpl-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain

  1. DL-7-azatryptophan and citrulline metabolism in the cyanobacterium Anabaena sp. strain 1F

    International Nuclear Information System (INIS)

    Chen, C.H.; Van Baalen, C.; Tabita, F.R.

    1987-01-01

    An alternative route for the primary assimilation of ammonia proceeds via glutamine synthetase-carbamyl phosphate synthetase and its inherent glutaminase activity in Anabaena sp. strain 1F, a marine filamentous, heterocystous cyanobacterium. Evidence for the presence of this possible alternative route to glutamate was provided by the use of amino acid analogs as specific enzyme inhibitors, enzymological studies, and radioistopic labeling experiments. The amino acid pool patterns of continuous cultures of Anabaena sp. strain 1F were markedly influenced by the nitrogen source. A relatively high concentration of glutamate was maintained in the amino acid pools of all cultures irrespective of the nitrogen source, reflecting the central role of glutamate in nitrogen metabolism. The addition of 1.0 microM azaserine increased the intracellular pools of glutamate and glutamine. All attempts to detect any enzymatic activity for glutamate synthase by measuring the formation of L-[ 14 C]glutamate from 2-keto-[1- 14 C]glutarate and glutamine failed. The addition of 10 microM DL-7-azatryptophan caused a transient accumulation of intracellular citrulline and alanine which was not affected by the presence of chloramphenicol. The in vitro activity of carbamyl phosphate synthetase and glutaminase increased severalfold in the presence of azatryptophan. Results from radioisotopic labeling experiments with [ 14 C]bicarbonate and L-[1- 14 C]ornithine also indicated that citrulline was formed via carbamyl phosphate synthetase and ornithine transcarbamylase. In addition to its effects on nitrogen metabolism, azatryptophan also affected carbon metabolism by inhibiting photosynthetic carbon assimilation and photosynthetic oxygen evolution

  2. Effects of glutamine on performance and intestinal mucosa morphometry of broiler chickens vaccinated against coccidiosis

    Directory of Open Access Journals (Sweden)

    Brenda Carla Luquetti

    2016-08-01

    Full Text Available ABSTRACT This study aimed to assess the effects of glutamine as feed additive on performance and intestinal mucosa morphometry of broiler chickens vaccinated against coccidiosis. A total of 400 day-old male chicks were randomly assigned to four treatments (NVNG – no vaccination, no glutamine supplementation; NVG – no vaccination, glutamine supplementation (10 g kg−1; VNG – vaccination, no glutamine supplementation; VG – vaccination, glutamine supplementation replicated four times with 25 birds per replicate. A commercial sprayed-on vaccine against coccidiosis containing Eimeria acervulina, E. maxima, E. mivati, and E. tenella was administered at the hatchery. Broiler performance was evaluated from 1-28 days, and morphometric parameters were analyzed at 14, 21, and 28 days of age. Body weight gain and feed intake were negatively affected by vaccination, but not by glutamine. Vaccination increased crypt depth in the duodenum and jejunum at 21 and 28 days. In conclusion, this study showed that glutamine was not able to increase weight gain of broiler chickens, irrespective of whether the animals were vaccinated or not against coccidiosis. Glutamine supplementation was able to improve feed conversion in vaccinated birds suggesting trophic effect on intestinal epithelium improving.

  3. Clinical and protein metabolic efficacy of glutamine granules-supplemented enteral nutrition in severely burned patients.

    Science.gov (United States)

    Peng, Xi; Yan, Hong; You, Zhongyi; Wang, Pei; Wang, Shiliang

    2005-05-01

    As an abundant amino acid in the human body, glutamine has many important metabolic roles that may protect or promote tissue integrity and enhance the immune system. A relative deficiency of glutamine in such patients could compromise recovery and result in prolonged illness and an increase in late mortality. The purpose of this clinical study is to observe the effects of enteral supplement with glutamine granules on protein metabolism in severely burned patients. Forty-eight severe burn patients (total burn surface area 30-75%, full thickness burn area 20-58%) who met the requirements of the protocol joined this double-blind randomized controlled clinical trial. Patients were randomly divided into two groups: burn control group (B group, 23 patients) and glutamine treated group (Gln group, 25 patients). There was isonitrogenous and isocaloric intake in both groups, glutamine and B group patents were supplemented with glutamine granules or placebo (glycine) at 0.5 g/kg per day for 14 days with oral feeding or tube feeding, respectively. The level of plasma glutamine, plasma protein content, urine nitrogen and urine 3-methylhistidine (3-MTH) excretion were determined, wound healing rate of the burned area and hospital stay were recorded. The results showed that there were significant reductions in plasma glutamine level and abnormal protein metabolism. After supplement with glutamine granules for 14 days, the plasma glutamine concentration was significantly higher than that in B group (607.86+/-147.25 micromol/L versus 447.63+/-132.38 micromol/L, P0.05). On the other hand, the amount of urine nitrogen and 3-MTH excreted in Gln group were significantly lower than that in B group. In addition, wound healing was faster and hospital stay days were shorter in Gln group than B group (46.59+/-12.98 days versus 55.68+/-17.36 days, P<0.05). These indicated that supplement glutamine granules with oral feeding or tube feeding could abate the degree of glutamine depletion

  4. Glutamine synthetase and alanine transaminase expression are decreased in livers of aged vs. young beef cows and GS can be upregulated by 17β-estradiol implants.

    Science.gov (United States)

    Miles, E D; McBride, B W; Jia, Y; Liao, S F; Boling, J A; Bridges, P J; Matthews, J C

    2015-09-01

    Aged beef cows (≥ 8 yr of age) produce calves with lower birth and weaning weights. In mammals, aging is associated with reduced hepatic expression of glutamine synthetase (GS) and alanine transaminase (ALT), thus impaired hepatic Gln-Glu cycle function. To determine if the relative protein content of GS, ALT, aspartate transaminase (AST), glutamate transporters (EAAC1, GLT-1), and their regulating protein (GTRAP3-18) differed in biopsied liver tissue of (a) aged vs. young (3 to 4 yr old) nonlactating, nongestating Angus cows (Exp. 1 and 2) and (b) aged mixed-breed cows with and without COMPUDOSE (17β-estradiol) ear implants (Exp. 3), Western blot analyses were performed. In Exp. 1, 12 young (3.62 ± 0.01 yr) and 13 aged (10.08 ± 0.42 yr) cows grazed the same mixed forage for 42 d (August-October). In Exp. 2, 12 young (3.36 ± 0.01 yr) and 12 aged (10.38 ± 0.47 yr) cows were individually fed (1.03% of BW) a corn-silage-based diet to maintain BW for 20 d. For both Exp. 1 and 2, the effect of cow age was assessed by ANOVA using the MIXED procedure of SAS. Cow BW did not change ( ≥ 0.17). Hepatic ALT (78% and 61%) and GS (52% and 71%) protein content (Exp. 1 and 2, respectively) was decreased ( ≤ 0.01), whereas GTRAP3-18 (an inhibitor of EAAC1 activity) increased ( ≤ 0.01; 170% and 136%) and AST, GLT-1, and EAAC1 contents did not differ ( ≥ 0.17) in aged vs. young cows. In Exp. 2, free concentrations (nmol/g) of Glu, Ala, Gln, Arg, and Orn in liver homogenates were determined. Aged cows tended to have less ( = 0.10) free Gln (15.0%) than young cows, whereas other AA concentrations did not differ ( 0.26). In Exp. 3, 14 aged (> 10 yr) cows were randomly allotted ( = 7) to sham or COMPUDOSE (25.7 mg of 17β-estradiol) implant treatment (TRT), and had ad libitum access to alfalfa hay for 28 d. Blood and liver biopsies were collected 14 and 28 d after implant treatment. Treatment, time after implant (DAY), and TRT × DAY effects were assessed by ANOVA using

  5. The early history of tRNA recognition by aminoacyl-tRNA synthetases

    Indian Academy of Sciences (India)

    Madhu

    2006-10-04

    Oct 4, 2006 ... Discovery of aminoacyl-tRNA synthetases and importance ... The pioneering work of Fritz Lipmann on the high-energy ... the peculiar structural and functional relationships tRNAs ... a bulk of only 20 families of tRNA molecules in contrast ...... balance of tRNA and aminoacyl-tRNA synthetase; Science 242.

  6. A highly conserved basidiomycete peptide synthetase produces a trimeric hydroxamate siderophore.

    Science.gov (United States)

    Brandenburger, Eileen; Gressler, Markus; Leonhardt, Robin; Lackner, Gerald; Habel, Andreas; Hertweck, Christian; Brock, Matthias; Hoffmeister, Dirk

    2017-08-25

    The model white-rot basidiomycete Ceriporiopsis ( Gelatoporia ) subvermispora B encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet uncharacterized fungal type VI siderophore synthetase family which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module and two thiolation/condensation (T-C) di-domain partial modules which, together, constitute an AT 1 C 1 T 2 C 2 T 3 C 3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as polyhistidine fusion in Aspergillus niger as soluble and active protein. N 5 -acetyl- N 5 -hydroxy-l-ornithine (l-AHO) and N 5 - cis -anhydromevalonyl- N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as substrates, as assessed in vitro using the substrate-dependent [ 32 P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo -CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as product in vitro , which was verified by LC-HRESIMS. Phylogenetic analyses suggest that type VI family siderophore synthetases are widespread in mushrooms and have evolved in a common ancestor of basidiomycetes. Importance : The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 supports further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the

  7. Solidago Vigaurea for Prostate Cancer Therapy

    Science.gov (United States)

    2011-04-01

    and modi fication including CTP synthetase, thymidylate synthase, dihydrofo late reductase, IMP dehydrogenase, ribonucleotide reductase, DNA polymerase...this context, it is worth noting that some metabolic abnormalities such as diabetes and even ageing are linked with higher incidence of cancers. However

  8. Current state of knowledge of hepatic encephalopathy (part I): newer treatment strategies for hyperammonemia in liver failure.

    Science.gov (United States)

    Kristiansen, Rune Gangsoy

    2016-12-01

    Alterations in interorgan metabolism of ammonia play an important role in the onset of hyperammonemia in liver failure. Glutamine synthetase (GS) in muscle is an important target for ammonia removal strategies in hyperammonemia. Ornithine Phenylacetate (OP) is hypothesized to remove ammonia by providing glutamate as a substrate for increased GS activity and hence glutamine production. The newly generated glutamine conjugates with phenylacetate forming phenylacetylglutamine which can be excreted in the urine, providing an excretion pathway for ammonia. We have also shown that OP targets glycine metabolism, providing an additional ammonia reducing effect.

  9. SCREENING OF ANTIMICROBIAL ACTIVITY AND GENES CODING POLYKETIDE SYNTHETASE AND NONRIBOSOMAL PEPTIDE SYNTHETASE OF ACTINOMYCETE ISOLATES

    Directory of Open Access Journals (Sweden)

    Silvia Kovácsová

    2013-12-01

    Full Text Available The aim of this study was to observe antimicrobial activity using agar plate diffusion method and screening genes coding polyketide synthetase (PKS-I and nonribosomal peptide synthetase (NRPS from actinomycetes. A total of 105 actinomycete strains were isolated from arable soil. Antimicrobial activity was demonstrated at 54 strains against at least 1 of total 12 indicator organisms. Antifungal properties were recorded more often than antibacterial properties. The presence of PKS-I and NRPS genes were founded at 61 of total 105 strains. The number of strains with mentioned biosynthetic enzyme gene fragments matching the anticipated length were 19 (18% and 50 (47% respectively. Overall, five actinomycete strains carried all the biosynthetical genes, yet no antimicrobial activity was found against any of tested pathogens. On the other hand, twenty-one strains showed antimicrobial activity even though we were not able to amplify any of the PKS or NRPS genes from them. Combination of the two methods showed broad-spectrum antimicrobial activity of actinomycetes isolated from arable soil, which indicate that actinomycetes are valuable reservoirs of novel bioactive compounds.

  10. Radiation-induced G/sub 2/-arrest is reduced by inhibitors of poly(adenosine diphosphoribose) synthetase

    International Nuclear Information System (INIS)

    Rowley, R.

    1985-01-01

    Experiments are in progress to test whether poly(adenosine diphosphoribose) synthesis is required for the induction of G/sub 2/-arrest in growing mammalian cells following X-irradiation. A variety of poly(ADPR) synthetase inhibitors have been tested to determine: 1) whether addition of an inhibitor to X-irradiated CHO cells reduces G/sub 2/-arrest; 2) whether compounds structurally similar to poly-(ADPR) synthetase inhibitors but inactive against this enzyme affect radiation-induced G/sub 2/-arrest and 3) whether the concentration dependence for poly(ADPR) synthetase inhibition matches that for G/sub 2/-arrest reduction. G/sub 2/-arrest was measured in X-irradiated (1.5 Gy) CHO cells using the mitotic cell selection technique. Poly(ADPR) synthetase activity was measured in permeabilized cells by /sup 3/H-NAD incorporation. The synthetase inhibitors used were 3-aminobenzamide, benzamide, nicotinamide, 4-acetyl pyridine, caffeine and theophylline. The inactive compounds used were 3-aminobenzoic acid, benzoic acid, nicotinic acid, adenine, adenosine and 3'-deoxyadenosine. Inhibitors of poly(ADPR) synthetase reduced G/sub 2/-arrest while related compounds which produced no enzyme inhibition did not. The concentration dependencies for G/sub 2/-arrest reduction and enzyme inhibition were similar only for methyl xanthines. Further analysis awaits the determination of intracellular drug concentrations

  11. GLUTAMIN MEMPERCEPAT WAKTU PEMULIHAN JUMLAH SEL LIMFOSIT LIEN SETELAH AKTIVITAS FISIK MAKSIMAL PADA MENCIT (GLUTAMINE SHORTENS RECOVERY TIME OF LIEN LYMPHOCYTES AFTER EXCESSIVE PHYSICAL ACTIVITY IN MICE

    Directory of Open Access Journals (Sweden)

    I Made Jawi

    2007-06-01

    Full Text Available The immunologic? system? of the body requires? suitable recovery time after? excessive physical? activity. The recovery? time of spleen lymphocytes after? excessive? physical activity? in one? investigation was? 3? days. The? aim? of this? research is to identify?the role of? glutamine? in shortening? the recovery time of? spleen? lymphocytes? after?excessive physical activity. The? study? was conducted? on? 70? adults? Balb/c mice which? were? divided? into? 7? groups, with? a randomized control? group post-test? only design. In this? study? an observation? was made on? spleen? lymphocytes? of control, after? excessive? physical activity(in the the? form of swimming? until? near? drowning with glutamine,? without glutamine ?and? after? the? recovery time of 1 and 2 days? of? each of? the 10 mice. Spleen? lymphocytes? were? counted in spleen preparation by mikroskop. The data obtained were tested? by? one way Anova. The findings? showed? that the number of spleen? lymphocytes significantly decrease after? excessive? physical activity?? in the?glutamine? and? non glutamine groups ( p < 0,05.The number of spleen? lymphocytes? was? not different as compered to control group or returned? to normal after recovery? time? of 1? day? in? the? glutamine group (p > 0,05 .?In the nonglutamine group the number of spleen lymphocytes was different from control group until 2 days (p<0,05 . From this finding it can be concluded that glutamine? shortens the recovery time of spleen lymphocytes? after? excessive? physical activity in mice.

  12. Pilot study with a glutamine-supplemented enteral formula in critically ill infants

    Directory of Open Access Journals (Sweden)

    Barbosa Eliana

    1999-01-01

    Full Text Available Seriously ill infants often display protein-calorie malnutrition due to the metabolic demands of sepsis and respiratory failure. Glutamine has been classified as a conditionally essential amino acid, with special usefulness in critical patients. Immunomodulation, gut protection, and prevention of protein depletion are mentioned among its positive effects in such circumstances. With the intent of evaluating the tolerance and clinical impact of a glutamine supplement in seriously ill infants, a prospective randomized study was done with nine patients. Anthropometric and biochemical determinations were made, and length of stay in the intensive care unit (ICU, in the hospital, and under artificial ventilation, and septic morbidity and mortality were tabulated. Infants in the treatment group (n=5 were enterally administered 0.3 g/kg of glutamine, whereas controls received 0.3 g/kg of casein during a standard period of five days. Septic complications occurred in 75% of the controls (3/4 versus 20% of the glutamine-treated group (1/5, p<=0.10, and two patients in the control group died of bacterial infections (50% vs. 0%, p<=0.10. Days in the ICU, in the hospital, and with ventilation numerically favored glutamine therapy, although without statistical significance. The supplements were usually well tolerated, and no patient required discontinuation of the program. The conclusion was that glutamine supplementation was safe and tended to be associated with less infectious morbidity and mortality in this high-risk population.

  13. Gamma-Glutamylpolyamine Synthetase GlnA3 Is Involved in the First Step of Polyamine Degradation Pathway in Streptomyces coelicolor M145

    Directory of Open Access Journals (Sweden)

    Agnieszka Bera

    2017-04-01

    Full Text Available Streptomyces coelicolor M145 was shown to be able to grow in the presence of high concentrations of polyamines, such as putrescine, cadaverine, spermidine, or spermine, as a sole nitrogen source. However, hardly anything is known about polyamine utilization and its regulation in streptomycetes. In this study, we demonstrated that only one of the three proteins annotated as glutamine synthetase-like protein, GlnA3 (SCO6962, was involved in the catabolism of polyamines. Transcriptional analysis revealed that the expression of glnA3 was strongly induced by exogenous polyamines and repressed in the presence of ammonium. The ΔglnA3 mutant was shown to be unable to grow on defined Evans agar supplemented with putrescine, cadaverine, spermidine, and spermine as sole nitrogen source. HPLC analysis demonstrated that the ΔglnA3 mutant accumulated polyamines intracellularly, but was unable to degrade them. In a rich complex medium supplemented with a mixture of the four different polyamines, the ΔglnA3 mutant grew poorly showing abnormal mycelium morphology and decreased life span in comparison to the parental strain. These observations indicated that the accumulation of polyamines was toxic for the cell. An in silico analysis of the GlnA3 protein model suggested that it might act as a gamma-glutamylpolyamine synthetase catalyzing the first step of polyamine degradation. GlnA3-catalyzed glutamylation of putrescine was confirmed in an enzymatic in vitro assay and the GlnA3 reaction product, gamma-glutamylputrescine, was detected by HPLC/ESI-MS. In this work, the first step of polyamine utilization in S. coelicolor has been elucidated and the putative polyamine utilization pathway has been deduced based on the sequence similarity and transcriptional analysis of homologous genes expressed in the presence of polyamines.

  14. Construction of hybrid peptide synthetases by module and domain fusions.

    Science.gov (United States)

    Mootz, H D; Schwarzer, D; Marahiel, M A

    2000-05-23

    Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.

  15. Elevated glutamine/glutamate ratio in cerebrospinal fluid of first episode and drug naive schizophrenic patients

    Directory of Open Access Journals (Sweden)

    Lindström Leif H

    2005-01-01

    Full Text Available Abstract Background Recent magnetic resonance spectroscopy (MRS studies report that glutamine is altered in the brains of schizophrenic patients. There were also conflicting findings on glutamate in cerebrospinal fluid (CSF of schizophrenic patients, and absent for glutamine. This study aims to clarify the question of glutamine and glutamate in CSF of first episode and drug naive schizophrenic patients. Method Levels of glutamine and glutamate in CSF of 25 first episode and drug-naive male schizophrenic patients and 17 age-matched male healthy controls were measured by a high performance liquid chromatography. Results The ratio (126.1 (median, 117.7 ± 27.4 (mean ± S.D. of glutamine to glutamate in the CSF of patients was significantly (z = -3.29, p = 0.001 higher than that (81.01 (median, 89.1 ± 22.5 (mean ± S.D. of normal controls although each level of glutamine and glutamate in patients was not different from that of normal controls. Conclusion Our data suggests that a disfunction in glutamate-glutamine cycle in the brain may play a role in the pathophysiology of schizophrenia.

  16. The re-assimilation of ammonia produced by photorespiration and the nitrogen economy of C3 higher plants.

    Science.gov (United States)

    Keys, Alfred J

    2006-02-01

    Photorespiration involves the conversion of glycine to serine with the release of ammonia and CO(2). In C(3) terrestrial higher plants the flux through glycine and serine is so large that it results in the production of ammonia at a rate far exceeding that from reduction of new nitrogen entering the plant. The photorespiratory nitrogen cycle re-assimilates this ammonia using the enzymes glutamine synthetase and glutamine:2-oxoglutarateaminotransferase.

  17. MARCH-1984

    Indian Academy of Sciences (India)

    A nuclear magnetic resonance spectroscopic study. S. Devarajan and K. R. K. Easwaran. 1 - 16. Studies on Aspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

  18. The effect of glutamine administration on urinary ammonium excretion in normal subjects and patients with renal disease.

    Science.gov (United States)

    Welbourne, T; Weber, M; Bank, N

    1972-07-01

    The effect of acute changes in the delivery rate of glutamine to the kidney on urinary ammonium excretion was studied in man. Healthy subjects and patients with intrinsic renal disease were studied under three different acid-base conditions: unaltered acid-base balance; NH(4)Cl-induced acidosis; and NaHCO(3)-induced alkalosis. Anhydrous L-glutamine was administered orally in a single dose of 260 mmoles during each of these three acid-base states. We found that endogenous venous plasma glutamine concentration fell during acidosis and rose during alkalosis in both healthy subjects and patients with renal disease. In healthy subjects, orally administered glutamine raised plasma glutamine concentration markedly over a 2-3 hr period. This was accompanied by an increase in urinary ammonium excretion and a rise in urine pH under normal acid-base conditions and during metabolic acidosis. No increase in ammonium excretion occurred when glutamine was administered during metabolic alkalosis in spite of an equivalent rise in plasma glutamine concentration. In patients with renal disease, endogenous venous plasma glutamine concentration was lower than in healthy subjects, perhaps as a result of mild metabolic acidosis. Acute oral glutamine loading failed to increase urinary ammonium excretion significantly during either unaltered acid-base conditions or after NH(4)Cl-induced acidosis, even though plasma glutamine rose as high as in healthy subjects. We conclude from these observations that glutamine delivery to the kidney is a rate-limiting factor for ammonium excretion in healthy subjects, both before and after cellular enzyme adaptation induced by metabolic acidosis. In contrast, in patients with renal disease, glutamine delivery is not rate-limiting for ammonium excretion. Presumably other factors, such as surviving renal mass and the activity of intracellular enzymes necessary for ammonia synthesis limit ammonium excretion in these patients.

  19. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  20. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    International Nuclear Information System (INIS)

    Nolan, N.L.; Kidwell, W.R.

    1982-01-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37 0 C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of γ-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of γ-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels

  1. Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

    Science.gov (United States)

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-02-24

    Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

  2. Moderate folic acid supplementation and MTHFD1-synthetase deficiency in mice, a model for the R653Q variant, result in embryonic defects and abnormal placental development.

    Science.gov (United States)

    Christensen, Karen E; Hou, Wenyang; Bahous, Renata H; Deng, Liyuan; Malysheva, Olga V; Arning, Erland; Bottiglieri, Teodoro; Caudill, Marie A; Jerome-Majewska, Loydie A; Rozen, Rima

    2016-11-01

    Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. Female Mthfd1S +/+ and Mthfd1S +/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S +/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater

  3. Influence of glutamine on the effect of resistance exercise on cardiac ANP in rats

    Directory of Open Access Journals (Sweden)

    Romeu Rodrigues de Souza

    2015-03-01

    Full Text Available Various nutritional supplements (herbs, vitamins, and micronutrients improve responses and adaptations to resistance exercise. ANP is a heart hormone that contributes to fluid, electrolyte and blood pressure homeostasis through its natriuretic and vasodilative actions. In the present study, the adaptation of ANP in response to resistance exercise was investigated in rats supplemented with glutamine for five weeks. The results showed that supplementation with glutamine did not influence the number of ANP granules per atrial cardiocyte in sedentary animals. In exercised-trained rats, the number and diameter of the granules was significantly higher in comparison with the control group and in exercised animals supplemented with glutamine there was significant increase in the number and diameter of ANP granules compared with controls. Altogether, these data indicated that in resistance exercise rats, glutamine significantly enhances cardiac ANP thus implicating the beneficial effects of glutamine supplementation to the ANP system.

  4. Assessing the extent of bone degradation using glutamine deamidation in collagen.

    Science.gov (United States)

    Wilson, Julie; van Doorn, Nienke L; Collins, Matthew J

    2012-11-06

    Collagen peptides are analyzed using a low-cost, high-throughput method for assessing deamidation using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For each chosen peptide, the theoretical distribution is calculated and the measured distribution for each sample compared with this to determine the extent of glutamine deamidation. The deamidation of glutamine (Q) to glutamic acid (E) results in a mass shift of +0.984 Da. Thus, from the resolution of our data, the second peak in the isotope distribution for a peptide containing one glutamine residue coincides with the first peak of the isotope distribution for the peptide in which the residue is deamidated. A genetic algorithm is used to determine the extent of deamidation that gives the best fit to the measured distribution. The method can be extended to peptides containing more than one glutamine residue. The extent of protein degradation assessed in this way could be used, for example, to assess the damage of collagen, and screen samples for radiocarbon dating and DNA analysis.

  5. Role of the Escherichia coli glnALG operon in regulation of ammonium transport

    International Nuclear Information System (INIS)

    Jayakuman, A.; Schulman, I.; MacNeil, D.; Barnes, E.M. Jr.

    1986-01-01

    Escherichia coli expresses a specific ammonium (methylammonium) transport system (Amt) when cultured with glutamate or glutamine as the nitrogen source. Over 95% of this Amt activity is repressed by growth of wild-type cells on media containing ammonia. The control of Amt expression was studied with strains containing specific mutations in the glnALG operon. GlnA - (glutamine synthetase deficient) mutants, which contain polar mutations on glnL and glnG genes and therefore have the Reg - phenotype (fail to turn on nitrogen-regulated operons such as histidase), expressed less than 10% of the Amt activity observed for the parental strain. Similarly, low levels of Amt were found in GlnG mutants having the GlnA + Reg - phenotype. However, GlnA - RegC mutants (a phenotype constitutive for histidase) contained over 70% of the parental Amt activity. At steady-state levels, GlnA - RegC mutants accumulated chemically unaltered [ 14 C]methylammonium against a 60- to 80-fold concentration gradient, whereas the labeled substrate was trapped within parental cells as γ-glutamylmethylamide. GlnL Reg - mutants (normal glutamine synthetase regulation) had less than 4% of the Amt activity observed for the parental strain. However, the Amt activity of GlnL RegC mutants was slightly higher than that of the parental strain and was not repressed during growth of cells in media containing ammonia. These findings demonstrate that glutamine synthetase is not required for Amt in E. coli. The loss of Amt in certain GlnA - strains is due to polar effects on glnL nd glnG genes, whose products are involved in expression of nitrogen-regulated genes, including that for Amt

  6. Plasma glutamine levels before cardiac surgery are related to post-surgery infections; an observational study

    Directory of Open Access Journals (Sweden)

    Hanneke Buter

    2016-11-01

    Full Text Available Abstract Background A low plasma glutamine level was found in 34% of patients after elective cardiothoracic surgery. This could be a result of the inflammation caused by surgical stress or the use of extracorporeal circulation (ECC. But it is also possible that plasma glutamine levels were already lowered before surgery and reflect an impaired metabolic state and a higher likelihood to develop complications. In the present study plasma glutamine levels were measured before and after cardiac surgery and we questioned whether there is a relation between plasma glutamine levels and duration of ECC and the occurrence of postoperative infections. Methods We performed a single-centre prospective, observational study in a closed-format, 20-bed, mixed ICU in a tertiary teaching hospital. We included consecutive patients after elective cardiac surgery with use of extracorporeal circulation. Blood samples were collected on the day prior to surgery and at admission on the ICU. The study was approved by the local Medical Ethics Committee (Regional Review Committee Patient-related Research, Medical Centre Leeuwarden, nWMO 115, April 28th 2015. Results Ninety patients were included. Pre-operative plasma glutamine level was 0.42 ± 0.10 mmol/l and post-operative 0.38 ± 0.09 mmol/l (p < 0.001. There was no relation between duration of extracorporeal circulation or aortic occlusion time and changes in plasma glutamine levels. A logistic regression analysis showed a significant correlation between the presence of a positive culture during the post-operative course and pre-operative plasma glutamine levels (p = 0.04. Conclusion Plasma glutamine levels are significantly lower just after cardiac surgery compared to pre-operative levels. We did not find a relation between the decrease in plasma glutamine levels and the duration of extracorporeal circulation or aortic clamp time. There was a correlation between pre-operative plasma glutamine levels

  7. Characterization of arachidonate 5-lipoxygenase and leukotriene A4 synthetase from RBL-1 cells

    International Nuclear Information System (INIS)

    Cook, M.; Hogaboom, G.K.; Sarau, H.M.; Foley, J.J.; Crooke, S.T.

    1986-01-01

    5-lipoxygenase (LO) and leukotriene (LT) A4 synthetase from RBL-1 high speed (105,000 x g for 60 min) supernatants were partially purified by protein-high performance liquid chromatography (HPLC) and characterized in detail. The partially purified preparation contained only 5-LO and LTA4 synthetase and was isolated from 12-LO, peroxidase and LTA4 hydrolase activities. Reaction products were separated by reversed phase HPLC and quantitated by absorption spectrophotometry and radiochemical detection. The enzyme preparation rapidly converted [ 14 C]arachidonate to [ 14 C]5-hydroperoxyeicosatetraenoic acid (HPETE) and [ 14 C]5,12-dihydroperoxyeicosatetraenoic acids (diHETEs). The 5,12-diHETEs were primarily non-enzymatic breakdown products of LTA4 (e.g., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Both the 5-LO and LTA4 synthetase activities were Ca 2+- and ATP-dependent. For both enzyme activities, the CA 2+ stimulation required the presence of ATP. The fatty acid hydroperoxides, 5-,12-, and 15-HPETE, both stimulated ([ 3 μM]) 5-LO and LTA4 synthetase activities. The rapid isolation and subsequent characterization of 5-LO and LTA4 synthetase provide the bases for the further understanding of the role of the LO pathway in biological processes

  8. Prevention of gastrointestinal injury by using glutamine in cardiac surgery

    Directory of Open Access Journals (Sweden)

    С. М. Ефремов

    2015-10-01

    Full Text Available A pilot double-blind, placebo-controlled, randomized study The aim of this study was to evaluate the efficiency of perioperative administration of glutamine to preserve intestinal integrity in patients undergoing cardiac surgery. 24 patients scheduled for elective coronary artery bypass surgery under cardiopulmonary bypass were included in this prospective, randomized, double-blind placebo controlled pilot study. 12 patients were randomized to receive glutamine (20% solution of N(2-L-alanyl-L-glutamine 0.4 g/kg a day, while the remaining 12 patients received an equivalent placebo dose (0.9% solution of NaCl. Infusion of glutamine/placebo was started after the induction of anesthesia and was continued for 24 hours. The primary end-point was dynamics of plasma concentration of a specific marker of intestinal damage, intestinal fatty acid binding protein (I-FABP. The secondary end-points were liver fatty acid binding protein (L-FABP, alpha glutathione s-transferase (aGST, heat shock protein 70 (HSP 70. There were no between-group differences of all the studied biochemical parameters at any stage of the study. Plasma I-FABP levels (median [25-75 percentile] were markedly elevated during CPB and remained the same postoperatively: 962 (577-2 067 and 883 (444-1 625 г/ml 5 min after un-clamping of aorta, 2203 (888-3 429 and 1 560 (506-2 657 г/ml 2 hours post-bypass, 897 (555-1 424 and 794 (505-951 г/ml 6 hours post-bypass in the GLN and control groups respectively. Perioperative administration of glutamine in dose of 0.4 g/kg a day does not appear to preserve intestinal integrity in low risk cardiac surgery patients.

  9. Brain MRS glutamine as a biomarker to guide therapy of hyperammonemic coma.

    Science.gov (United States)

    O'Donnell-Luria, Anne H; Lin, Alexander P; Merugumala, Sai K; Rohr, Frances; Waisbren, Susan E; Lynch, Rebecca; Tchekmedyian, Vatche; Goldberg, Aaron D; Bellinger, Andrew; McFaline-Figueroa, J Ricardo; Simon, Tracey; Gershanik, Esteban F; Levy, Bruce D; Cohen, David E; Samuels, Martin A; Berry, Gerard T; Frank, Natasha Y

    2017-05-01

    Acute idiopathic hyperammonemia in an adult patient is a life-threatening condition often resulting in a rapid progression to irreversible cerebral edema and death. While ammonia-scavenging therapies lower blood ammonia levels, in comparison, clearance of waste nitrogen from the brain may be delayed. Therefore, we used magnetic resonance spectroscopy (MRS) to monitor cerebral glutamine levels, the major reservoir of ammonia, in a gastric bypass patient with hyperammonemic coma undergoing therapy with N-carbamoyl glutamate and the ammonia-scavenging agents, sodium phenylacetate and sodium benzoate. Improvement in mental status mirrored brain glutamine levels, as coma persisted for 48h after plasma ammonia normalized. We hypothesize that the slower clearance for brain glutamine levels accounts for the delay in improvement following initiation of treatment in cases of chronic hyperammonemia. We propose MRS to monitor brain glutamine as a noninvasive approach to be utilized for diagnostic and therapeutic monitoring purposes in adult patients presenting with idiopathic hyperammonemia. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  11. Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

    Science.gov (United States)

    Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

    1976-07-01

    Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

  12. The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

    Science.gov (United States)

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2002-07-16

    Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

  13. Utilization of glutamine by the retina: an autoradiographic and metabolic study

    Energy Technology Data Exchange (ETDEWEB)

    Voaden, M J; Lake, N; Marshall, J; Morjaria, B [Institute of Ophthalmology, London (UK). Dept. of Visual Science

    1978-10-01

    The cells able to accumulate exogenously applied (/sup 3/H) glutamine in rat, cat, frog, pigeon and guinea pig retinas have been located by autoradiography, and the fate of the labelled glutamine, as regards its incorporation into aspartic, glutamic and ..gamma..-amino-butyric acids, followed for 60 min. The results support the notion of glutamine as a precursor of transmitter amino acids in some neurones. In particular, it would appear to be a source of a relatively stable pool of GABA which may be located, with species variation, in amacrine or ganglion cells. In the pigeon retina glutamate pool incorporates and retains a major percentage of the label, and perikarya in the middle of the inner nuclear layer of the tissue are predominantly labelled.

  14. Comparative proteomic analyses reveal the proteome response to short-term drought in Italian ryegrass (Lolium multiflorum.

    Directory of Open Access Journals (Sweden)

    Ling Pan

    Full Text Available Drought is a major abiotic stress that impairs growth and productivity of Italian ryegrass. Comparative analysis of drought responsive proteins will provide insight into molecular mechanism in Lolium multiflorum drought tolerance. Using the iTRAQ-based approach, proteomic changes in tolerant and susceptible lines were examined in response to drought condition. A total of 950 differentially accumulated proteins was found to be involved in carbohydrate metabolism, amino acid metabolism, biosynthesis of secondary metabolites, and signal transduction pathway, such as β-D-xylosidase, β-D-glucan glucohydrolase, glycerate dehydrogenase, Cobalamin-independent methionine synthase, glutamine synthetase 1a, Farnesyl pyrophosphate synthase, diacylglycerol, and inositol 1, 4, 5-trisphosphate, which might contributed to enhance drought tolerance or adaption in Lolium multiflorum. Interestingly, the two specific metabolic pathways, arachidonic acid and inositol phosphate metabolism including differentially accumulated proteins, were observed only in the tolerant lines. Cysteine protease cathepsin B, Cysteine proteinase, lipid transfer protein and Aquaporin were observed as drought-regulated proteins participating in hydrolysis and transmembrane transport. The activities of phospholipid hydroperoxide glutathione peroxidase, peroxiredoxin, dehydroascorbate reductase, peroxisomal ascorbate peroxidase and monodehydroascorbate reductase associated with alleviating the accumulation of reactive oxygen species in stress inducing environments. Our results showed that drought-responsive proteins were closely related to metabolic processes including signal transduction, antioxidant defenses, hydrolysis, and transmembrane transport.

  15. An 15N study of the effects of nitrate, ammonium, and nitrate + ammonium nutrition on nitrogen assimilation in Zea mays L

    International Nuclear Information System (INIS)

    Murphy, A.T.

    1984-10-01

    A brief review of the literature on the effects of nitrate and ammonium nitrogen sources on plant growth, and the assimilation of those nitrogen sources, has been presented. It was concluded that ammonium nutrition produces optimum growth, with nitrate + ammonium being a better nitrogen source than only nitrate. Leaf blade nitrate reductase activity exceeded that of the root in nitrate-fed plants, suggesting that the shoot is the major region of nitrate assimilation. This is further supported by the results of xylem exudate analysis, where 93% of the newly-absorbed nitrogen exported by the roots was detected as nitrate. Evidence in support of this hypothesis was also obtained by studying the distribution of 15 N in the various nitrogenous compounds. The effects of nitrogen source on plant growth, organic nitrogen and inorganic nitrogen contents, and the rates of incorporation into nitrogenous compounds were studied. The observed differences were explained with reference to the effects of the various nitrogen sources on the physiology of the plants. The experimental techniques included assays of the enzymes nitrate reductase and glutamine synthetase, whole plant growth studies, and the analysis of nitrogenous compounds of xylem exudate and those extracted from the leaf blade, leaf base, and root regions of maize plants after feeding with a nutrient solution containing nitrogen as 15 N

  16. Response to Dietary Supplementation of Glutamine in Broiler Chickens Subjected to Transportation Stress

    Directory of Open Access Journals (Sweden)

    Majid SHAKERI

    2016-07-01

    Full Text Available The main purpose of this study was to determine effects of glutamine supplementation on performance and blood parameters including Hsp70 and acute phase protein when chicken were subjected to transportation stress. A total of four hundred day-old-male cobb-500 chicks were obtained directly from a local hatchery. The chicks were allotted to two groups as: immediate placement (1 hour after hatching with access to feed and water and placement after 24h transportation without access to feed and water. The experiment consisted of a factorial arrangement of 2 different diets and 2 different time of placement. Chicks from each placement group were fed either basal diet or basal diet + 1% glutamine from 1 to 21 days of age. The results indicated that dietary glutamine improved the body weight gain and feed conversion ratio significantly when chicks were subjected to delayed or immediate placement. In conclusion, supplementing chicken with glutamine in diet can reduce negative effects of delayed access to feed and water during transportation. Moreover, APP concentration and HSP70 level were positively affected when chicks supplemented with glutamine in the diet.

  17. Lack of functional benefit with glutamine versus placebo in Duchenne muscular dystrophy: a randomized crossover trial.

    Directory of Open Access Journals (Sweden)

    Elise Mok

    Full Text Available Oral glutamine decreases whole body protein breakdown in Duchenne muscular dystrophy (DMD. We evaluated the functional benefit of 4 months oral glutamine in DMD.30 ambulant DMD boys were included in this double-blind, randomized crossover trial with 2 intervention periods: glutamine (0.5 g/kg/d and placebo, 4 months each, separated by a 1-month wash-out, at 3 outpatient clinical investigation centers in France. Functional benefit was tested by comparing glutamine versus placebo on change in walking speed at 4 months. Secondary outcome measures were: 2-minute walk test, work, power, muscle mass (urinary creatinine, markers of myofibrillar protein breakdown (urinary 3-methyl-histidine/creatinine, serum creatine phospho-kinase, body composition (fat free mass, fat mass percentage, safety and oral nutrient intake. There was no improvement in the primary end point (walking speed or in secondary measures of muscle function (2-minute walk test, work, power in the glutamine group compared with placebo. However, subjects receiving glutamine or placebo showed no deterioration in functional measures over the course of the 9-month trial. No differences in muscle mass, markers of protein breakdown or serum creatine phosho-kinase were observed, except for a blunted increase in fat free mass in the glutamine group which led to a greater increase in fat mass percentage. Glutamine was safe and well-tolerated.This trial did not identify additional benefit of 4 months oral glutamine over placebo on muscle mass or function in ambulatory DMD boys. Although apparently safe, current data cannot support routine supplementation in this population as a whole, until further research proves otherwise.(ClinicalTrials.gov NCT00296621.

  18. The effect of free glutamine and peptide ingestion on the rate of muscle glycogen resynthesis in man

    DEFF Research Database (Denmark)

    Van Hall, Gerrit; Saris, W H; van de Schoor, P A

    2000-01-01

    hydrolysate (26% glutamine) and 3) a whey hydrolysate (6.6% glutamine). Plasma glutamine, decreased by approximately 20% during recovery with ingestion of the control drink, no changes with ingestion of the protein hydrolysates drinks, and a 2-fold increase with ingestion of the free glutamine drinks....... The rate of glycogen resynthesis was not significantly different in the four tests: 28 +/- 5, 26 +/- 6, 33 +/- 4, and 34 +/- 3 mmol glucosyl units x kg(-1) dry weight muscle x h(-1) for the control, glutamine, wheat- and whey hydrolysate ingestion, respectively. It is concluded that ingestion...... of a glutamine/carbohydrate mixture does not increase the rate of glycogen resynthesis in muscle. Glycogen resynthesis rates were higher, although not statistically significant, after ingestion of the drink containing the wheat (21 +/- 8%) and whey protein hydrolysate (20 +/- 6%) compared to ingestion...

  19. Maternal L-glutamine supplementation prevents prenatal alcohol exposure-induced fetal growth restriction in an ovine model.

    Science.gov (United States)

    Sawant, Onkar B; Wu, Guoyao; Washburn, Shannon E

    2015-06-01

    Prenatal alcohol exposure is known to cause fetal growth restriction and disturbances in amino acid bioavailability. Alterations in these parameters can persist into adulthood and low birth weight can lead to altered fetal programming. Glutamine has been associated with the synthesis of other amino acids, an increase in protein synthesis and it is used clinically as a nutrient supplement for low birth weight infants. The aim of this study was to explore the effect of repeated maternal alcohol exposure and L-glutamine supplementation on fetal growth and amino acid bioavailability during the third trimester-equivalent period in an ovine model. Pregnant sheep were randomly assigned to four groups, saline control, alcohol (1.75-2.5 g/kg), glutamine (100 mg/kg, three times daily) or alcohol + glutamine. In this study, a weekend binge drinking model was followed where treatment was done 3 days per week in succession from gestational day (GD) 109-132 (normal term ~147). Maternal alcohol exposure significantly reduced fetal body weight, height, length, thoracic girth and brain weight, and resulted in decreased amino acid bioavailability in fetal plasma and placental fluids. Maternal glutamine supplementation successfully mitigated alcohol-induced fetal growth restriction and improved the bioavailability of glutamine and glutamine-related amino acids such as glycine, arginine, and asparagine in the fetal compartment. All together, these findings show that L-glutamine supplementation enhances amino acid availability in the fetus and prevents alcohol-induced fetal growth restriction.

  20. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

    Science.gov (United States)

    Metallo, Christian M.; Gameiro, Paulo A.; Bell, Eric L.; Mattaini, Katherine R.; Yang, Juanjuan; Hiller, Karsten; Jewell, Christopher M.; Johnson, Zachary R.; Irvine, Darrell J.; Guarente, Leonard; Kelleher, Joanne K.; Vander Heiden, Matthew G.; Iliopoulos, Othon; Stephanopoulos, Gregory

    2013-01-01

    Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol1-3. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle (TCA) and fatty acid synthesis4. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis5, the regulation and utilization of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells employ reductive metabolism of alpha-ketoglutarate (αKG) to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1) dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived αKG for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially utilize reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon utilization in order to produce AcCoA and support lipid synthesis in mammalian cells. PMID:22101433

  1. Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

    Science.gov (United States)

    ... belongs to a class of genetic diseases called urea cycle disorders. In this condition, the carbamoyl phosphate synthetase I ... Management Resources (4 links) Baby's First Test GeneReview: Urea Cycle Disorders Overview MedlinePlus Encyclopedia: Hereditary Urea Cycle Abnormality National ...

  2. Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes.

    Science.gov (United States)

    Tzagoloff, A; Shtanko, A

    1995-06-01

    Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

  3. Randomised trial of glutamine and selenium supplemented parenteral nutrition for critically ill patients. Protocol Version 9, 19 February 2007 known as SIGNET (Scottish Intensive care Glutamine or seleNium Evaluative Trial

    Directory of Open Access Journals (Sweden)

    Vale Luke D

    2007-09-01

    Full Text Available Abstract Background Mortality rates in the Intensive Care Unit and subsequent hospital mortality rates in the UK remain high. Infections in Intensive Care are associated with a 2–3 times increased risk of death. It is thought that under conditions of severe metabolic stress glutamine becomes "conditionally essential". Selenium is an essential trace element that has antioxidant and anti-inflammatory properties. Approximately 23% of patients in Intensive Care require parenteral nutrition and glutamine and selenium are either absent or present in low amounts. Both glutamine and selenium have the potential to influence the immune system through independent biochemical pathways. Systematic reviews suggest that supplementing parenteral nutrition in critical illness with glutamine or selenium may reduce infections and mortality. Pilot data has shown that more than 50% of participants developed infections, typically resistant organisms. We are powered to show definitively whether supplementation of PN with either glutamine or selenium is effective at reducing new infections in critically ill patients. Methods/design 2 × 2 factorial, pragmatic, multicentre, double-blind, randomised controlled trial. The trial has an enrolment target of 500 patients. Inclusion criteria include: expected to be in critical care for at least 48 hours, aged 16 years or over, patients who require parenteral nutrition and are expected to have at least half their daily nutritional requirements given by that route. Allocation is to one of four iso-caloric, iso-nitrogenous groups: glutamine, selenium, both glutamine & selenium or no additional glutamine or selenium. Trial supplementation is given for up to seven days on the Intensive Care Unit and subsequent wards if practicable. The primary outcomes are episodes of infection in the 14 days after starting trial nutrition and mortality. Secondary outcomes include antibiotic usage, length of hospital stay, quality of life and

  4. Randomised trial of glutamine and selenium supplemented parenteral nutrition for critically ill patients. Protocol Version 9, 19 February 2007 known as SIGNET (Scottish Intensive care Glutamine or seleNium Evaluative Trial).

    Science.gov (United States)

    Andrews, Peter J D; Avenell, Alison; Noble, David W; Campbell, Marion K; Battison, Claire G; Croal, Bernard L; Simpson, William G; Norrie, John; Vale, Luke D; Cook, Jonathon; de Verteuil, Robyn; Milne, Anne C

    2007-09-20

    Mortality rates in the Intensive Care Unit and subsequent hospital mortality rates in the UK remain high. Infections in Intensive Care are associated with a 2-3 times increased risk of death. It is thought that under conditions of severe metabolic stress glutamine becomes "conditionally essential". Selenium is an essential trace element that has antioxidant and anti-inflammatory properties. Approximately 23% of patients in Intensive Care require parenteral nutrition and glutamine and selenium are either absent or present in low amounts. Both glutamine and selenium have the potential to influence the immune system through independent biochemical pathways. Systematic reviews suggest that supplementing parenteral nutrition in critical illness with glutamine or selenium may reduce infections and mortality. Pilot data has shown that more than 50% of participants developed infections, typically resistant organisms. We are powered to show definitively whether supplementation of PN with either glutamine or selenium is effective at reducing new infections in critically ill patients. 2 x 2 factorial, pragmatic, multicentre, double-blind, randomised controlled trial. The trial has an enrollment target of 500 patients. Inclusion criteria include: expected to be in critical care for at least 48 hours, aged 16 years or over, patients who require parenteral nutrition and are expected to have at least half their daily nutritional requirements given by that route. Allocation is to one of four iso-caloric, iso-nitrogenous groups: glutamine, selenium, both glutamine & selenium or no additional glutamine or selenium. Trial supplementation is given for up to seven days on the Intensive Care Unit and subsequent wards if practicable. The primary outcomes are episodes of infection in the 14 days after starting trial nutrition and mortality. Secondary outcomes include antibiotic usage, length of hospital stay, quality of life and cost-effectiveness. To date more than 285 patients have been

  5. Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

    Directory of Open Access Journals (Sweden)

    Thai Q Tran

    2017-11-01

    Full Text Available Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

  6. Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

    Science.gov (United States)

    Tran, Thai Q; Ishak Gabra, Mari B; Lowman, Xazmin H; Yang, Ying; Reid, Michael A; Pan, Min; O'Connor, Timothy R; Kong, Mei

    2017-11-01

    Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

  7. Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

    2011-01-01

    Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

  8. effects of enteral glutamine supplementation on reduction

    African Journals Online (AJOL)

    Objective: To determine the effect of enteral glutamine in reducing the incidence of ... in use. These modalities include among others; topical antibacterial agents, early excision of eschar, and ... in the burns unit and plastic surgery ward 4D of.

  9. The influence of prenatal X-irradiation on the activity of SRNA-aminoacyl synthetases in the developing rabbit brain

    International Nuclear Information System (INIS)

    Wender, M.; Zgorzalewicz, B.

    1976-01-01

    The activities of sRNA-aminoacyl synthetases were investigated in the cerebral white and grey matter of rabbits subjected during their prenatal life to a single x-ray dose of 150 rad. The results of investigations have shown that ionizing radiation acting during intrauterine development of the experimental animal brings about a distinct depression of all sRNA-aminoacyl synthetase activities in the newborn irradiated litter. During the postnatal development of these animals the activities of some of the synthetases further decreased and even at adulthood, where they are normally very low, their activities were below the control values. The activities of some other synthetases, after the initial depression, showed no further decrease and at adulthood had values comparable to controls. The results indicate clearly that prenatal exposure to ionizing radiation also affects the steps of protein biosynthesis which depend on the activity of sRNA-aminoacyl synthetases. (author)

  10. Quantitation and localisation of (in vitro) transglutaminase-catalysed glutamine hydroxylation using mass spectrometry

    NARCIS (Netherlands)

    Piersma, S.R.; Pijpekamp, A. van de; Wijngaards, G.; Gruppen, H.; Boumans, H.

    2002-01-01

    A mass spectrometric approach was chosen to quantify and localise in vitro enzymatically modified glutamine (Gln) residues in a glutamine-rich protein. This protein (named dB1), a cloned domain of the high molecular weight wheat glutenin subunit Dx5, was modified by microbial transglutaminase

  11. Effects of UV-B irradiation on growth, survival, pigmentation and nitrogen metabolism enzymes in Cyanobacteria

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, R.P.; Hader, D.P. [Institut fuer Botanik und Pharmazeutische Biologie, Friedrich-Alexander Universitaet, Erlangen (Germany); Kumar, H.D.; Kumar, A. [Banaras Hindu University, Varanasi (India)

    1995-12-31

    The effects of artificial UV-B irradiation on growth, survival, pigmentation, nitrate reductase (NR), glutamine synthetase (GS) and total protein profile have been studied in a number of N{sub 2}-fixing cyanobacterial strains isolated from rice (paddy) fields in India. Different organisms show different effects in terms of growth and survival. Complete killing of Anabaena sp. and Nostoc carmium occurs after 120 min of UV-B exposure, whereas the same occurs only after 150 min of exposure in the case of Nostoc commune and Scytonema sp. Growth patterns of the cells treated with UV-B revealed that Nostoc commune and Scytonema sp. are comparatively more tolerant than Anabaena sp. and Nostoc carmium. Pigment content, particularly phycocyanin, was severely decreased following UV-B irradiation in all strains tested so far. In vivo NR activity was found to increase, while in vivo GS activity was decreased following exposure to UV-B for different durations in all test organisms; although complete inhibition of GS activity did not occur even after 120 min of UV-B exposure. (author). 37 refs, 6 figs.

  12. Effects of UV-B irradiation on growth, survival, pigmentation and nitrogen metabolism enzymes in Cyanobacteria

    International Nuclear Information System (INIS)

    Sinha, R.P.; Hader, D.P.; Kumar, H.D.; Kumar, A.

    1995-01-01

    The effects of artificial UV-B irradiation on growth, survival, pigmentation, nitrate reductase (NR), glutamine synthetase (GS) and total protein profile have been studied in a number of N 2 -fixing cyanobacterial strains isolated from rice (paddy) fields in India. Different organisms show different effects in terms of growth and survival. Complete killing of Anabaena sp. and Nostoc carmium occurs after 120 min of UV-B exposure, whereas the same occurs only after 150 min of exposure in the case of Nostoc commune and Scytonema sp. Growth patterns of the cells treated with UV-B revealed that Nostoc commune and Scytonema sp. are comparatively more tolerant than Anabaena sp. and Nostoc carmium. Pigment content, particularly phycocyanin, was severely decreased following UV-B irradiation in all strains tested so far. In vivo NR activity was found to increase, while in vivo GS activity was decreased following exposure to UV-B for different durations in all test organisms; although complete inhibition of GS activity did not occur even after 120 min of UV-B exposure. (author)

  13. Nitrogen Starvation Acclimation in Synechococcus elongatus: Redox-Control and the Role of Nitrate Reduction as an Electron Sink

    Directory of Open Access Journals (Sweden)

    Alexander Klotz

    2015-03-01

    Full Text Available Nitrogen starvation acclimation in non-diazotrophic cyanobacteria is characterized by a process termed chlorosis, where the light harvesting pigments are degraded and the cells gradually tune down photosynthetic and metabolic activities. The chlorosis response is governed by a complex and poorly understood regulatory network, which converges at the expression of the nblA gene, the triggering factor for phycobiliprotein degradation. This study established a method that allows uncoupling metabolic and redox-signals involved in nitrogen-starvation acclimation. Inhibition of glutamine synthetase (GS by a precise dosage of l-methionine-sulfoximine (MSX mimics the metabolic situation of nitrogen starvation. Addition of nitrate to such MSX-inhibited cells eliminates the associated redox-stress by enabling electron flow towards nitrate/nitrite reduction and thereby, prevents the induction of nblA expression and the associated chlorosis response. This study demonstrates that nitrogen starvation is perceived not only through metabolic signals, but requires a redox signal indicating over-reduction of PSI-reduced electron acceptors. It further establishes a cryptic role of nitrate/nitrite reductases as electron sinks to balance conditions of over-reduction.

  14. Nitrogen Starvation Acclimation in Synechococcus elongatus: Redox-Control and the Role of Nitrate Reduction as an Electron Sink

    Science.gov (United States)

    Klotz, Alexander; Reinhold, Edgar; Doello, Sofía; Forchhammer, Karl

    2015-01-01

    Nitrogen starvation acclimation in non-diazotrophic cyanobacteria is characterized by a process termed chlorosis, where the light harvesting pigments are degraded and the cells gradually tune down photosynthetic and metabolic activities. The chlorosis response is governed by a complex and poorly understood regulatory network, which converges at the expression of the nblA gene, the triggering factor for phycobiliprotein degradation. This study established a method that allows uncoupling metabolic and redox-signals involved in nitrogen-starvation acclimation. Inhibition of glutamine synthetase (GS) by a precise dosage of l-methionine-sulfoximine (MSX) mimics the metabolic situation of nitrogen starvation. Addition of nitrate to such MSX-inhibited cells eliminates the associated redox-stress by enabling electron flow towards nitrate/nitrite reduction and thereby, prevents the induction of nblA expression and the associated chlorosis response. This study demonstrates that nitrogen starvation is perceived not only through metabolic signals, but requires a redox signal indicating over-reduction of PSI-reduced electron acceptors. It further establishes a cryptic role of nitrate/nitrite reductases as electron sinks to balance conditions of over-reduction. PMID:25780959

  15. An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

    DEFF Research Database (Denmark)

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Hausmann, Corinne D

    2011-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translatio...... of a complex between MtSerRS and MtArgRS provides a means by which methanogenic archaea can optimize an early step in translation under a wide range of extreme environmental conditions....

  16. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    Directory of Open Access Journals (Sweden)

    Laakso Kati

    2007-10-01

    Full Text Available Abstract Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1 and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

  17. Evolutionary anomalies among the aminoacyl-tRNA synthetases

    Science.gov (United States)

    Doolittle, R. F.; Handy, J.; Bada, J. L. (Principal Investigator)

    1998-01-01

    Unexpected relationships among the various aminoacyl-tRNA synthetases continue to be uncovered. The question arises - is this mainly the result of promiscuous exchange, or is the confusion really a reflection of the differential loss of past duplications? Phylogenetic analysis may yet provide the answer.

  18. The aldo-keto reductase superfamily homepage.

    Science.gov (United States)

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  19. Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

    International Nuclear Information System (INIS)

    Hinojosa-Leon, M.; Dubourdieu, M.; Sanchez-Crispin, J.A.; Chippaux, M.

    1986-01-01

    Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities

  20. Serum Glutamine Levels as a Potential Diagnostic Biomarker in Sepsis following Surgery for Peritonitis.

    Science.gov (United States)

    Yang, Chun-Ju; Huang, Ting-Shuo; Lee, Tung-Liang; Yang, Kang-Chung; Yuan, Shin-Sheng; Lu, Ruey-Hwa; Hsieh, Chung-Ho; Shyu, Yu-Chiau

    2017-12-31

    Few diagnostic biomarkers for sepsis after emergency peritonitis surgery are available to clinicians, and, thus, it is important to develop new biomarkers for patients undergoing this procedure. We investigated whether serum glutamine and selenium levels could be diagnostic biomarkers of sepsis in individuals recovering from emergency peritonitis surgery. From February 2012 to March 2013, patients who had peritonitis diagnosed at the emergency department and underwent emergency surgery were screened for eligibility. Serum glutamine and selenium levels were obtained at pre-operative, post-operative and recovery time points. The average level of pre-operation serum glutamine was significantly different from that on the recovery day (0.317 ± 0.168 vs. 0.532 ± 0.155 mM, P peritonitis. We recommend including glutamine as a biomarker for sepsis severity assessment in addition to the commonly used clinical indicators.

  1. The utilization of glutamine by the retina: an autoradiographic and metabolic study

    International Nuclear Information System (INIS)

    Voaden, M.J.; Lake, N.; Marshall, J.; Morjaria, B.

    1978-01-01

    The cells able to accumulate exogenously applied [ 3 H] glutamine in rat, cat, frog, pigeon and guinea pig retinas have been located by autoradiography, and the fate of the labelled glutamine, as regards its incorporation into aspartic, glutamic and γ-amino-butyric acids, followed for 60 min. The results support the notion of glutamine as a precursor of transmitter amino acids in some neurones. In particular, it would appear to be a source of a relatively stable pool of GABA which may be located, with species variation, in amacrine or ganglion cells. In the pigeon retina glutamate pool incorporates and retains a major percentage of the label, and perikarya in the middle of the inner nuclear layer of the tissue are predominantly labelled. (author)

  2. Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

  3. Physical studies of adenylosuccinate synthetase

    International Nuclear Information System (INIS)

    Bass, M.B.

    1987-01-01

    To determine the chemical mechanism of the reaction catalyzed by adenylosuccinate synthetase, positional isotope exchange studies were performed. Positional isotope exchange from the β-γ bridge to the β nonbridge position of [γ- 18 O]GTP was followed using 31 P NMR. The positional isotope exchange was found to occur in the presence of either IMP or IMP and succinate. The exchange did not occur in the presence of asparate. These results support a reaction mechanism which involves formation of a 6-phosphoryl-IMP intermediate with subsequent attack by aspartate to form adenylosuccinate as originally proposed by Lieberman in 1956. In order to resolve the NMR resonances for positional isotope exchange, it was necessary to find a chelator which would limit exchange broadening. trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid was found to be a suitable chelator at neutral and acidic pH. Studies of adenylosuccinate synthetase from Escherichia coli have been limited by the low concentrations of enzyme present in the cell and the difficulty in purifying the enzyme to homogeneity. Overproduction of the enzyme by cloning the purA gene into a runaway replication plasmid allowed the cells to produce a much higher concentration of enzyme. A new purification scheme is reported that takes advantage of the overproduced enzyme. Yields of 75 mg of homogeneous enzyme have been obtained from 76 g of E. coli cell paste

  4. Prefrontal changes in the glutamate-glutamine cycle and neuronal/glial glutamate transporters in depression with and without suicide

    NARCIS (Netherlands)

    Zhao, J.; Verwer, R.W.H.; van Wamelen, D.J.; Qi, X.R.; Gao, S.F.; Lucassen, P.J.; Swaab, D.F.

    2016-01-01

    There are indications for changes in glutamate metabolism in relation to depression or suicide. The glutamate-glutamine cycle and neuronal/glial glutamate transporters mediate the uptake of the glutamate and glutamine. The expression of various components of the glutamate-glutamine cycle and the

  5. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Science.gov (United States)

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

  6. Henoch-Schönlein purpura nephritis occurring postpartum in a patient with anti-PL-7 anti-synthetase syndrome.

    Science.gov (United States)

    Nagai, Kojiro; Kishi, Jun; Morizumi, Shun; Minakuchi, Jun; Bando, Yoshimi; Nishioka, Yasuhiko; Doi, Toshio

    2017-09-01

    A 37-year-old pregnant woman developed purpura which was subsequently diagnosed as Henoch-Schönlein purpura (HSP). After childbirth, the patient developed proteinuria and hematuria. Further examination revealed that the HSP nephritis (HSPN) was associated with anti-threonyl-tRNA synthetase anti-synthetase syndrome. The onset of HSPN during pregnancy or after childbirth is rare. Moreover, to our knowledge, this is the first case to describe renal involvement in anti-synthetase syndrome.

  7. A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi.

    Science.gov (United States)

    Byers, D M; Holmes, C G

    1990-01-01

    An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.

  8. Microbial nitrilases: versatile, spiral forming, industrial enzymes

    CSIR Research Space (South Africa)

    Thuku, RN

    2009-03-01

    Full Text Available such case is the NAD+ synthetase from Mycobacterium tuberculosis (Bellinzoni et al., 2005). This enzyme relies on an associated amino-terminal amidase domain in order to utilize glutamine as a source of nitrogen and liberate ammonia which is required...

  9. Oxygen and xenobiotic reductase activities of cytochrome P450.

    NARCIS (Netherlands)

    Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

    1995-01-01

    The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

  10. Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

    International Nuclear Information System (INIS)

    Herguedas, Beatriz; Martínez-Júlvez, Marta; Frago, Susana; Medina, Milagros; Hermoso, Juan A.

    2009-01-01

    Native and selenomethionine-labelled FAD synthetase from C. ammoniagenes have been crystallized by the hanging-drop vapour-diffusion method. A MAD data set for SeMet-labelled FAD synthetase was collected to 2.42 Å resolution, while data sets were collected to 1.95 Å resolution for the native crystals. FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokaryotic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P2 1 3, with unit-cell parameters a = b = c = 133.47 Å and a = b = c = 133.40 Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 Å resolution, respectively

  11. Structure and mechanism of dimethylsulfoxide reductase, a molybdopterin-containing enzyme of DMSO reductase family

    International Nuclear Information System (INIS)

    McEwan, A.G.; Ridge, J.P.; McDevitt, C.A.; Hanson, G.R.

    2001-01-01

    Full text: Apart from nitrogenase, enzymes containing molybdenum are members of a superfamily, the molybdopterin-containing enzymes. Most of these enzymes catalyse an oxygen atom transfer and two electron transfer reaction. During catalysis the Mo at the active site cycles between the Mo(VI) and Mo(IV) states. The DMSO reductase family of molybdopterin-containing enzymes all contain a bis(molybdopterin guanine dinucleotide)Mo cofactor and over thirty examples have now been described. Over the last five years crystal structures of dimethylsulfoxide (DMSO) reductase and four other enzymes of the DMSO reductase family have revealed that enzymes of this family have a similar tertiary structure. The Mo atom at the active site is coordinated by four thiolate ligands provided by the dithiolene side chains of the two MGD molecules of the bis(MGD)Mo cofactor as well as a ligand provided by an amino acid side chain. In addition, an oxygen atom in the form of an oxo, hydroxo or aqua group is also coordinated to the Mo atom. In the case of dimethylsulfoxide reductase X-ray crystallography of the product-reduced species and Raman spectroscopy has demonstrated that the enzyme contains a single exchangeable oxo group that is H-bonded to W116

  12. Comparative genome analysis of central nitrogen metabolism and its control by GlnR in the class Bacilli

    Directory of Open Access Journals (Sweden)

    Kormelink Tom

    2012-05-01

    Full Text Available Abstract Background The assimilation of nitrogen in bacteria is achieved through only a few metabolic conversions between alpha-ketoglutarate, glutamate and glutamine. The enzymes that catalyze these conversions are glutamine synthetase, glutaminase, glutamate dehydrogenase and glutamine alpha-ketoglutarate aminotransferase. In low-GC Gram-positive bacteria the transcriptional control over the levels of the related enzymes is mediated by four regulators: GlnR, TnrA, GltC and CodY. We have analyzed the genomes of all species belonging to the taxonomic families Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae to determine the diversity in central nitrogen metabolism and reconstructed the regulation by GlnR. Results Although we observed a substantial difference in the extent of central nitrogen metabolism in the various species, the basic GlnR regulon was remarkably constant and appeared not affected by the presence or absence of the other three main regulators. We found a conserved regulatory association of GlnR with glutamine synthetase (glnRA operon, and the transport of ammonium (amtB-glnK and glutamine/glutamate (i.e. via glnQHMP, glnPHQ, gltT, alsT. In addition less-conserved associations were found with, for instance, glutamate dehydrogenase in Streptococcaceae, purine catabolism and the reduction of nitrite in Bacillaceae, and aspartate/asparagine deamination in Lactobacillaceae. Conclusions Our analyses imply GlnR-mediated regulation in constraining the import of ammonia/amino-containing compounds and the production of intracellular ammonia under conditions of high nitrogen availability. Such a role fits with the intrinsic need for tight control of ammonia levels to limit futile cycling.

  13. Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation.

    Science.gov (United States)

    Pardo, Beatriz; Rodrigues, Tiago B; Contreras, Laura; Garzón, Miguel; Llorente-Folch, Irene; Kobayashi, Keiko; Saheki, Takeyori; Cerdan, Sebastian; Satrústegui, Jorgina

    2011-01-01

    The glutamate-glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate-glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo (13)C nuclear magnetic resonance labeling with (13)C(2)acetate or (1-(13)C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.

  14. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.

    1986-01-01

    of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined......Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...

  15. Exogenous trehalose improves growth under limiting nitrogen through upregulation of nitrogen metabolism.

    Science.gov (United States)

    Lin, Yingchao; Zhang, Jie; Gao, Weichang; Chen, Yi; Li, Hongxun; Lawlor, David W; Paul, Matthew J; Pan, Wenjie

    2017-12-19

    The trehalose (Tre) pathway has strong effects on growth and development in plants through regulation of carbon metabolism. Altering either Tre or trehalose 6-phosphate (T6P) can improve growth and productivity of plants as observed under different water availability. As yet, there are no reports of the effects of modification of Tre orT6P on plant performance under limiting nutrition. Here we report that nitrogen (N) metabolism is positively affected by exogenous application of Tre in nitrogen-deficient growing conditions. Spraying foliage of tobacco (Nicotiana tabacum) with trehalose partially alleviated symptoms of nitrogen deficiency through upregulation of nitrate and ammonia assimilation and increasing activities of nitrate reductase (NR), glycolate oxidase (GO), glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) with concomitant changes in ammonium (NH 4 + ) and nitrate (NO 3 - ) concentrations, glutamine and amino acids. Chlorophyll and total nitrogen content of leaves and rates of photosynthesis were increased compared to nitrogen-deficient plants without applied Tre. Total plant biomass accumulation was also higher in Tre -fed nitrogen-deficient plants, with a smaller proportion of dry weight partitioned to roots, compared to nitrogen-deficient plants without applied Tre. Consistent with higher nitrogen assimilation and growth, Tre application reduced foliar starch. Minimal effects of Tre feeding were observed on nitrogen-sufficient plants. The data show, for the first time, significant stimulatory effects of exogenous Tre on nitrogen metabolism and growth in plants growing under deficient nitrogen. Under such adverse conditions metabolism is regulated for survival rather than productivity. Application of Tre can alter this regulation towards maintenance of productive functions under low nitrogen. This has implications for considering approaches to modifying the Tre pathway for to improve crop nitrogen-use efficiency and

  16. Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

    Science.gov (United States)

    Esmaeili, Fatemeh; Ghaheri, Matin; Kahrizi, Danial; Mansouri, Mohsen; Safavi, Seyed Mehdi; Ghorbani, Tayebeh; Muhammadi, Sarre; Rahmanian, Elham; Vaziri, Siavash

    2018-02-10

    Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

  17. Self-protection of Pseudomonas syringae pv. tabaci from its toxin, tabtoxinine-β-lactam

    International Nuclear Information System (INIS)

    Knight, T.J.; Durbin, R.D.; Langston-Unkefer, P.J.

    1987-01-01

    An extracellular toxin, tabtoxinine-β-lactam (TβL), is produced by Pseudomonas syringae pv. tabaci. This toxin irreversibly inhibits its target, glutamine synthetase; yet P. syringae pv. tabaci retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection of P. syringae pv. tabaci, the authors compared the effects of TβL on Tox + (TβL-producing, insensitive to TβL) and Tox - (TβL nonproducing, sensitive to TΛ) strains. The extent of protection afforded to the Tox - strain when induced to adenylylate glutamine synthetase was tested. It was concluded that an additional protection mechanism was required. A detoxification activity was found in the Tox + strain which opens the ε-lactam ring to TβL to produce the inactive, open-chain form, tabtoxinine. Whole cells of the Tox + strain incubated for 24 h with [ 14 C]TβL (0.276 μmol/3 x 10 10 cells) contained [ 14 C]tabtoxinine (0.056 μmol), and the medium contained TβL (0.226 μmol). Extracts of spheroplasts of the Tox + stain also converted TβL to tabtoxinine, whereas extracts of the Tox - strain did not alter TβL. The conversion was time dependent and stoichiometric and was destroyed by boiling for 30 min or by the addition of 5mM EDTA. Penicillin, a possible substrate and competitive inhibitor of this lactamase activity, inhibited the conversion of TΛ to tabtoxinine. Periplasmic fluid did not catalyze the conversion of TβL

  18. Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania).

    Science.gov (United States)

    Brattsten, L B; Wilkinson, C F

    1975-07-01

    1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase.

  19. Protective effects of L-glutamine against toxicity of deltamethrin in the cerebral tissue

    Directory of Open Access Journals (Sweden)

    Varol S

    2016-04-01

    Full Text Available Sefer Varol, Hasan Hüseyin Özdemir, Mehmet Uğur Çevik, Yaşar Altun, Ibrahim Ibiloğlu, Aysun Ekinci, Aslıhan Okan Ibiloğlu, Metin Balduz, Demet Arslan, Recep Tekin, Fesih Aktar, Mehmet Ufuk Aluçlu Department of Neurology, Faculty of Medicine, Dicle University, Diyarbakir, Turkey Background: Deltamethrin (DLM is a broad-spectrum synthetic dibromo-pyrethroid pesticide that is widely used for agricultural and veterinary purposes. However, human exposure to the pesticide leads to neurotoxicity. Glutamine is one of the principal, free intracellular amino acids and may also be an antioxidant. This study was undertaken in order to examine the neuroprotective and antioxidant potential of L-glutamine against DLM toxicity in female Wistar albino rats. Materials and methods: The rats were divided into the following groups (n=10: Group I: control (distilled water; 10 mL/kg, po one dose, Group II: L-glutamine (1.5 g/kg, po one dose, Group III: DLM (35 mg/kg, po one dose, and Group IV: DLM (35 mg/kg, po one dose and L-glutamine (1.5 g/kg, po one dose after 4 hours. Total oxidant status (TOS, total antioxidant status (TAS, tumor necrosis factor-α, interleukin (IL-1β, and IL-6 levels and apoptosis were evaluated in brain tissue. Results: DLM-treated animals had a significant increase in brain biochemical parameters, as well as TOS and TAS. Furthermore, the histopathological examination showed neuronal cell degeneration in the cerebral tissue. L-Glutamine treatment decreased the elevated brain levels of TOS and neuronal cell degeneration. There was no difference in tumor necrosis factor-α, IL-1β, and IL-6 levels between the groups. Conclusion: L-Glutamine may reduce the toxic effects of DLM in the cerebral tissue through antioxidant properties. Keywords: deltamethrin, L-glutamine, rat

  20. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

    International Nuclear Information System (INIS)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2007-01-01

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K 2 ) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222 1 , with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit

  1. Plasma glutamine is a minor precursor for the synthesis of citrulline: A multispecies study

    Science.gov (United States)

    Glutamine is considered the main precursor for citrulline synthesis in many species, including humans. The transfer of 15N from 2[15N]-glutamine to citrulline has been used as evidence for this precursor-product relationship. However, work in mice has shown that nitrogen and carbon tracers follow di...

  2. N-Linked Glycosylation is an Important Parameter for Optimal Selection of Cell Lines Producing Biopharmaceutical Human IgG

    NARCIS (Netherlands)

    van Berkel, Patrick H. C.; Gerritsen, Jolanda; Perdok, Gerrard; Valbjorn, Jesper; Vink, Tom; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

    2009-01-01

    We studied the variations in N-linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO-KISV cells. The glycans

  3. Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

    DEFF Research Database (Denmark)

    Nilsson, Dan; Hove-Jensen, Bjarne

    1987-01-01

    The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting...... in a DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up...... to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA....

  4. Glutamine reduces postprandial glycemia and augments the glucagon-like peptide-1 response in type 2 diabetes patients

    DEFF Research Database (Denmark)

    Samocha-Bonet, Dorit; Wong, Olivia; Synnott, Emma-Leigh

    2011-01-01

    Impaired glucagon-like peptide (GLP-1) secretion or response may contribute to ineffective insulin release in type 2 diabetes. The conditionally essential amino acid glutamine stimulates GLP-1 secretion in vitro and in vivo. In a randomized, crossover study, we evaluated the effect of oral...... glutamine, with or without sitagliptin (SIT), on postprandial glycemia and GLP-1 concentration in 15 type 2 diabetes patients (glycated hemoglobin 6.5 ± 0.6%). Participants ingested a low-fat meal (5% fat) after receiving either water (control), 30 g l-glutamine (Gln-30), 15 g L-glutamine (Gln-15), 100 mg...... concentration and limiting postprandial glycemia in type 2 diabetes....

  5. Effect of rapid weight loss and glutamine supplementation on immunosuppression of combat athletes: a double-blind, placebo-controlled study.

    Science.gov (United States)

    Tritto, Aline C C; Amano, Mariane T; De Cillo, Maria E; Oliveira, Vinicius A; Mendes, Sandro H; Yoshioka, Caroline; Roschel, Hamilton; Camara, Niels Olsen S; Gualano, Bruno; Artioli, Guilherme G

    2018-02-01

    The role of plasma glutamine concentration and glutamine supplementation on immunosuppression was investigated in combat athletes. Twenty-three male athletes were randomly assigned to receive glutamine (21 g/day, n=12) or placebo (ovalbumin, n=11) for 10 days. Six athletes who did not lose weight served as controls. Athletes were assessed 21 days before (-21d), 1 day before (-1d) and 5 days after (+5d) a competition. Weight reduction was similar between glutamine (-8.2%± 4.1%) and placebo (-8.5%±2.4%) and negligible in control (-0.6%±1.4%). In both weight-loss groups, the majority of athletes reported symptoms of upper respiratory symptoms, as assessed by the Wisconsin upper respiratory symptom survey questionnaire. Only two athletes reported symptoms in the control group. Immune cell function remained unchanged throughout the study except for an increase in neutrophil phagocytic activity (placebo: -21d=5,251±2,986; -1d=17,428±22,374; +5d=21,125±21,934; glutamine: -21d=6,096±3,549; -1d=11,029±17,113; +5d=28,186±21,032 FI) and a minor change in monocyte phagocytic activity (placebo: -21d=4,421±3,634; -1d=3,329±6,283; +5d=3,243± 2,553; glutamine: -21d=4,051±3,186; -1d=3,106±2,625; +5d=4,981± 4,598) in both glutamine and placebo after weight loss. Plasma glutamine and cortisol remained unchanged across the study. creatine kinase levels were increased in placebo (-21d=125.2±54.1; -1d=187.2± 73.5; +5d=111.3±59.1 U/L) but not in glutamine (-21d=136.2±58.2; -1d= 168.8±65.0; +5d=129.7±64.0 U/L). Rapid weight loss increased the frequency and severity of infection symptoms, but this was neither associated with plasma glutamine depletion nor counteracted by glutamine supplementation.

  6. Effects of L-glutamine supplementation on maternal and fetal hemodynamics in gestating ewes exposed to alcohol

    OpenAIRE

    Sawant, Onkar B.; Ramadoss, Jayanth; Hankins, Gary D.; Wu, Guoyao; Washburn, Shannon E.

    2014-01-01

    Not much is known about effects of gestational alcohol exposure on maternal and fetal cardiovascular adaptations. This study determined whether maternal binge alcohol exposure and L-glutamine supplementation could affect maternal-fetal hemodynamics and fetal regional brain blood flow during the brain growth spurt period. Pregnant sheep were randomly assigned to one of four groups: saline control, alcohol (1.75–2.5 g/kg body weight), glutamine (100 mg/kg body weight) or alcohol + glutamine. A ...

  7. Co-operation between Polymerases and Nucleotide Synthetases in the RNA World.

    Directory of Open Access Journals (Sweden)

    Ye Eun Kim

    2016-11-01

    Full Text Available It is believed that life passed through an RNA World stage in which replication was sustained by catalytic RNAs (ribozymes. The two most obvious types of ribozymes are a polymerase, which uses a neighbouring strand as a template to make a complementary sequence to the template, and a nucleotide synthetase, which synthesizes monomers for use by the polymerase. When a chemical source of monomers is available, the polymerase can survive on its own. When the chemical supply of monomers is too low, nucleotide production by the synthetase is essential and the two ribozymes can only survive when they are together. Here we consider a computational model to investigate conditions under which coexistence and cooperation of these two types of ribozymes is possible. The model considers six types of strands: the two functional sequences, the complementary strands to these sequences (which are required as templates, and non-functional mutants of the two sequences (which act as parasites. Strands are distributed on a two-dimensional lattice. Polymerases replicate strands on neighbouring sites and synthetases produce monomers that diffuse in the local neighbourhood. We show that coexistence of unlinked polymerases and synthetases is possible in this spatial model under conditions in which neither sequence could survive alone; hence, there is a selective force for increasing complexity. Coexistence is dependent on the relative lengths of the two functional strands, the strand diffusion rate, the monomer diffusion rate, and the rate of deleterious mutations. The sensitivity of this two-ribozyme system suggests that evolution of a system of many types of ribozymes would be difficult in a purely spatial model with unlinked genes. We therefore speculate that linkage of genes onto mini-chromosomes and encapsulation of strands in protocells would have been important fairly early in the history of life as a means of enabling more complex systems to evolve.

  8. Cerebral glucose metabolism and the glutamine cycle as detected by in vivo and in vitro 13C NMR spectroscopy.

    Science.gov (United States)

    García-Espinosa, María A; Rodrigues, Tiago B; Sierra, Alejandra; Benito, Marina; Fonseca, Carla; Gray, Heather L; Bartnik, Brenda L; García-Martín, María L; Ballesteros, Paloma; Cerdán, Sebastián

    2004-01-01

    We review briefly 13C NMR studies of cerebral glucose metabolism with an emphasis on the roles of glial energetics and the glutamine cycle. Mathematical modeling analysis of in vivo 13C turnover experiments from the C4 carbons of glutamate and glutamine are consistent with: (i) the glutamine cycle being the major cerebral metabolic route supporting glutamatergic neurotransmission, (ii) glial glutamine synthesis being stoichiometrically coupled to glycolytic ATP production, (iii) glutamine serving as the main precursor of neurotransmitter glutamate and (iv) glutamatergic neurotransmission being supported by lactate oxidation in the neurons in a process accounting for 60-80% of the energy derived from glucose catabolism. However, more recent experimental approaches using inhibitors of the glial tricarboxylic acid (TCA) cycle (trifluoroacetic acid, TFA) or of glutamine synthase (methionine sulfoximine, MSO) reveal that a considerable portion of the energy required to support glutamine synthesis is derived from the oxidative metabolism of glucose in the astroglia and that a significant amount of the neurotransmitter glutamate is produced from neuronal glucose or lactate rather than from glial glutamine. Moreover, a redox switch has been proposed that allows the neurons to use either glucose or lactate as substrates for oxidation, depending on the relative availability of these fuels under resting or activation conditions, respectively. Together, these results suggest that the coupling mechanisms between neuronal and glial metabolism are more complex than initially envisioned.

  9. Ammonium absorption mechanism of rice seedling roots and 15N-labelling pattern of their glutamine-amide group, 2

    International Nuclear Information System (INIS)

    Arima, Yasuhiro; Kumazawa, Kikuo

    1975-01-01

    The processes of producing glutamine and asparagine at the initial stage of the absorption and assimilation of ammonia in rice seedling roots were examined in relation to glutamic acid, aspartic acid and ammonia by 15 N-labelling method. When ( 15 NH 4 ) 2 SO 4 was absorbed into the roots, 15 N concentration appeared very high in glutamine-amide radical and ammonia. It was also higher in amide radical than in amino radical in both glutamine and asparagine, while 15 N concentration in the amino radical of glutamine and asparagine were far lower than that of corresponding glutamine acid and aspartic acid. From these facts, glutamine-amide radical seems to be produced directly from the ammonia in culture media at the contact point of root cells and the culture media, while there is some possibility that asparagine-amide radical is formed from other amino compounds than ammonia. Also the amino radical of aspartic acid seems to be produced not only by the transamination from glutamic acid but also by the reductive amination of oxalautic acid by ammonium. (Kobatake, H.)

  10. Intravenous glutamine enhances COX-2 activity giving cardioprotection.

    LENUS (Irish Health Repository)

    McGuinness, Jonathan

    2009-03-01

    Preconditioning, a highly evolutionary conserved endogenous protective response, provides the most powerful form of anti-infarct protection known. We investigated whether acute intravenous glutamine, through an effect on cyclooxygenase (COX)-2 and heat shock protein (HSP) 72, might induce preconditioning.

  11. Modeling the role of covalent enzyme modification in Escherichia coli nitrogen metabolism

    International Nuclear Information System (INIS)

    Kidd, Philip B; Wingreen, Ned S

    2010-01-01

    In the bacterium Escherichia coli, the enzyme glutamine synthetase (GS) converts ammonium into the amino acid glutamine. GS is principally active when the cell is experiencing nitrogen limitation, and its activity is regulated by a bicyclic covalent modification cascade. The advantages of this bicyclic-cascade architecture are poorly understood. We analyze a simple model of the GS cascade in comparison to other regulatory schemes and conclude that the bicyclic cascade is suboptimal for maintaining metabolic homeostasis of the free glutamine pool. Instead, we argue that the lag inherent in the covalent modification of GS slows the response to an ammonium shock and thereby allows GS to transiently detoxify the cell, while maintaining homeostasis over longer times

  12. [Effect of glutamine on small intestinal repair in weanling rats after chronic diarrhea].

    Science.gov (United States)

    Huang, Zu-xiong; Ye, Li-yan; Zheng, Zhi-yong; Chen, Xin-min; Ren, Rong-na; Tong, Guo-yuan

    2005-05-01

    To investigate the nutrient effect of glutamine on small intestinal repair in weanling rats after chronic diarrhea. Forty 21-day-old wistar rats were randomly divided into five groups (8 in each). Animal model of chronic diarrhea was induced by a lactose enriched diet in the weanling Wistar rat, normal control group was fed with a standard semipurified diet, and after 14 days the rats in both groups were killed to test the establishment of the model. After the establishment of the model, the other groups were fed with the standard semipurified diet to recover for 7 days, and were randomly divided into three groups: non-intervention group, glutamine (Gln)-intervention group and control group. Glutamine concentrations in blood was detected by high-performance liquid chromatography (HPLC). Morphological changes including villus height and villus surface area of the jejunum were measured under a light microscope and electron microscope, expression of proliferating cell nuclear antigen (PCNA) as an index of cell proliferation was observed using immunohistochemical staining and image analysis. The diarrhea rate in model group was 100 percent, average diarrhea index was 1.16 +/- 0.06, but both diarrhea rate and average diarrhea index in control group were 0 (P body weight, plasma Gln concentration, villus height, villus surface area and expression of PCNA in non-intervened group compared with the control group (P body weight, villus height and villus surface area in Gln-intervened group compared with control group (P 0.05). And compared with non-intervened group, except for body weight (P > 0.05), plasma glutamine, villus height, villus surface area and expression of PCNA were all significantly increased in Gln-intervened group. Chronic diarrhea can induce malnutrition and reduce the villus height, villus surface area, expression of PCNA and plasm glutamine concentration. Oral glutamine could improve the proliferation of crypt cell and promote repair of intestinal mucosa

  13. The effect of glutamine-enriched enteral nutrition on intestinal permeability in very-low-birth-weight infants : A randomized controlled trial

    NARCIS (Netherlands)

    van den Berg, Anemone; Fetter, Willem P. F.; Westerbeek, Elisabeth A. M.; van der Vegt, Ina M.; van der Molen, Hilda R. A.; van Elburg, Ruurd M.

    2006-01-01

    Background: Very-low-birth-weight (VLBW) infants are susceptible to glutamine depletion. Glutamine depletion has negative effects on intestinal integrity. The lower infection rate in VLBW infants receiving glutamine-enriched enteral nutrition may originate from improved intestinal integrity, as

  14. The effect of glutamine-enriched enteral nutrition on intestinal permeability in very-low-birth-weight infants: A randomized controlled trial

    NARCIS (Netherlands)

    van den Berg, Anemone; Fetter, Willem P. F.; Westerbeek, Elisabeth A. M.; van der Vegt, Ina M.; van der Molen, Hilda R. A.; van Elburg, Ruurd M.

    2006-01-01

    Background: Very-low-birth-weight (VLBW) infants are susceptible to glutamine depletion. Glutamine depletion has negative effects on intestinal integrity. The lower infection rate in VLBW infants receiving glutamine-enriched enteral nutrition may originate from improved intestinal integrity, as

  15. Proteome reference maps of the Lotus japonicus nodule and root

    DEFF Research Database (Denmark)

    Dam, Svend Secher; Dyrlund, Thomas F.; Ussatjuk, Anna

    2014-01-01

    formation mutant (snf1) was determined. From nodules and roots, 780 and 790 protein spots from 2D gels were identified and approximately 45% of the corresponding unique gene accessions were common. Including a previous proteomics set from Lotus pod and seed, the common gene accessions were decreased to 7...... stress level at this developmental stage. In contrast, protein spots corresponding to nodulins such as leghemoglobin, asparagine synthetase, sucrose synthase, and glutamine synthetase were prevalent in red nodules. The distinct biochemical state of nodules was further highlighted by the conspicuous...

  16. [Cardioprotective effects of glutamine in patients with ischemic heart disease operated under conditions of extracorporeal blood circulation].

    Science.gov (United States)

    Lomivorotov, V V; Efremov, S M; Shmyrev, V A; Ponomarev, D N; Sviatchenko, A V; Kniaz'kova, L G

    2012-01-01

    It was conducted a study of glutamine cardioptotective effects during perioperative use in patients with ischemic heart disease, operated under CB. Exclusion criteria were: left ventricular ejection fraction less than 50%, diabetes melitus, myocardial infarction less than 3 months ago, Patients of the study group (n=25) had glutamine (20% solution N(2)-L-alanine-L-glutamine ("Dipeptiven" Fresenius Kabi, Germany); 0.4 g/kg/day. Patients of control group (n=25) received placebo (0.9% NaCl solution). The main indicators were the dynamics of troponin I, as well as central hemodynamics parameters. On the 1-st day after operation the concentration of troponin I was significantly lower in the glutamine-group compared placebo-group (1.280 (0.840-2.230) 2.410 (1.060-6.600) ng/ml; p=0.035). 4 hours after CB in a glutamine-group also had significantly large indicators of cardiac index (2.58 (2.34-2.91) l/min/m2 vs 2.03 (1.76-2.32)) l/min/m2; p=0,002) and stroke index (32.8 (27.8-36.0.) ml/m2 vs 26.1 (22.6-31.8) ml/m2; p=0.023). Systemic vascular resistance index was significantly lower in glutamine-group (1942 (1828-2209) dyn x s/cm(-5)/m2 vs 2456 (2400-3265) dyn x s/cm(-5)/m2; p=0.001). Conclusion. Perioperative use of N(2)-L-alanine-L-glutamine during the first 24 hours ofperioperative period gives cardioprotective effect in patients with ischemic heart disease operated under CB.

  17. Determination of glutamine and glutamic acid in mammalian cell cultures using tetrathiafulvalene modified enzyme electrodes.

    Science.gov (United States)

    Mulchandani, A; Bassi, A S

    1996-01-01

    Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures. The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase. The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol. Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system. When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.

  18. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end produc...

  19. Glutamine Supplemented Parenteral Nutrition to Prevent Ventilator-Associated Pneumonia in the Intensive Care Unit

    Directory of Open Access Journals (Sweden)

    Meltem Türkay Aydoğmuş

    2012-12-01

    Full Text Available Objective: Ventilator-associated pneumonia (VAP is a form of nosocomial pneumonia that increases patient morbidity and mortality, length of hospital stay, and healthcare costs. Glutamine preserves the intestinal mucosal structure, increases immune function, and reduces harmful changes in gut permeability in patients receiving total parenteral nutrition (TPN. We hypothesized that TPN supplemented by glutamine might prevent the development of VAP in patients on mechanical ventilator support in the intensive care unit (ICU. Material and Methods: With the approval of the ethics committee and informed consent from relatives, 60 patients who were followed in the ICU with mechanical ventilator support were included in our study. Patients were divided into three groups. The first group received enteral nutrition (n=20, and the second was prescribed TPN (n=20 while the third group was given glutamine-supplemented TPN (n=20. C-reactive protein (CRP, sedimentation rate, body temperature, development of purulent secretions, increase in the amount of secretions, changes in the characteristics of secretions and an increase in requirement of deep tracheal aspiration were monitored for seven days by daily examination and radiographs. Results: No statistically significant difference was found among groups in terms of development of VAP (p=0.622. Conclusion: Although VAP developed at a lower rate in the glutamine-supplemented TPN group, no statistically significant difference was found among any of the groups. Glutamine-supplemented TPN may have no superiority over unsupplemented enteral and TPN in preventing VAP.

  20. Glutamine supplemented parenteral nutrition to prevent ventilator-associated pneumonia in the intensive care unit.

    Science.gov (United States)

    Aydoğmuş, Meltem Türkay; Tomak, Yakup; Tekin, Murat; Katı, Ismail; Hüseyinoğlu, Urfettin

    2012-12-01

    Ventilator-associated pneumonia (VAP) is a form of nosocomial pneumonia that increases patient morbidity and mortality, length of hospital stay, and healthcare costs. Glutamine preserves the intestinal mucosal structure, increases immune function, and reduces harmful changes in gut permeability in patients receiving total parenteral nutrition (TPN). We hypothesized that TPN supplemented by glutamine might prevent the development of VAP in patients on mechanical ventilator support in the intensive care unit (ICU). With the approval of the ethics committee and informed consent from relatives, 60 patients who were followed in the ICU with mechanical ventilator support were included in our study. Patients were divided into three groups. The first group received enteral nutrition (n=20), and the second was prescribed TPN (n=20) while the third group was given glutamine-supplemented TPN (n=20). C-reactive protein (CRP), sedimentation rate, body temperature, development of purulent secretions, increase in the amount of secretions, changes in the characteristics of secretions and an increase in requirement of deep tracheal aspiration were monitored for seven days by daily examination and radiographs. No statistically significant difference was found among groups in terms of development of VAP (p=0.622). Although VAP developed at a lower rate in the glutamine-supplemented TPN group, no statistically significant difference was found among any of the groups. Glutamine-supplemented TPN may have no superiority over unsupplemented enteral and TPN in preventing VAP.

  1. L-glutamine supplementation prevents the development of experimental diabetic cardiomyopathy in streptozotocin-nicotinamide induced diabetic rats.

    Directory of Open Access Journals (Sweden)

    Sachin L Badole

    Full Text Available The objective of the present investigation was to evaluate the effect of L-glutamine on cardiac myopathy in streptozotocin-nicotinamide induced diabetic rats. Diabetes was induced in overnight fasted Sprague Dawely rats by using intraperitonial injection of streptozotocin (55 mg/kg. Nicotinamide (100 mg/kg, i.p. was administered 20 min before administration of streptozotocin. Experimental rats were divided into Group I: non-diabetic control (distilled water; 10 ml/kg, p.o., II: diabetic control (distilled water, 10 ml/kg, p.o., III: L-glutamine (500 mg/kg, p.o. and IV: L-glutamine (1000 mg/kg, p.o.. All groups were diabetic except group I. The plasma glucose level, body weight, electrocardiographic abnormalities, hemodynamic changes and left ventricular contractile function, biological markers of cardiotoxicity, antioxidant markers were determined after 4 months after STZ with nicotinamide injection. Histopathological changes of heart tissue were carried out by using H and E stain. L-glutamine treatment improved the electrocardiographic, hemodynamic changes; LV contractile function; biological markers; oxidative stress parameters and histological changes in STZ induced diabetic rats. Results from the present investigation demonstrated that L-glutamine has seemed a cardioprotective activity.

  2. The standard enthalpies of formation of L-asparagine and L-{alpha}-glutamine

    Energy Technology Data Exchange (ETDEWEB)

    Contineanu, Iulia, E-mail: icontineanu@yahoo.com [Institute of Physical Chemistry ' I.G. Murgulescu' , Spl. Independentei 202, Bucharest (Romania); Neacsu, Ana, E-mail: anna_matache@yahoo.com [Institute of Physical Chemistry ' I.G. Murgulescu' , Spl. Independentei 202, Bucharest (Romania); Perisanu, Stefan T., E-mail: stefan.perisanu@upb.ro [Laboratory of General Chemistry, Polytechnic University of Bucharest, str. Polizu nr. 1, Bucuresti (Romania)

    2010-01-10

    The energies of combustion of L-asparagine and L-{alpha}-glutamine were measured in a static bomb adiabatic calorimeter. Corrections were made for the heats due to the ignition of sample and for the nitric acid formation. The derived enthalpies of formation in solid state of asparagine monohydrate, nonhydrated asparagine and glutamine are respectively -1084.1 {+-} 3.0, -788.1 {+-} 4.7 and -834.3 {+-} 4.6 kJ mol{sup -1}. The data of enthalpy of formation are compared with the literature values and with estimated values by means of group additivity, using parameters recommended by Domalski and Hearing. The discrepancies between experimental and calculated values are explained by considering the number and strength of intermolecular hydrogen bonds. The dehydration of asparagine monohydrate and the possible melting of the three amino acids were investigated by means of DSC. Glutamine melts without considerable decomposition at about 182 {sup o}C, while asparagines decompose during the fusion process (208 {sup o}C).

  3. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  4. Glutamine deprivation enhances antitumor activity of 3-bromopyruvate through the stabilization of monocarboxylate transporter-1.

    Science.gov (United States)

    Cardaci, Simone; Rizza, Salvatore; Filomeni, Giuseppe; Bernardini, Roberta; Bertocchi, Fabio; Mattei, Maurizio; Paci, Maurizio; Rotilio, Giuseppe; Ciriolo, Maria Rosa

    2012-09-01

    Anticancer drug efficacy might be leveraged by strategies to target certain biochemical adaptations of tumors. Here we show how depriving cancer cells of glutamine can enhance the anticancer properties of 3-bromopyruvate, a halogenated analog of pyruvic acid. Glutamine deprival potentiated 3-bromopyruvate chemotherapy by increasing the stability of the monocarboxylate transporter-1, an effect that sensitized cells to metabolic oxidative stress and autophagic cell death. We further elucidated mechanisms through which resistance to chemopotentiation by glutamine deprival could be circumvented. Overall, our findings offer a preclinical proof-of-concept for how to employ 3-bromopyruvate or other monocarboxylic-based drugs to sensitize tumors to chemotherapy. ©2012 AACR.

  5. Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells

    DEFF Research Database (Denmark)

    Grav, Lise Marie; Julie la Cour Karottki, Karen; Lee, Jae Seong

    2017-01-01

    and yields. In this chapter, we present our protocol on how to use the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knockout engineering target genes in CHO cells. As an example, we refer to the glutamine synthetase (GS...

  6. Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop.

    Science.gov (United States)

    Stephen, Preyesh; Ye, Sheng; Zhou, Ming; Song, Jian; Zhang, Rongguang; Wang, En-Duo; Giegé, Richard; Lin, Sheng-Xiang

    2018-05-25

    Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNA Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Glutamine-enriched enteral diet increases renal arginine production

    NARCIS (Netherlands)

    Houdijk, A. P.; van Leeuwen, P. A.; Teerlink, T.; FLINKERBUSCH, E. L.; Boermeester, M. A.; Sauerwein, H. P.; Wesdorp, R. I.

    1994-01-01

    Arginine (Arg) is generated in the kidney by the conversion of circulating citrulline. The most important source for circulating citrulline is the metabolism of glutamine (Gln) by the gut. In this study, we investigated the influence of an enteral diet enriched with Gln on renal Arg synthesis in the

  8. Bioinformatics approach of three partial polyprenol reductase genes in Kandelia obovata

    Science.gov (United States)

    Basyuni, M.; Wati, R.; Sagami, H.; Oku, H.; Baba, S.

    2018-03-01

    This present study describesthe bioinformatics approach to analyze three partial polyprenol reductase genes from mangrove plant, Kandeliaobovataas well aspredictedphysical and chemical properties, potential peptide, subcellular localization, and phylogenetic. The diversity was noted in the physical and chemical properties of three partial polyprenol reductase genes. The values of chloroplast were relatively high, showed that chloroplast transit peptide occurred in mangrove polyprenol reductase. The target peptide value of mitochondria varied from 0.088 to 0.198 indicated it was possible to be present. These results suggested the importance of understanding the diversity of physicochemical properties of the different amino acids in polyprenol reductase. The subcellular localization of two partial genes located in the plasma membrane. To confirm the homology among the polyprenol reductase in the database, a dendrogram was drawn. The phylogenetic tree depicts that there are three clusters, the partial genes of K. obovata joined the largest one: C23157 was close to Ricinus communis polyprenol reductase. Whereas, C23901 and C24171 were grouped with Ipomoea nil polyprenol reductase, suggested that these polyprenol reductase genes form distinct separation into tropical habitat plants.

  9. The impact of use of Glutamine on patients with head and neck tumors in radiotherapy and chemotherapy treatment

    International Nuclear Information System (INIS)

    Boligon, Caroline Schardong; Huth, Adriane

    2011-01-01

    Introduction: patients with head and neck neoplasia usually show malnutrition or a nutritional risk, because of common symptoms like: dysphagia, odynophagia and xerostomia. Objective: this study aimed to verify the impact of using amino glutamine in patients with head and neck neoplasia and under radiotherapy and chemotherapy treatment concomitantly. Methods: the research was quantitative, cross-sectional, descriptive and exploratory. The data was collected from nutritional evaluation, and patients chart consultation. The patients were divided in a control group (without use of glutamine) and a test group (with use of glutamine). 16 patients, 13 of which were men and three were women, participated in the research. Results: The control group presented mucositis grades I to IV while patients who used the amino glutamine showed mucositis grades I to II only. It could be observed that the Nutritional Risk Index decreased, which represents higher nutritional risk in patients from the control group only. In patients who used glutamine, this decrease was not significant. Conclusion: these results suggest that the use of glutamine in patients with head and neck tumors and under antineoplastic therapy helps to maintain their nutritional stage and to prevent mucositis throughout their treatment, mainly grades III and IV, which prevents adequate and regular eating and nourishment. (author)

  10. Reduced brain glutamine in female varsity rugby athletes after concussion and in non-concussed athletes after a season of play.

    Science.gov (United States)

    Schranz, Amy L; Manning, Kathryn Y; Dekaban, Gregory A; Fischer, Lisa; Jevremovic, Tatiana; Blackney, Kevin; Barreira, Christy; Doherty, Timothy J; Fraser, Douglas D; Brown, Arthur; Holmes, Jeff; Menon, Ravi S; Bartha, Robert

    2018-04-01

    The purpose of this study was to use non-invasive proton magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI) to monitor changes in prefrontal white matter metabolite levels and tissue microstructure in female rugby players with and without concussion (ages 18-23, n = 64). Evaluations including clinical tests and 3 T MRI were performed at the beginning of a season (in-season) and followed up at the end of the season (off-season). Concussed athletes were additionally evaluated 24-72 hr (n = 14), three months (n = 11), and six months (n = 8) post-concussion. Reduced glutamine at 24-72 hr and three months post-concussion, and reduced glutamine/creatine at three months post-concussion were observed. In non-concussed athletes (n = 46) both glutamine and glutamine/creatine were lower in the off-season compared to in-season. Within the MRS voxel, an increase in fractional anisotropy (FA) and decrease in radial diffusivity (RD) were also observed in the non-concussed athletes, and correlated with changes in glutamine and glutamine/creatine. Decreases in glutamine and glutamine/creatine suggest reduced oxidative metabolism. Changes in FA and RD may indicate neuroinflammation or re-myelination. The observed changes did not correlate with clinical test scores suggesting these imaging metrics may be more sensitive to brain injury and could aid in assessing recovery of brain injury from concussion. © 2017 Wiley Periodicals, Inc.

  11. Supplementation with a combination of beta-hydroxy-beta-methylbutyrate (HMB), arginine, and glutamine is safe and could improve hematological parameters.

    Science.gov (United States)

    Rathmacher, J A; Nissen, S; Panton, L; Clark, R H; Eubanks May, P; Barber, A E; D'Olimpio, J; Abumrad, N N

    2004-01-01

    Combining the amino acids arginine and glutamine with the leucine metabolite beta-hydroxy-beta-methylbutyrate (HMB) has been shown to reverse lean tissue loss in cancer and acquired immunodeficiency syndrome (AIDS) patients. Although each of these nutrients has been shown to be safe, the safety of this mixture has not been reported. Three double-blind studies examined the safety of the combination of HMB, arginine and glutamine on blood chemistries, hematology, emotional profile, and adverse events. Study 1 was conducted in healthy adult males (n = 34), study 2 was in HIV patients with AIDS-associated weight loss (n = 43), and study 3 was in cancer patients with wasting (n = 32). Volunteers were assigned to either a placebo or a mixture of 3 g HMB, 14 g arginine, and 14 g glutamine per day. Across the 3 studies, HMB, arginine, and glutamine supplementation was not associated with any adverse indicators of health. The only significant changes noted were positive indicators of health status. HMB, arginine, and glutamine supplementation was associated with an improvement in emotional profile (p = .05), a decreased feeling of weakness (p = .03), and increased red blood cells, hemoglobin, hematocrit, lymphocytes, and eosinophils (p HMB, arginine, and glutamine supplementation, which was possibly caused by the additional nitrogen consumed or to the fact that ureagenesis is influenced by arginine and glutamine supplementation. These results show that HMB, arginine, and glutamine can be safely used to treat muscle wasting associated with AIDS and cancer.

  12. The effect of glutamine infusion on the inflammatory response and HSP70 during human experimental endotoxaemia

    DEFF Research Database (Denmark)

    Andreasen, Anne Sofie; Pedersen-Skovsgaard, Theis; Mortensen, Ole Hartvig

    2009-01-01

    was associated with alterations in white blood cell and differential counts, tumour necrosis factor-alpha, IL-6, temperature and heart rate, but glutamine affected neither the endotoxin-induced change in these variables nor the expression of HSP70 in BMNCs. CONCLUSIONS: Endotoxin reduced plasma glutamine...

  13. Use of 13N in studies of fixation of dinitrogen and assimilation of ammonium by cyanobacteria

    International Nuclear Information System (INIS)

    Meeks, J.C.; Wolk, C.P.; Thomas, J.; Austin, S.M.; Galonsky, A.; Michigan State Univ., East Lansing

    1978-01-01

    13 N (tsub(1/2)=10min) has been used to identify the initial products of assimilation of N 2 and NH 4 + by intact filaments of a number of cyanobacteria and by heterocysts isolated from Anabaena cylindrica. Ammonium, the amide nitrogen of glutamine, and the α-amino nitrogen of glutamate, in that order, were the first observed products of fixation of [ 13 N]N 2 . Amide-labelled glutamine was the initial product of metabolism of 13 NH 4 + by A. cylindrica grown with either NH 4 + or N 2 as the nitrogen source. Glutamate was the second major product of 13 NH 4 + assimilation. Isolated heterocysts form [ 13 N]glutamine but not [ 13 N]glutamate from [ 13 N]N 2 or 13 NH 4 + . Formation of [ 13 N]glutamine from [ 13 N]N 2 was inhibited by acetylene, indicating metabolic coupling of the activity of glutamine synthetase to that of nitrogenase in these cells. A diffusible substance produced by heterocysts inhibits nearby cells of the same filament from differentiating into heterocysts. Glutamine (or a derivative of glutamine) may be involved in inhibiting differentiation of vegetative cells. (author)

  14. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

    2003-10-21

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  15. Effects of Glutamine on Gastric Emptying of Low- and High-Nutrient Drinks in Healthy Young Subjects—Impact on Glycaemia

    Directory of Open Access Journals (Sweden)

    Yang T. Du

    2018-06-01

    Full Text Available Glutamine is a potent stimulus for the release of glucagon-like peptide-1, which increases postprandial insulin and slows gastric emptying (GE. We determined the effects of glutamine on GE of, and glycaemic responses to, low- and high-nutrient drinks in eight healthy males (mean age 21.6 ± 0.7 years and BMI 22.9 ± 0.7 kg/m2. Participants were studied on four occasions on which they consumed either a low-nutrient (beef soup; 18 kcal or high-nutrient (75 g dextrose; 255 kcal drink, each with or without 30 g of glutamine (120 kcal, in a randomised, crossover design. GE (2D ultrasound, blood glucose and plasma insulin concentrations were measured concurrently. Glutamine slowed GE (half emptying time (T50 of both low- (45 ± 3 min vs. 26 ± 2 min, p < 0.001, and high-nutrient, (100 ± 5 min vs. 77 ± 5 min, p = 0.03 drinks, however, there was no effect on GE of the high nutrient drinks when expressed as kcal/min (3.39 ± 0.21 kcal/min vs. 3.81 ± 0.20 kcal/min, p = 0.25. There was no change in blood glucose after the low-nutrient drinks with or without glutamine, despite a slight increase in plasma insulin with glutamine (p = 0.007. The rise in blood glucose following the high-nutrient drink (p = 0.0001 was attenuated during the first 60 min by glutamine (p = 0.007. We conclude that in healthy subjects, glutamine slows GE of both low- and high-nutrient drinks comparably and attenuates the rise in blood glucose after the high-nutrient glucose drink.

  16. Perioperative Alanyl-Glutamine-Supplemented Parenteral Nutrition in Chronic Radiation Enteritis Patients With Surgical Intestinal Obstruction: A Prospective, Randomized, Controlled Study.

    Science.gov (United States)

    Yao, Danhua; Zheng, Lei; Wang, Jian; Guo, Mingxiao; Yin, Jianyi; Li, Yousheng

    2016-04-01

    A prospective, randomized, controlled study was performed to evaluate the effects of perioperative alanyl-glutamine-supplemented parenteral nutrition (PN) support on the immunologic function, intestinal permeability, and nutrition status of surgical patients with chronic radiation enteritis (CRE)-induced intestinal obstruction. Patients who received 0.4 g/kg/d alanyl-glutamine and isonitrogenous PN were assigned to an alanyl-glutamine-supplemented PN (Gln-PN) group and a control group, respectively. Serum levels of alanine aminotransferase and glutamine, body fat mass (FM), immunologic function, and intestinal permeability were measured before and after surgery. Serum glutamine levels of the Gln-PN group significantly exceeded that of the control group (P nutrition state and intestinal motility of surgical patients with CRE-induced intestinal obstruction. © 2015 American Society for Parenteral and Enteral Nutrition.

  17. Effects of L-glutamine supplementation on the myenteric neurons from the duodenum and cecum of diabetic rats

    Directory of Open Access Journals (Sweden)

    Jacqueline Nelisis Zanoni

    2011-03-01

    Full Text Available CONTEXT: Peripheral neuropathy is one of the chronic complications of diabetes mellitus and is directly related to gastrointestinal consequences of the disease. Myenteric neurons are affected in some pathological conditions such as diabetic neuropathy. The imbalance between cellular antioxidants and free radicals, leading to an increase in oxidative stress, is considered one of the main factors responsible for neuronal damages in diabetes. Drugs that reduce the oxidative stress may play a significant role in the treatment of neurological complications of diabetes mellitus. OBJECTIVE: To evaluate the effect of L-glutamine supplementation on the myenteric neurons from the cecum and duodenum of Wistar rats with streptozotocin-induced diabetes mellitus. METHODS: The animals were divided in four groups (n = 5: non-treated normoglycemics, normoglycemics treated with L-glutamine, non-treated diabetics and diabetics treated with L-glutamine from the 4th day of diabetes induction on. The amino acid L-glutamine was added to their diet at 1%. Giemsa's technique was employed to stain the myenteric neurons. We determined the cell body area of 500 neurons in each group studied. The quantitative analysis was performed by sampling in an area of 16.6 mm² in the cecum and 3.6 mm² in the duodenum of each animal. RESULTS: After the supplementation with L-glutamine in the duodenum, we observed a preservation of neuronal density in groups normoglycemic and diabetic (P<0.05. We also observed a preservation of the cell bodies area in diabetic animals (group treated with L-glutamine (P<0.05. In the cecum, that preservation was not evident. CONCLUSION: Supplementation with L-glutamine (1% promoted a neuroprotective effect on the myenteric neurons from the duodenum of rats, both in terms of natural aging and of diabetes mellitus.

  18. Rapid approach for cloning bacterial single-genes directly from soils ...

    African Journals Online (AJOL)

    Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of ...

  19. Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies

    Science.gov (United States)

    Gonsalves, Wilson I.; Hitosugi, Taro; Ghosh, Toshi; Jevremovic, Dragan; Petterson, Xuan-Mai; Wellik, Linda; Kumar, Shaji K.; Nair, K. Sreekumaran

    2018-01-01

    The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention. PMID:29321378

  20. Biochemical, Physiological and Transcriptomic Comparison between Burley and Flue-Cured Tobacco Seedlings in Relation to Carbohydrates and Nitrate Content

    Directory of Open Access Journals (Sweden)

    Yafei Li

    2017-12-01

    Full Text Available Burley tobacco is a genotype of chloroplast-deficient mutant with accumulates high levels of tobacco-specific nitrosamines (TSNAs which would induce malignant tumors in animals. Nitrate is a principle precursor of tobacco-specific nitrosamines. Nitrate content in burley tobacco was significantly higher than that in flue-cured tobacco. The present study investigated differences between the two tobacco types to explore the mechanisms of nitrate accumulation in burley tobacco. transcripts (3079 related to the nitrogen and carbon metabolism were observed. Expression of genes involved in carbon fixation, glucose and starch biosynthesis, nitrate translocation and assimilation were significantly low in burley tobacco than flue-cured tobacco. Being relative to flue-cured tobacco, burley tobacco was significantly lower at total nitrogen and carbohydrate content, nitrate reductase and glutamine synthetase activities, chlorophyll content and photosynthetic rate (Pn, but higher nitrate content. Burley tobacco required six-fold more nitrogen fertilizers than flue-cured tobacco, but both tobaccos had a similar leaf biomass. Reduced chlorophyll content and photosynthetic rate (Pn might result in low carbohydrate formation, and low capacity of nitrogen assimilation and translocation might lead to nitrate accumulation in burley tobacco.

  1. Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

    Science.gov (United States)

    Ghoshroy, Sohini; Robertson, Deborah L

    2015-01-01

    Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

  2. Effects of polyamine biosynthesis inhibitors on S-adenosylmethionine synthetase and S-adenosylmethionine decarboxylase activities in carrot cell cultures

    Science.gov (United States)

    S.C. Minocha; R. Minocha; A. Komamine

    1991-01-01

    Changes in the activites of S-adcnosylmethionine (SAM) synthetase (methionine adenosyltransferase, EC 2.5.1.6.) and SAM decarboxylase (EC 4.1.1.50) were studied in carrot (Daucus carota) cell cultures in response to 2,4-dichlorophenoxyacetic acid (2,4-D) and several inhibitors of polyamine biosynthesis. Activity of SAM synthetase increased...

  3. Structural Analysis of the Active Site Geometry of N5-Carboxyaminoimidazole Ribonucleotide Synthetase from Escherichia coli

    International Nuclear Information System (INIS)

    Thoden, James B.; Holden, Hazel M.; Firestine, Steven M.

    2008-01-01

    N 5 -Carboxyaminoimidazole ribonucleotide synthetase (N 5 -CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N 5 -CAIR, MgADP, and P i . The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N 5 -CAIR synthetase from Escherichia coli with either MgATP or MgADP/P i bound in the active site cleft. These structures, determined to 1.6-(angstrom) resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu238 bridges them. For the structure of the MgADP/P i complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P i rather than ATP. The bound P i shifts by approximately 3 (angstrom) relative to the γ-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg242 and His244. Since the reaction mechanism of N 5 -CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N 5 -CAIR synthetase.

  4. Synthesis of Biobased Succinonitrile from Glutamic Acid and Glutamine

    NARCIS (Netherlands)

    Lammens, T.M.; Nôtre, Le J.; Franssen, M.C.R.; Scott, E.L.; Sanders, J.P.M.

    2011-01-01

    Succinonitrile is the precursor of 1,4-diaminobutane, which is used for the industrial production of polyamides. This paper describes the synthesis of biobased succinonitrile from glutamic acid and glutamine, amino acids that are abundantly present in many plant proteins. Synthesis of the

  5. Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

    DEFF Research Database (Denmark)

    Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

    1992-01-01

    can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show......Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression....... Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose...

  6. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

    Science.gov (United States)

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-06-18

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. A Tyrosine-Dependent Riboswitch Controls the Expression of a Tyrosyl-tRNA Synthetase from Acidithiobacillus ferrooxidans

    Directory of Open Access Journals (Sweden)

    Paula Bustamante

    2016-06-01

    Full Text Available Expression of aminoacyl-tRNA synthetases is regulated by a variety of mechanisms at the level of transcription or translation. A T-box dependent transcription termination / antitermination riboswitch system that responds to charged / uncharged tRNA regulates expression of aminoacyl tRNA synthetase genes in Gram-positive bacteria. TyrZ, the gene encoding tyrosyl-tRNA synthetase from Acidithiobacillus ferrooxidans, a Gram-negative acidophilic bacterium that participates in bioleaching of minerals, resembles the gene from Bacillus subtilis including the 5´-untranslated region encoding the riboswitch. Transcription of A. ferrooxidans tyrZ is induced by the presence of tyrosine by a mechanism involving antitermination of transcription. This mechanism is probably adapted to the low supply of amino acids of acidic environments of autotrophic bioleaching microorganisms. This work is licensed under a Creative Commons Attribution 4.0 International License.

  8. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui; Deveryshetty, Jaigeeth; Agarwal, Vinayak; Cronan, John E.; Nair, Satish K.

    2017-04-17

    Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

  9. Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue

    International Nuclear Information System (INIS)

    Baba, T.; Muth, J.; Allen, C.M.

    1985-01-01

    The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [ 3 H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cations. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl 2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [ 14 C]isopentenyl pyrophosphate, and MgCl 2 , the labeled polypeptide gave a ratio of 14 C/ 3 H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cations are present

  10. Introduction of a leucine half-zipper engenders multiple high-quality crystals of a recalcitrant tRNA synthetase

    International Nuclear Information System (INIS)

    Guo, Min; Shapiro, Ryan; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-01

    E. coli alanyl-tRNA synthetase is recalcitrant to crystallization. A group of leucine substitutions has transformed the protein. Although Escherichia coli alanyl-tRNA synthetase was among the first tRNA synthetases to be sequenced and extensively studied by functional analysis, it has proved to be recalcitrant to crystallization. This challenge remained even for crystallization of the catalytic fragment. By mutationally introducing three stacked leucines onto the solvent-exposed side of an α-helix, an engineered catalytic fragment of the synthetase was obtained that yielded multiple high-quality crystals and cocrystals with different ligands. The engineered α-helix did not form a leucine zipper that interlocked with the same α-helix from another molecule. Instead, using the created hydrophobic spine, it interacted with other surfaces of the protein as a leucine half-zipper (LHZ) to enhance the crystal lattice interactions. The LHZ made crystal lattice contacts in all crystals of different space groups. These results illustrate the power of introducing an LHZ into helices to facilitate crystallization. The authors propose that the method can be unified with surface-entropy reduction and can be broadly used for protein-surface optimization in crystallization

  11. Lack of protective effect of thromboxane synthetase inhibitor (CGS-13080) on single dose radiated canine intestine

    International Nuclear Information System (INIS)

    Barter, J.F.; Marlow, D.; Kamath, R.K.; Harbert, J.; Torrisi, J.R.; Barnes, W.A.; Potkul, R.K.; Newsome, J.T.; Delgado, G.

    1991-01-01

    The effect of a thromboxane A2 synthetase inhibitor (CGS-13080) on canine intestine was studied using a single dose of radiation, and radioactive microspheres were used to determine resultant blood flow. Thromboxane A2 causes vasospasm and platelet aggregation and may play a dominant role in radiation injury. However, there was no effect on the intestinal blood flow diminution occurring after radiation in this laboratory model using this thromboxane A2 synthetase inhibitor

  12. Physiological hypercortisolemia increases proteolysis, glutamine, and alanine production

    International Nuclear Information System (INIS)

    Darmaun, D.; Matthews, D.E.; Bier, D.M.

    1988-01-01

    Physiological elevations of plasma cortisol levels, as are encountered in stress and severe trauma, were produced in six normal subjects by infusing them with hydrocortisone for 64 h. Amino acid kinetics were measured in the postabsorptive state using three 4-h infusions of L-[1- 13 C]leucine, L-phenyl[ 2 H 5 ]phenylalanine, L-[2- 15 N]glutamine, and L-[1- 13 C]alanine tracers (1) before, (2) at 12 h, and (3) at 60 h of cortisol infusion. Before and throughout the study, the subjects ate a normal diet of adequate protein and energy intake. The cortisol infusion raised plasma cortisol levels significantly from 10 ± 1 to 32 ± 4 μg/dl, leucine flux from 83 ± 3 to 97 ± 3 μmol·kg -1 ·h -1 , and phenylalanine flux from 34 ± 1 to 39 ± 1 (SE) μmol·kg -1 ·h -1 after 12 h of cortisol infusion. These increases were maintained until the cortisol infusion was terminated. These nearly identical 15% increases in two different essential amino acid appearance rates are reflective of increased whole body protein breakdown. Glutamine flux rose by 12 h of cortisol infusion and remained elevated at the same level at 64 h. The increase in flux was primarily due to a 55% increase in glutamine de novo synthesis. Alanine flux increased with acute hypercortisolemia and increased further at 60 h of cortisol infusion, a result primarily of increased alanine de novo synthesis. Insulin, alanine, and lactate plasma levels responded similarly with significant rises between the acute and chronic periods of cortisol infusion. Thus hypercortisolemia increases both protein breakdown and the turnover of important nonessential amino acids for periods of up to 64 h

  13. Effect of carbohydrate supplementation on plasma glutamine during prolonged exercise and recovery

    DEFF Research Database (Denmark)

    Van Hall, Gerrit; Saris, W H; Wagenmakers, A J

    1998-01-01

    . Eight well-trained subjects cycled at an alternating workload of 50 and 80% Wmax until exhaustion (59 to 140 min). During the exercise bout the subjects received either water (control) or a carbohydrate (CHO) drink (83 g CHO x l(-1), 2 ml x kg(-1) per kg body weight every 15 min). Plasma glutamine......%) decreased below the pre-exercise level. The plasma alanine and the total amino acid concentration was still suppressed after 7 h of recovery. In conclusion, carbohydrate supplementation had neither an effect during exercise nor during recovery on the concentration of plasma glutamine or other amino acids...

  14. Enzymatic evidence for the key role of arginine in nitrogen translocation by arbuscular mycorrhiza fungi

    DEFF Research Database (Denmark)

    Cruz, C.; Egsgaard, Helge; Trujillo, C.

    2007-01-01

    - compartment petri dishes and three ammonium levels were supplied to the compartment containing the extraradical mycelium (ERM), but no roots. Time courses of specific enzyme activity were obtained for glutamine synthetase, argininosuccinate synthetase, arginase, and urease in the ERM and AM roots. 15 NH 4 1...... was used to follow the dynamics of nitrogen incorporation into and turnover of Arg. Both the absence of external nitrogen and the presence of L- norvaline, an inhibitor of Arg synthesis, prevented the synthesis of Arg in the ERM and resulted in decreased activity of arginase and urease in the AM root...

  15. Regional tumour glutamine supply affects chromatin and cell identity

    DEFF Research Database (Denmark)

    Højfeldt, Jonas W; Helin, Kristian

    2016-01-01

    Limited perfusion of solid tumours produces a nutrient-deprived tumour core microenvironment. Low glutamine levels in the tumour core are now shown to lead to reduced levels of α-ketoglutarate and decreased histone demethylase activity, thereby promoting a less differentiated and more therapy-res...

  16. Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate.

    Science.gov (United States)

    Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D

    2008-11-25

    o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase

  17. Impaired Hippocampal Glutamate and Glutamine Metabolism in the db/db Mouse Model of Type 2 Diabetes Mellitus

    DEFF Research Database (Denmark)

    Andersen, Jens Velde; Nissen, Jakob Dahl; Christensen, Sofie Kjellerup

    2017-01-01

    Type 2 diabetes mellitus (T2DM) is a risk factor for the development of Alzheimer's disease, and changes in brain energy metabolism have been suggested as a causative mechanism. The aim of this study was to investigate the cerebral metabolism of the important amino acids glutamate and glutamine...... significantly reduced 13C labeling in glutamate, glutamine, GABA, citrate, and aspartate from metabolism of [U-13C]glutamate. Additionally, reduced 13C labeling were observed in GABA, citrate, and aspartate from [U-13C]glutamine metabolism in hippocampal slices of db/db mice when compared to controls. None...

  18. Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

    Science.gov (United States)

    Fiebig, David; Schmelz, Stefan; Zindel, Stephan; Ehret, Vera; Beck, Jan; Ebenig, Aileen; Ehret, Marina; Fröls, Sabrina; Pfeifer, Felicitas; Kolmar, Harald; Fuchsbauer, Hans-Lothar; Scrima, Andrea

    2016-09-23

    Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Cloning and nitrate induction of nitrate reductase mRNA

    OpenAIRE

    Cheng, Chi-Lien; Dewdney, Julia; Kleinhofs, Andris; Goodman, Howard M.

    1986-01-01

    Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase p...

  20. Alanyl-glutamine dipeptide restores the cytoprotective stress proteome of mesothelial cells exposed to peritoneal dialysis fluids.

    Science.gov (United States)

    Kratochwill, Klaus; Boehm, Michael; Herzog, Rebecca; Lichtenauer, Anton Michael; Salzer, Elisabeth; Lechner, Michael; Kuster, Lilian; Bergmeister, Konstantin; Rizzi, Andreas; Mayer, Bernd; Aufricht, Christoph

    2012-03-01

    Exposure of mesothelial cells to peritoneal dialysis fluids (PDF) results in cytoprotective cellular stress responses (CSR) that counteract PDF-induced damage. In this study, we tested the hypothesis that the CSR may be inadequate in relevant models of peritoneal dialysis (PD) due to insufficient levels of glutamine, resulting in increased vulnerability against PDF cytotoxicity. We particularly investigated the role of alanyl-glutamine (Ala-Gln) dipeptide on the cytoprotective PDF stress proteome. Adequacy of CSR was investigated in two human in vitro models (immortalized cell line MeT-5A and mesothelial cells derived from peritoneal effluent of uraemic patients) following exposure to heat-sterilized glucose-based PDF (PD4-Dianeal, Baxter) diluted with medium and, in a comparative proteomics approach, at different levels of glutamine ranging from depletion (0 mM) via physiological (0.7 mM) to pharmacological levels (8 mM administered as Ala-Gln). Despite severe cellular injury, expression of cytoprotective proteins was dampened upon PDF exposure at physiological glutamine levels, indicating an inadequate CSR. Depletion of glutamine aggravated cell injury and further reduced the CSR, whereas addition of Ala-Gln at pharmacological level restored an adequate CSR, decreasing cellular damage in both PDF exposure systems. Ala-Gln specifically stimulated chaperoning activity, and cytoprotective processes were markedly enhanced in the PDF stress proteome. Taken together, this study demonstrates an inadequate CSR of mesothelial cells following PDF exposure associated with low and physiological levels of glutamine, indicating a new and potentially relevant pathomechanism. Supplementation of PDF with pharmacological doses of Ala-Gln restored the cytoprotective stress proteome, resulting in improved resistance of mesothelial cells to exposure to PDF. Future work will study the clinical relevance of CSR-mediated cytoprotection.

  1. Effects of L-glutamine supplementation on maternal and fetal hemodynamics in gestating ewes exposed to alcohol.

    Science.gov (United States)

    Sawant, Onkar B; Ramadoss, Jayanth; Hankins, Gary D; Wu, Guoyao; Washburn, Shannon E

    2014-08-01

    Not much is known about effects of gestational alcohol exposure on maternal and fetal cardiovascular adaptations. This study determined whether maternal binge alcohol exposure and L-glutamine supplementation could affect maternal-fetal hemodynamics and fetal regional brain blood flow during the brain growth spurt period. Pregnant sheep were randomly assigned to one of four groups: saline control, alcohol (1.75-2.5 g/kg body weight), glutamine (100 mg/kg body weight) or alcohol + glutamine. A chronic weekend binge drinking paradigm between gestational days (GD) 99 and 115 was utilized. Fetuses were surgically instrumented on GD 117 ± 1 and studied on GD 120 ± 1. Binge alcohol exposure caused maternal acidemia, hypercapnea, and hypoxemia. Fetuses were acidemic and hypercapnic, but not hypoxemic. Alcohol exposure increased fetal mean arterial pressure, whereas fetal heart rate was unaltered. Alcohol exposure resulted in ~40 % reduction in maternal uterine artery blood flow. Labeled microsphere analyses showed that alcohol induced >2-fold increases in fetal whole brain blood flow. The elevation in fetal brain blood flow was region-specific, particularly affecting the developing cerebellum, brain stem, and olfactory bulb. Maternal L-glutamine supplementation attenuated alcohol-induced maternal hypercapnea, fetal acidemia and increases in fetal brain blood flow. L-Glutamine supplementation did not affect uterine blood flow. Collectively, alcohol exposure alters maternal and fetal acid-base balance, decreases uterine blood flow, and alters fetal regional brain blood flow. Importantly, L-glutamine supplementation mitigates alcohol-induced acid-base imbalances and alterations in fetal regional brain blood flow. Further studies are warranted to elucidate mechanisms responsible for alcohol-induced programming of maternal uterine artery and fetal circulation adaptations in pregnancy.

  2. Effects of honey, glutamine and their combination on canine small ...

    African Journals Online (AJOL)

    olayemitoyin

    and adaptation, evidenced by increased in residual bowel Villi height (27.71µm), ... advantage over glutamine or honey and may be a preferred treatment for short bowel syndrome patients. .... good general health based on complete clinical.

  3. 5α-reductase activity in rat adipose tissue

    International Nuclear Information System (INIS)

    Zyirek, M.; Flood, C.; Longcope, C.

    1987-01-01

    We measured the 5 α-reductase activity in isolated cell preparations of rat adipose tissue using the formation of [ 3 H] dihydrotestosterone from [ 3 H] testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5α-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10 -8 M), when added to the medium, caused a 90% decrease in 5α-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5α-reductase activity in each tissue studied

  4. Characterization of mitochondrial thioredoxin reductase from C. elegans

    International Nuclear Information System (INIS)

    Lacey, Brian M.; Hondal, Robert J.

    2006-01-01

    Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a k cat of 610 min -1 and a K m of 610 μM using E. coli thioredoxin as substrate. The reported k cat is 25% of the k cat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate

  5. Exogenous glutamine increases lipid accumulation in developing seeds of castor bean (Ricinus communis L. cultured in vitro

    Directory of Open Access Journals (Sweden)

    Zhang Yang

    2015-01-01

    Full Text Available This report describes biomass production and compositional changes of developing castor seeds in response to change in the nitrogen resource (glutamine of the medium. During the early developmental period (24-36 days after pollination, oil was found to initially accumulate in the developing seeds. Carbohydrates and oil were inversely related after glutamine provision (35 mM, in the culture medium. [U-14C] sucrose labeling was used to investigate the effect of metabolic fluxes among different storage materials. Addition of glutamine led to a 7% increase of labeling in lipids and an inverse decrease of labeling in carbohydrates. It was postulated that changes in the glutamine concentration in the medium are likely to influence the partitioning of resources between the various storage products, especially carbohydrates and oil. These observations will contribute to a better understanding of assimilate partitioning in developing castor seeds and the development of molecular strategies to improve castor bean seed quality and plant breeding studies.

  6. Dietary sources of aldose reductase inhibitors: prospects for alleviating diabetic complications.

    Science.gov (United States)

    Saraswat, Megha; Muthenna, P; Suryanarayana, P; Petrash, J Mark; Reddy, G Bhanuprakash

    2008-01-01

    Activation of polyol pathway due to increased aldose reductase activity is one of the several mechanisms that have been implicated in the development of various secondary complications of diabetes. Though numerous synthetic aldose reductase inhibitors have been tested, these have not been very successful clinically. Therefore, a number of common plant/ natural products used in Indian culinary have been evaluated for their aldose reductase inhibitory potential in the present study. The aqueous extracts of 22 plant-derived materials were prepared and evaluated for the inhibitory property against rat lens and human recombinant aldose reductase. Specificity of these extracts towards aldose reductase was established by testing their ability to inhibit a closely related enzyme viz, aldehyde reductase. The ex vivo incubation of erythrocytes in high glucose containing medium was used to underscore the significance in terms of prevention of intracellular sorbitol accumulation. Among the 22 dietary sources tested, 10 showed considerable inhibitory potential against both rat lens and human recombinant aldose reductase. Prominent inhibitory property was found in spinach, cumin, fennel, lemon, basil and black pepper with an approximate IC50 of 0.2 mg/mL with an excellent selectivity towards aldose reductase. As against this, 10 to 20 times higher concentrations were required for 50% inhibition of aldehyde reductase. Reduction in the accumulation of intracellular sorbitol by the dietary extracts further substantiated their in vivo efficacy. The findings reported here indicate the scope of adapting life-style modifications in the form of inclusion of certain common sources in the diet for the management of diabetic complications.

  7. Effects of adding different levels of Glutamine to modified Beltsville extender on the survival of frozen rooster semen.

    Science.gov (United States)

    Khiabani, Aytak Bakhshayesh; Moghaddam, Gholamali; Kia, Hossein Daghigh

    2017-09-01

    The aim of the present study was to investigate the effects of l-glutamine on the quality of frozen-thawed rooster semen. Semen samples were collected from eight mature roosters (Ross 308). After initial semen assessments, samples of adequate quality were mixed together and diluted with modified Beltsville extender without l-glutamine (control) and supplemented with 2.5, 5, and 7.5mM l-glutamine. Semen straws were subjected to cryopreservation and evaluated twice at 15-day intervals. After thawing, sperm viability, total and progressive sperm motilities were measured by Eosin-Nigrosine and Computer-Aided Sperm Analysis (CASA), respectively. The results showed that sperm functions decreased on day 30 compared to day 15. The extender supplemented with 5mM glutamine improved (prooster sperm to be frozen for longer. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

    DEFF Research Database (Denmark)

    Roy, Hervé; Zou, S Betty; Bullwinkle, Tammy J

    2011-01-01

    The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF......-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded a-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases....

  9. 5α-reductases in human physiology: an unfolding story.

    Science.gov (United States)

    Traish, Abdulmaged M

    2012-01-01

    5α-reductases are a family of isozymes expressed in a wide host of tissues including the central nervous system (CNS) and play a pivotal role in male sexual differentiation, development and physiology. A comprehensive literature search from 1970 to 2011 was made through PubMed and the relevant information was summarized. 5α reductases convert testosterone, progesterone, deoxycorticosterone, aldosterone and corticosterone into their respective 5α-dihydro-derivatives, which serve as substrates for 3α-hydroxysteroid dehydrogenase enzymes. The latter transforms these 5α-reduced metabolites into a subclass of neuroactive steroid hormones with distinct physiological functions. The neuroactive steroid hormones modulate a multitude of functions in human physiology encompassing regulation of sexual differentiation, neuroprotection, memory enhancement, anxiety, sleep and stress, among others. In addition, 5α -reductase type 3 is also implicated in the N-glycosylation of proteins via formation of dolichol phosphate. The family of 5α-reductases was targeted for drug development to treat pathophysiological conditions, such as benign prostatic hyperplasia and androgenetic alopecia. While the clinical use of 5α-reductase inhibitors was well established, the scope and the magnitude of the adverse side effects of such drugs, especially on the CNS, is still unrecognized due to lack of knowledge of the various physiological functions of this family of enzymes, especially in the CNS. There is an urgent need to better understand the function of 5α-reductases and the role of neuroactive steroids in human physiology in order to minimize the potential adverse side effects of inhibitors targeting 5α-reductases to treat benign prostatic hyperplasia and androgenic alopecia.

  10. Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2.

    Science.gov (United States)

    Keresztessy, Zsolt; Csosz, Eva; Hársfalvi, Jolán; Csomós, Krisztián; Gray, Joe; Lightowlers, Robert N; Lakey, Jeremy H; Balajthy, Zoltán; Fésüs, László

    2006-11-01

    Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q(6), Q(8), and Q(22) are modified by TG2. Kinetic parameters of SnQ1 transamidation (K(M)(app) = 250 microM, k(cat) = 18.3 sec(-1), and k(cat)/K(M)(app) = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

  11. General metabolism of Laribacter hongkongensis: a genome-wide analysis

    Directory of Open Access Journals (Sweden)

    Curreem Shirly O

    2011-04-01

    Full Text Available Abstract Background Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes and pathways of the general metabolism of L. hongkongensis and correlated them with its phenotypic characteristics. Results The L. hongkongensis genome possesses the pentose phosphate and gluconeogenesis pathways and tricarboxylic acid and glyoxylate cycles, but incomplete Embden-Meyerhof-Parnas and Entner-Doudoroff pathways, in agreement with its asaccharolytic phenotype. It contains enzymes for biosynthesis and β-oxidation of saturated fatty acids, biosynthesis of all 20 universal amino acids and selenocysteine, the latter not observed in Neisseria gonorrhoeae, Neisseria meningitidis and Chromobacterium violaceum. The genome contains a variety of dehydrogenases, enabling it to utilize different substrates as electron donors. It encodes three terminal cytochrome oxidases for respiration using oxygen as the electron acceptor under aerobic and microaerophilic conditions and four reductases for respiration with alternative electron acceptors under anaerobic conditions. The presence of complete tetrathionate reductase operon may confer survival advantage in mammalian host in association with diarrhea. The genome contains CDSs for incorporating sulfur and nitrogen by sulfate assimilation, ammonia assimilation and nitrate reduction. The existence of both glutamate dehydrogenase and glutamine synthetase/glutamate synthase pathways suggests an importance of ammonia metabolism in the living environments that it may encounter. Conclusions The L. hongkongensis genome possesses a variety of genes and pathways for carbohydrate, amino acid and lipid metabolism, respiratory chain and sulfur and nitrogen metabolism. These allow the bacterium to utilize various substrates for energy production and survive in different environmental niches.

  12. Combined Effects of Tauroursodeoxycholic Acid and Glutamine on Bacterial Translocation in Obstructive Jaundiced Rats

    Directory of Open Access Journals (Sweden)

    Ahmet Rahmi Hatipoğlu

    2013-12-01

    Full Text Available Background: Bacterial Translocation is believed to be an important factor on mortality and morbidity in Obstructive Jaundiced. Aims: We investigated the probable or estimated positive effects of tauroursodeoxycholic acid, which has antibacterial and regulatory effects on intestinal flora, together with glutamine on BT in an experimental obstructive jaundiced rat model. Study Design: Animal experimentation. Methods: Forty adult, male, Sprague Dawley rats were used in this study. Animals were randomised and divided into five groups of eight each: sham (Sh; control (common bile duct ligation, CBDL; and supplementation groups administered tauroursodeoxycholic acid (CBDL+T, glutamine (CBDL+G, or tauroursodeoxycholic acid plus glutamine (CBDL+TG. Blood and liver, spleen, MLN, and ileal samples were taken via laparotomy under sterile conditions for investigation of bacterial translocation and intestinal mucosal integrity and hepatic function tests on the tenth postoperative day. Results: There were statistically significant differences in BT rates in all samples except the spleen of the CBDL+TG group compared with the CBDL group (p=0.041, p=0.026, and p=0.041, respectively. Conclusion: It is essential to protect hepatic functions besides maintaining intestinal mucosal integrity in the active struggle against BT occurring in obstructive jaundice. The positive effect on intestinal mucosal integrity can be increased if glutamine is used with tauroursodeoxycholic acid, which also has hepatoprotective and immunomodulatory features.

  13. Some amino acids levels: glutamine,glutamate, and homocysteine, in plasma of children with chronic kidney disease.

    Science.gov (United States)

    Fadel, Fatina I; Elshamaa, Manal F; Essam, Rascha G; Elghoroury, Eman A; El-Saeed, Gamila S M; El-Toukhy, Safinaz E; Ibrahim, Mona Hamed

    2014-03-01

    The high prevalence of protein-energy malnutrition is a critical issue for patients with chronic kidney disease (CKD). Serum albumin is the most commonly used nutritional marker. Another index is plasma amino acid (AA) profile. Of these, the plasma levels of glutamine, glutamate and homocysteine, correlate well with nutritional status. We measured some plasma AAs in children with different stages CKD to provide information in monitoring the therapeutic strategy, particularly in AA supplementary therapy or protein restriction. Three amino acids were evaluated along with albumin and high sensitivity C-reactive protein (hs-CRP) in 30 patients with advanced CKD stages 4 and 5. They were divided into two groups undergoing conservative treatment (CT) (n=15) or hemodialysis (HD) (n=15). An additional group of patients with nephrotic syndrome [CKD stage 2] was also studied to assess the alterations of plasma free amino acids with the early stage of CKD. Another 30 age- and sex-matched healthy children served as controls. A significant increase in plasma concentration of amino acid glutamine was observed in children with advanced CKD stages 4 and 5 when compared with controls (P=0.02).Plasma glutamine level was significantly higher in ESRD children on HD than in children with nephrotic syndrome (P=0.02). We did not find a significant difference between HD children and CT children as regard to glutamine level. Notable differences were in the plasma homocysteine level detected in the CKD groups patients, which was greater than that in controls (P=0.0001). Plasma homocysteine level was significantly higher in children on HD than in children with nephrotic syndrome (P=0.01). A significant differences was observed in hs-CRP levels between the CKD groups and the controls (P=0.04). Albumin levels were lower in CKD groups than in controls (p=0.01). Glutamine showed significant positive correlations with blood urea level (r=0.84, P=0.002) and blood ammonia level (r=0.72, P=0

  14. Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results  The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  15. Increased hepatic glycogen synthetase and decreased phosphorylase in trained rats

    DEFF Research Database (Denmark)

    Galbo, H; Saugmann, P; Richter, Erik

    1979-01-01

    Rats were either physically trained by a 12 wk swimming program or were freely eating or weight matched, sedentary controls. Trained rats had a higher relative liver weight and total hepatic glycogen synthetase (EC 2.4.1.11) activity and a lower phosphorylase (EC 2.4.1.1) activity than the other...

  16. Radioprotective effect of cysteamine in glutathione synthetase-deficient cells

    International Nuclear Information System (INIS)

    Deschavanne, P.J.; Debieu, D.; Malaise, E.P.; Midander, J.; Revesz, L.

    1986-01-01

    The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect. (author)

  17. Quantitative analysis of 15N labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives

    Science.gov (United States)

    The enteral metabolism of glutamine and citrulline are intertwined because, while glutamine is one of the main fuel sources for the enterocyte, citrulline is one of its products. It has been shown that the administration of 15N labeled glutamine results in the incorporation of the 15N label into cit...

  18. Quantitative analysis of 15N-labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives.

    Science.gov (United States)

    Marini, Juan C

    2011-05-15

    The enteral metabolisms of glutamine and citrulline are intertwined because, while glutamine is one of the main fuel sources for the enterocyte, citrulline is one of its products. It has been shown that the administration of (15)N-labeled glutamine results in the incorporation of the (15)N label into citrulline, but it is not clear which of the three nitrogen groups of citrulline is actually labeled. To determine the (15)N-enrichment of the positional isomers of glutamine and citrulline, a rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed. The amino acids were analyzed as their dansyl derivatives. The product ion resulting from the loss of NH(3) from the omega carbon allows for the determination of the enrichment of the ureido (citrulline) or amido groups (glutamine). The protonated pyrrolidine (citrulline) or 5-oxopyrrolidine (glutamine) product ion contains the 2-N (amino group) and is used to determine its enrichment. The method described showed no ion suppression and a wide dynamic range ranging from 1.3 picomoles to 2 nanomoles for citrulline. Background samples and standards resulted in enrichments not different from those theoretically expected. The enrichment curves for the different glutamine and citrulline isotopomers were linear (R(2)  > 0.998) over the range of enrichments studied. The method developed provides an additional insight into the metabolism of glutamine and citrulline tracing the precursor-product relationship between these two amino acids. Copyright © 2011 John Wiley & Sons, Ltd.

  19. Development of an Improved Mammalian Overexpression Method for Human CD62L

    Science.gov (United States)

    Brown, Haley A.; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D.

    2014-01-01

    We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402

  20. Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

    Science.gov (United States)

    Sharma, Arvind; Sharma, Amit

    2015-02-01

    The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

  1. Seryl-tRNA Synthetases in Translation and Beyond

    Directory of Open Access Journals (Sweden)

    Marko Močibob

    2016-06-01

    Full Text Available For a long time seryl-tRNA synthetases (SerRSs stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS, exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases. These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place

  2. Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases

    DEFF Research Database (Denmark)

    Knudsen, Michael; Søndergaard, Dan Ariel; Tofting-Olesen, Claus

    2016-01-01

    Motivation: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g.~antibiotics. There ...

  3. Initial organic products of fixation of [13N]dinitrogen by root nodules of soybean (Glycine max)

    International Nuclear Information System (INIS)

    Meeks, J.C.; Wolk, C.P.; Schilling, N.; Shaffer, P.W.; Avissar, Y.; Chien, W.S.

    1978-01-01

    When detached soybean Glycine max (L.) Merr. cv. Hark, nodules assimilate ( 13 N)N 2 , the initial organic product of fixation is glutamine; glutamate becomes more highly radioactive than glutamine within 1 minute; 13 N in alanine becomes detectable at 1 minute of fixation and increases rapidly between 1 and 2 minutes. After 15 minutes of fixation, the major 13 N-labeled organic products in both detached and attached nodules are glutamate and alanine, plus, in the case of attached nodules, an unidentified substance, whereas ( 13 N)glutamine comprises only a small fraction of organic 13 N, and very little 13 N is detected in asparagine. The fixation of ( 13 N)N 2 into organic products was inhibited more than 99 percent by C 2 H 2 (10 percent, v/v). The results support the idea that the glutamine synthetase-glutamate synthase pathway is the primary route for assimilation of fixed nitrogen in soybean nodules

  4. Glutamine-Elicited Secretion of Glucagon-Like Peptide 1 Is Governed by an Activated Glutamate Dehydrogenase.

    Science.gov (United States)

    Andersson, Lotta E; Shcherbina, Liliya; Al-Majdoub, Mahmoud; Vishnu, Neelanjan; Arroyo, Claudia Balderas; Aste Carrara, Jonathan; Wollheim, Claes B; Fex, Malin; Mulder, Hindrik; Wierup, Nils; Spégel, Peter

    2018-03-01

    Glucagon-like peptide 1 (GLP-1), secreted from intestinal L cells, glucose dependently stimulates insulin secretion from β-cells. This glucose dependence prevents hypoglycemia, rendering GLP-1 analogs a useful and safe treatment modality in type 2 diabetes. Although the amino acid glutamine is a potent elicitor of GLP-1 secretion, the responsible mechanism remains unclear. We investigated how GLP-1 secretion is metabolically coupled in L cells (GLUTag) and in vivo in mice using the insulin-secreting cell line INS-1 832/13 as reference. A membrane-permeable glutamate analog (dimethylglutamate [DMG]), acting downstream of electrogenic transporters, elicited similar alterations in metabolism as glutamine in both cell lines. Both DMG and glutamine alone elicited GLP-1 secretion in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH) was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG. In conclusion, our results suggest that nonelectrogenic nutrient uptake and metabolism play an important role in L cell stimulus-secretion coupling. Metabolism of glutamine and related analogs by GDH in the L cell may explain why GLP-1 secretion, but not that of insulin, is activated by these secretagogues in vivo. © 2017 by the American Diabetes Association.

  5. Antioxidant defence of L-glutamine on mitochondrial function in ...

    African Journals Online (AJOL)

    Myocardial infarction is a major public health concern and the leading cause of death all over the world. A better understanding of the processes involved in myocardial infarction has stimulated the search for biomolecules, which could limit the myocardial injury. We determined the protective activity of L-glutamine on ...

  6. Contribution of extracellular glutamine as an anaplerotic substrate to neuronal metabolism: a re-evaluation by multinuclear NMR spectroscopy in primary cultured neurons.

    Science.gov (United States)

    Shokati, Touraj; Zwingmann, Claudia; Leibfritz, Dieter

    2005-10-01

    Multinuclear NMR spectroscopy is used to investigate the effect of glutamine on neuronal glucose metabolism. Primary neurons were incubated with [1-(13C)]glucose in the absence or presence of glutamine (2 mM) and/or NH4Cl (5 mM). After ammonia-treatment, the concentrations of high-energy phosphates decreased up to 84% of control, which was aggravated in glutamine-containing medium (up to 42% of control). These effects could not be attributed to changes in mitochondrial glucose oxidation. Withdrawal of glutamine decreased amino acid concentrations, e.g. of glutamate to 53%, but also considerably lessened the 13C enrichment in [4-(13C)]glutamate to 8.3% of control, and decreased the 13C-enrichment in acetyl-CoA entering the Krebs cycle (P neuronal glutamate stores, glutamate formation is mainly attributed to its de novo synthesis from glucose. Furthermore, mitochondrial glucose metabolism strongly depends on the supply of carbons from glutamine, indicating that exogenous glutamine is a well-suited substrate to replenish neuronal Krebs cycle intermediates.

  7. Crystallization and diffraction analysis of thioredoxin reductase from Streptomyces coelicolor

    International Nuclear Information System (INIS)

    Koháryová, Michaela; Brynda, Jiří; Řezáčová, Pavlína; Kollárová, Marta

    2011-01-01

    Thioredoxin reductase from S. coelicolor was crystallized and diffraction data were collected to 2.4 Å resolution. Thioredoxin reductases are homodimeric flavoenzymes that catalyze the transfer of electrons from NADPH to oxidized thioredoxin substrate. Bacterial thioredoxin reductases represent a promising target for the development of new antibiotics. Recombinant thioredoxin reductase TrxB from Streptomyces coelicolor was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from cryocooled crystals to 2.4 Å resolution using a synchrotron-radiation source. The crystals belonged to the primitive monoclinic space group P2 1 , with unit-cell parameters a = 82.9, b = 60.6, c = 135.4 Å, α = γ = 90.0, β = 96.5°

  8. Lymphocyte Glucose and Glutamine Metabolism as Targets of the Anti-Inflammatory and Immunomodulatory Effects of Exercise

    Directory of Open Access Journals (Sweden)

    Frederick Wasinski

    2014-01-01

    Full Text Available Glucose and glutamine are important energetic and biosynthetic nutrients for T and B lymphocytes. These cells consume both nutrients at high rates in a function-dependent manner. In other words, the pathways that control lymphocyte function and survival directly control the glucose and glutamine metabolic pathways. Therefore, lymphocytes in different functional states reprogram their glucose and glutamine metabolism to balance their requirement for ATP and macromolecule production. The tight association between metabolism and function in these cells was suggested to introduce the possibility of several pathologies resulting from the inability of lymphocytes to meet their nutrient demands under a given condition. In fact, disruptions in lymphocyte metabolism and function have been observed in different inflammatory, metabolic, and autoimmune pathologies. Regular physical exercise and physical activity offer protection against several chronic pathologies, and this benefit has been associated with the anti-inflammatory and immunomodulatory effects of exercise/physical activity. Chronic exercise induces changes in lymphocyte functionality and substrate metabolism. In the present review, we discuss whether the beneficial effects of exercise on lymphocyte function in health and disease are associated with modulation of the glucose and glutamine metabolic pathways.

  9. Light Sensitivity of Lactococcus lactis Thioredoxin Reductase

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas

    The thioredoxin system has evolved in all kingdoms of life acting as a key antioxidant system in the defense against oxidative stress. The thioredoxin system utilizes reducing equivalents from NADPH to reduce protein disulfide targets. The reducing equivalents are shuttled via a flavin and redox...... active dithiol motif in thioredoxin reductase (TrxR) to reduce the small ubiquitous thioredoxin (Trx). Trx in turn regulates the protein dithiol/disulfide balance by reduction of protein disulfide targets in e.g. ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductase. The glutathione......, thus expected to rely mainly on the Trx system for thiol-disulfide control. L. lactis is an important industrial microorganism used as starter culture in the dairy production of cheese, buttermilk etc. and known to be sensitive to oxidative stress. The L. lactis TrxR (LlTrxR) is a homodimeric...

  10. Glutamine-enriched enteral nutrition in very low-birth-weight infants

    NARCIS (Netherlands)

    van den Berg, Anemone; van Zwol, Annelies; Moll, Henriëtte A.; Fetter, Willem P. F.; van Elburg, Ruurd M.

    2007-01-01

    Objective: To determine the effect of glutamine-enriched enteral nutrition in very low- birth- weight infants on the incidence of allergic and infectious diseases during the first year of life. Design: Follow- up study. Setting: Tertiary care hospital. Participants: All surviving infants who

  11. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  12. Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

    Science.gov (United States)

    Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

    2002-03-01

    The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

  13. Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM

    DEFF Research Database (Denmark)

    Celebioglu, Hasan Ufuk; Olesen, Sita Vaag; Prehn, Kennie

    2017-01-01

    .2-2.1 fold reduced relative abundance comprised elongation factor G, phosphoglycerate kinase, BipAEFTU family GTP-binding protein, ribonucleoside triphosphate reductase, adenylosuccinate synthetase, 30S ribosomal protein S1, and manganese-dependent inorganic pyrophosphatase. Surface proteome of cellobiose...

  14. The role of biliverdin reductase in colorectal cancer

    International Nuclear Information System (INIS)

    Bauer, M.

    2010-01-01

    In recent years, the effects of biliverdin and bilirubin have been studied extensively, and an inhibitory effect of bile pigments in cancer progression has been proposed. In this study we focused on the effects of biliverdin reductase, the enzyme that converts biliverdin to bilirubin, in colorectal cancer. For in vitro experiments we used a human colorectal carcinoma cell line and transfected it with an expression construct of shRNA specific for biliverdin reductase, to create cells with stable knock-down of enzyme expression. Cell proliferation was analyzed using the CASY model TT cell counting device. Western blot protein analysis was performed to study intracellular signaling cascades. Samples of human colorectal cancer were analyzed using immunohistochemistry. We were able to confirm the antiproliferative effects of bile pigments on cancer cells in vitro. However, this effect was attenuated in biliverdin reductase knock down cells. ERK and Akt activation seen under biliverdin and bilirubin treatment was also reduced in biliverdin reductase deficient cells. Immunohistochemical analysis of tumor samples from patients with colorectal cancer showed elevated biliverdin reductase levels. High enzyme expression was associated with lower overall and disease free patient survival. We conclude that BVR is required for bile pigment mediated effects regarding cancer cell proliferation and modulation of intracellular signaling cascades. The role of BVR overexpression in vivo and its exact influence on cancer progression and patient survival need to be further investigated. (author) [de

  15. Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes

    International Nuclear Information System (INIS)

    Zielke, H.R.; Tildon, J.T.; Landry, M.E.; Max, S.R.

    1990-01-01

    Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP) + 1 microM dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP + DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U- 14 C]glutamate was recovered in the medium as radioactive glutamine, 2-3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate

  16. Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Justin R. Prigge

    2017-06-01

    Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

  17. Kinetics of carbonyl reductase from human brain.

    OpenAIRE

    Bohren, K M; von Wartburg, J P; Wermuth, B

    1987-01-01

    Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. D...

  18. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  19. Oral supplementations with L-glutamine or L-alanyl-L-glutamine do not change metabolic alterations induced by long-term high-fat diet in the B6.129F2/J mouse model of insulin resistance.

    Science.gov (United States)

    Bock, Patricia Martins; Krause, Mauricio; Schroeder, Helena Trevisan; Hahn, Gabriela Fernandes; Takahashi, Hilton Kenji; Schöler, Cinthia Maria; Nicoletti, Graziella; Neto, Luiz Domingos Zavarize; Rodrigues, Maria Inês Lavina; Bruxel, Maciel Alencar; Homem de Bittencourt, Paulo Ivo

    2016-01-01

    In this work, we aimed to investigate the effects of long-term supplementations with L-glutamine or L-alanyl-L-glutamine in the high-fat diet (HFD)-fed B6.129SF2/J mouse model over insulin sensitivity response and signaling, oxidative stress markers, metabolism and HSP70 expression. Mice were fed in a standard low-fat diet (STA) or a HFD for 20 weeks. In the 21th week, mice from the HFD group were allocated in five groups and supplemented for additional 8 weeks with different amino acids: HFD control group (HFD-Con), HFD + dipeptide L-alanyl-L-glutamine group (HFD-Dip), HFD + L-alanine group (HFD-Ala), HFD + L-glutamine group (HFD-Gln), or the HFD + L-alanine + L-glutamine (in their free forms) group (HFD-Ala + Gln). HFD induced higher body weight, fat pad, fasted glucose, and total cholesterol in comparison with STA group. Amino acid supplementations did not induce any modifications in these parameters. Although insulin tolerance tests indicated insulin resistance in all HFD groups, amino acid supplementations did not improve insulin sensitivity in the present model. There were also no significant differences in the immunocontents of insulin receptor, Akt, and Toll-like receptor-4. Notably, total 70 kDa heat shock protein (HSP72 + HSP73) contents in the liver was markedly increased in HFD-Con group as compared to STA group, which might suggest that insulin resistance is only in the beginning. Apparently, B6.129SF2/J mice are more resistant to the harmful effects of HFD through a mechanism that may include gut adaptation, reducing the absorption of nutrients, including amino acids, which may explain the lack of improvements in our intervention.

  20. Oral glutamine increases circulating glucagon-like peptide 1, glucagon, and insulin concentrations in lean, obese, and type 2 diabetic subjects

    DEFF Research Database (Denmark)

    Greenfield, Jerry R; Farooqi, I Sadaf; Keogh, Julia M

    2008-01-01

    objective was to determine whether glutamine increases circulating GLP-1 and GIP concentrations in vivo and, if so, whether this is associated with an increase in plasma insulin. DESIGN: We recruited 8 healthy normal-weight volunteers (LEAN), 8 obese individuals with type 2 diabetes or impaired glucose...... plasma insulin concentrations. Glutamine stimulated glucagon secretion in all 3 study groups. CONCLUSION: Glutamine effectively increases circulating GLP-1, GIP, and insulin concentrations in vivo and may represent a novel therapeutic approach to stimulating insulin secretion in obesity and type 2......BACKGROUND: Incretin hormones, such as glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play an important role in meal-related insulin secretion. We previously demonstrated that glutamine is a potent stimulus of GLP-1 secretion in vitro. OBJECTIVE: Our...

  1. Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

    Science.gov (United States)

    Alvarez, Maricel; Huygens, Dries; Olivares, Erick; Saavedra, Isabel; Alberdi, Miren; Valenzuela, Eduardo

    2009-08-01

    Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.

  2. Dose intercomparison for 400–500 keV electrons using FWT-60 film and glutamine (spectrophotometric readout) dosimeters

    DEFF Research Database (Denmark)

    Gupta, B. L.; Nilekani, S. R.; Gehringer, P.

    1986-01-01

    This paper describes the dose and the depth dose measurements with FWT-60 film and glutamine (Spectrophotometric readout) dosimeters for 400–500 keV electrons. The glutamine powder was spread uniformly in polyethylene bags and the powder thickness in each bag was 5 mg cm−2. Both techniques show...

  3. A nonribosomal peptide synthetase (Pes1) confers protection against oxidative stress in Aspergillus fumigatus.

    Science.gov (United States)

    Reeves, Emer P; Reiber, Kathrin; Neville, Claire; Scheibner, Olaf; Kavanagh, Kevin; Doyle, Sean

    2006-07-01

    Aspergillus fumigatus is an important human fungal pathogen. The Aspergillus fumigatus genome contains 14 nonribosomal peptide synthetase genes, potentially responsible for generating metabolites that contribute to organismal virulence. Differential expression of the nonribosomal peptide synthetase gene, pes1, in four strains of Aspergillus fumigatus was observed. The pattern of pes1 expression differed from that of a putative siderophore synthetase gene, sidD, and so is unlikely to be involved in iron acquisition. The Pes1 protein (expected molecular mass 698 kDa) was partially purified and identified by immunoreactivity, peptide mass fingerprinting (36% sequence coverage) and MALDI LIFT-TOF/TOF MS (four internal peptides sequenced). A pes1 disruption mutant (delta pes1) of Aspergillus fumigatus strain 293.1 was generated and confirmed by Southern and western analysis, in addition to RT-PCR. The delta pes1 mutant also showed significantly reduced virulence in the Galleria mellonella model system (P < 0.001) and increased sensitivity to oxidative stress (P = 0.002) in culture and during neutrophil-mediated phagocytosis. In addition, the mutant exhibited altered conidial surface morphology and hydrophilicity, compared to Aspergillus fumigatus 293.1. It is concluded that pes1 contributes to improved fungal tolerance against oxidative stress, mediated by the conidial phenotype, during the infection process.

  4. Nitrate reductase activity and its relationship with applied nitrogen in soybean

    International Nuclear Information System (INIS)

    Ge Wenting; Jin Xijun; Ma Chunmei; Dong Shoukun; Gong Zhenping; Zhang Lei

    2011-01-01

    Field experiments were conducted to study the nitrate reductase activity and its relationship to nitrogen by using frame tests (pot without bottom), sand culture and 15 N-urea at transplanting in soybean variety Suinong 14. Results showed that the activity of nitrate reductase in leaf changed as a signal peak curve with the soybean growth, lower in vegetative growth phase, higher in reproductive growth period and reached the peak in blooming period, then decreased gradually. Nitrogen application showed obvious effect on the nitrate reductase activity. The activities of nitrate reductase in leaves followed the order of N 135 > N 90 > N 45 > N 0 in vegetative growth stage, no clear regularity was found during the whole reproductive growth period. The activities of nitrate reductase in leaves were accorded with the order of upper leaves > mid leaves > lower leaves, and it was very significant differences (P 15 N labeling method during beginning seed stage and full seed stage shown that 15 N abundance in various organs at different node position also followed the same order, suggesting that high level of nitrate reductase activity at upper leaves of soybean promoted the assimilation of NO 3 - . (authors)

  5. Natural aminoacyl tRNA synthetase fragment enhances cardiac function after myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Margaret E McCormick

    Full Text Available A naturally-occurring fragment of tyrosyl-tRNA synthetase (TyrRS has been shown in higher eukaryotes to 'moonlight' as a pro-angiogenic cytokine in addition to its primary role in protein translation. Pro-angiogenic cytokines have previously been proposed to be promising therapeutic mechanisms for the treatment of myocardial infarction. Here, we show that systemic delivery of the natural fragment of TyRS, mini-TyrRS, improves heart function in mice after myocardial infarction. This improvement is associated with reduced formation of scar tissue, increased angiogenesis of cardiac capillaries, recruitment of c-kitpos cells and proliferation of myocardial fibroblasts. This work demonstrates that mini-TyrRS has beneficial effects on cardiac repair and regeneration and offers support for the notion that elucidation of the ever expanding repertoire of noncanonical functions of aminoacyl tRNA synthetases offers unique opportunities for development of novel therapeutics.

  6. Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala.

    Directory of Open Access Journals (Sweden)

    Alberto Guevara-Flores

    Full Text Available A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR, thioredoxin-glutathione reductase (TGR, and a putative thioredoxin reductase (TrxR was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

  7. Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

    Directory of Open Access Journals (Sweden)

    Jolanta Marciniak

    2014-01-01

    Full Text Available Soluble nitrate reductase from cucumber roots (Cucumis sativus L. was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR from cotyledon roots and leaves.

  8. Effects of Standard and/or Glutamine Dipeptide and/or Omega-3 ...

    African Journals Online (AJOL)

    Supplemented Parenteral Nutrition on Neutrophil Functions, Interleukin-8 Level and Length of ... Standard TPN and glutamine and lipid emulsion with omega 3 fatty acids were given to colorectal cancer patients and the effects of these to neutrophil ...

  9. Expression and site-directed mutagenesis of human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-05-17

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

  10. Expression and site-directed mutagenesis of human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-01-01

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 → Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by α-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme

  11. Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine

    DEFF Research Database (Denmark)

    Gassler, Nikolaus; Kopitz, Jürgen; Tehrani, Arman

    2004-01-01

    Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology......-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine....

  12. Irradiation temperature effect on glutamine (spectrophotometric readout) dosimeter

    International Nuclear Information System (INIS)

    Gupta, B.L.; Narayan, G.R.; Nilekani, S.R.; Bhat, R.M.

    2001-01-01

    For the dose estimation using glutamine dosimeter, 20 mg L-glutamine powder is dissolved in 10 ml of a solution which contains 2x10 -3 mol dm -3 ferrous ammonium sulphate and 10 -4 mol dm -3 xylenol orange in aerated aqueous 0.033 mol dm -3 sulphuric acid (FX). The plot of absorbance at 549 nm against dose is non-linear, however, the plot of 1/absorbance vs. 1/dose is linear. The slope of this linear plot changes above an absorbance of 0.3 corresponding to a dose of about 15 kGy. The response of the dosimeter is independent of irradiation temperature in the temperature range of 23-30 deg. C within the uncertainty of dose measurement. Below 23 deg. C, the absorbance decreases by 1.23% per deg. sign C decrease in temperature while between 30 and 40 deg. C, the absorbance increases by 0.75% per deg. C increase in temperature. Above 40 deg. C, the absorbance increases by 0.2% per deg. C only. The absorbance is corrected for the irradiation temperature and the dose is read from a calibration graph at 25 deg. C. Either a linear or polynomial relation can be used for the dose calculation

  13. Fatty acid biosynthesis VII. Substrate control of chain-length of products synthesised by rat liver fatty acid synthetase

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

    1970-01-01

    - 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. - 2....... A wide range (C4:0–C18:0) of fatty acids was synthesised and the proportions were modified by substrate concentrations in the same manner as for purified rabbit mammary gland fatty acid synthetase. - 3. The relative amount of radioactivity incorporated from added acetyl-CoA and malonyl-CoA depended...... of long-chain fatty acids was synthesised from carboxylated acetyl-CoA than from added malonyl-CoA. - 5. It is suggested that acetyl-CoA carboxylase may carboxylate acetate bound to fatty acid synthetase....

  14. Holocarboxylase synthetase deficiency pre and post newborn screening

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2016-06-01

    Full Text Available Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

  15. Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2 defects predicts differential effects on aminoacylation

    Directory of Open Access Journals (Sweden)

    Liliya eEuro

    2015-02-01

    Full Text Available The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19 is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations.The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change p.R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

  16. Isotopic studies on structure-function relationships of nucleic acids and enzymes. Three year progress report, May 1972--October 1975

    International Nuclear Information System (INIS)

    Boyer, P.D.

    1975-01-01

    The most important accomplishments and major contributions are tabulated with citations to published work. The more important unpublished contributions deal with the early events in ATP formation by chloroplasts, energy linkage in reaction steps of oxidative phosphorylation, molecular integrity of parental DNA, bound pyrophosphate and 18 O-exchanges by inorganic pyrophosphatase, and glutamine synthetase exchanges and mechanisms. These are being prepared for publication

  17. The Effectiveness of Combined Use of Antioxidant and Glutamine in Abdominal Sepsis

    Directory of Open Access Journals (Sweden)

    V. V. Nazaretyan

    2017-01-01

    Full Text Available Aim of the study: the effectiveness of concomitant use of antioxidant therapy with antioxidant 2-ethyl-6- methyl-3-hydroxypyridine succinate (mexidol and intensive nutritional support with glutamine in patients with abdominal sepsis (AS. Materials and methods. 170 patients with abdominal sepsis (AS involved in the study were separated into two groups. Patients of group 1 (control group, n=70 received basic treatment. Patients from group 2 (n=100 were divided into 2 subgroups. Patients from the subgroup 21 (n=70, in additon to the basic treatment, received intravenously, by drop infusion, mexidol (2000 mg per day and dipeptiven (27.5 g per day, patients from subgroup 22 (n=30 additionally to that received per os glutamine. Survival analysis was carried out according to the Kaplan-Meier method with using of the Cox's F-test and Mantel-Cox test for testing of statistical hypotheses. Results. Treatment outcomes analysis showed that in the basic group 2, mortality was lower than in the control group 1. A statistically significant increase of cumulative part in the survivors was revealed using mexidol and glutamine. Conclusion. Concomitant intravenous administration of medications had positive effects on treatment outcomes. Following on from the analysis results, we may suggest that the pair mexidol + dipeptiven interrupts the cascade of development of abdominal sepsis and contributes to avoiding a critical condition during sepsis.

  18. Evolution of the Maillard Reaction in Glutamine or Arginine-Dextrinomaltose Model Systems

    Directory of Open Access Journals (Sweden)

    Silvia Pastoriza

    2016-12-01

    Full Text Available Enteral formulas are foods designed for medical uses to feed patients who are unable to eat normally. They are prepared by mixing proteins, amino acids, carbohydrates and fats and submitted to sterilization. During thermal treatment, the Maillard reaction takes place through the reaction of animo acids with reducing sugars. Thus, although glutamine and arginine are usually added to improve the nutritional value of enteral formulas, their final concentration may vary. Thus, in the present paper the early, intermediate, and advanced states of the Maillard reaction were studied in model systems by measuring loss of free amino acids through the decrease of fluorescence intensity with o-phtaldialdehyde (OPA, 5-Hydroximethylfurfural (HMF, furfural, glucosylisomaltol, fluorescence, and absorbance at 420 nm. The systems were prepared by mixing glutamine or arginine with dextrinomaltose (similar ingredients to those used in special enteral formula, and heated at 100 °C, 120 °C and 140 °C for 0 to 30 min. The recorded changes in the concentration of furanic compounds was only useful for longer heating times of high temperatures, while absorbance and fluorescence measurements were useful in all the assayed conditions. In addition, easiness and sensitivity of absorbance and fluorescence make them useful techniques that could be implemented as indicators for monitoring the manufacture of special enteral formulas. Glucosylisomaltol is a useful indicator to monitor the manufacture of glutamine-enriched enteral formulas.

  19. Evolution of the Maillard Reaction in Glutamine or Arginine-Dextrinomaltose Model Systems.

    Science.gov (United States)

    Pastoriza, Silvia; Rufián-Henares, José Ángel; García-Villanova, Belén; Guerra-Hernández, Eduardo

    2016-12-07

    Enteral formulas are foods designed for medical uses to feed patients who are unable to eat normally. They are prepared by mixing proteins, amino acids, carbohydrates and fats and submitted to sterilization. During thermal treatment, the Maillard reaction takes place through the reaction of animo acids with reducing sugars. Thus, although glutamine and arginine are usually added to improve the nutritional value of enteral formulas, their final concentration may vary. Thus, in the present paper the early, intermediate, and advanced states of the Maillard reaction were studied in model systems by measuring loss of free amino acids through the decrease of fluorescence intensity with o -phtaldialdehyde (OPA), 5-Hydroximethylfurfural (HMF), furfural, glucosylisomaltol, fluorescence, and absorbance at 420 nm. The systems were prepared by mixing glutamine or arginine with dextrinomaltose (similar ingredients to those used in special enteral formula), and heated at 100 °C, 120 °C and 140 °C for 0 to 30 min. The recorded changes in the concentration of furanic compounds was only useful for longer heating times of high temperatures, while absorbance and fluorescence measurements were useful in all the assayed conditions. In addition, easiness and sensitivity of absorbance and fluorescence make them useful techniques that could be implemented as indicators for monitoring the manufacture of special enteral formulas. Glucosylisomaltol is a useful indicator to monitor the manufacture of glutamine-enriched enteral formulas.

  20. Nutrition Modulation of Cardiotoxicity and Anticancer Efficacy Related to Doxorubicin Chemotherapy by Glutamine and ω-3 Polyunsaturated Fatty Acids

    Science.gov (United States)

    Xue, Hongyu; Ren, Wenhua; Denkinger, Melanie; Schlotzer, Ewald; Wischmeyer, Paul E.

    2015-01-01

    Background Doxorubicin (DOX) has been one of the most effective antitumor agents against a broad spectrum of malignancies. However, DOX-induced cardiotoxicity forms the major cumulative dose-limiting factor. Glutamine and ω-3 polyunsaturated fatty acids (PUFAs) are putatively cardioprotective during various stresses and/or have potential chemosensitizing effects during cancer chemotherapy. Methods Antitumor activity and cardiotoxicity of DOX treatment were evaluated simultaneously in a MatBIII mammary adenocarcinoma tumor-bearing rat model treated with DOX (cumulative dose 12 mg/kg). Single or combined treatment of parenteral glutamine (0.35 g/kg) and ω-3 PUFAs (0.19 g/kg eicosapentaenoic acid and 0.18 g/kg docosahexaenoic acid) was administered every other day, starting 6 days before chemotherapy initiation until the end of study (day 50). Results Glutamine alone significantly prevented DOX-related deterioration of cardiac function, reduced serum cardiac troponin I levels, and diminished cardiac lipid peroxidation while not affecting tumor inhibition kinetics. Single ω-3 PUFA treatment significantly enhanced antitumor activity of DOX associated with intensified tumoral oxidative stress and enhanced tumoral DOX concentration while not potentiating cardiac dysfunction or increasing cardiac oxidative stress. Intriguingly, providing glutamine and ω-3 PUFAs together did not consistently confer a greater benefit; conversely, individual benefits on cardiotoxicity and chemosensitization were mostly attenuated or completely lost when combined. Conclusions Our data demonstrate an interesting differentiality or even dichotomy in the response of tumor and host to single parenteral glutamine and ω-3 PUFA treatments. The intriguing glutamine × ω-3 PUFA interaction observed draws into question the common assumption that there are additive benefits of combinations of nutrients that are beneficial on an individual basis. PMID:25888676

  1. 15N-enrichments of ammonia and glutamine in blood after infusion of 15N-ammonia in chickens fed low or high protein diet

    International Nuclear Information System (INIS)

    Karasawa, Yutaka; Koh, Katsuki

    1985-01-01

    In this experment, the blood ammonia and glutamine amide came from infused ammonia were determined when N-15 labeled ammonium acetate was intraportally infused into the chickens fed 5 or 20 % protein diet. The data obtained indicated that the infused ammonia was taken into blood glutamine amide, and also accumulated in blood as it is, in both dietary groups. 10 to 12 months old White Leghorn male birds were used. The experimental diet was fed once a day for 5 days to the birds weighting about 1.2 kg by 35 g per kg body weight. The experimental diet was consumed within 40 min in all cases. Cardiac and portal catheterization were performed for blood collection and ammonia infusion, respectively. After finishing the infusion, blood samples were taken to analyze the ammonia and glutamine contents and their N-15 enrichment. Statistical difference was not observed in the appearance of N-15 in ammonia and glutamine amide between two dietary groups. The N-15 enrichment in blood ammonia and the amide of plasma glutamine, and the calculated exogenous nitrogen in the ammonia and glutamine amide tended to be more in the 5 % protein diet group than the other. (Kako, I.)

  2. Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

    Science.gov (United States)

    Hadd, Andrew; Perona, John J

    2014-12-19

    We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

  3. Interactions of glutamine dipeptides with sodium dodecyl sulfate in aqueous solution measured by volume, conductivity, and fluorescence spectra

    Energy Technology Data Exchange (ETDEWEB)

    Yan Zhenning, E-mail: yanzzn@zzu.edu.cn [Department of Chemistry, Zhengzhou University, Zhengzhou, Henan 450001 (China); Sun Ximeng; Li Weiwei; Li Yu [Department of Chemistry, Zhengzhou University, Zhengzhou, Henan 450001 (China); Wang Jianji [Department of Chemistry, Henan Normal University, Xinxiang, Henan 453007 (China)

    2011-10-15

    Highlights: > Ion-ion and ion-polar group interactions are dominant interactions. > The SDS addition and temperature increase cause a dehydration effect on dipeptides. > The addition of dipeptide in water decreases the c{sub cmc} of SDS. > Enthalpy-entropy compensation takes place during micellization. > Micelle aggregation number was decreased by addition of glutamine dipeptides. - Abstract: Densities, conductivities, and fluorescence spectra of {l_brace}sodium dodecyl sulfate (SDS) + glutamine dipeptide + water{r_brace} mixtures were measured as a function of temperature. The density data have been utilized to calculate apparent molar volumes, standard partial molar volumes (V{sub 2,{phi}}{sup o}), standard partial molar volumes of transfer from water to aqueous SDS solutions ({Delta}{sub t}V{sup o}), the hydration number, partial molar expansibility (E{sub {phi}}{sup o}), and Hepler's constant of glutamine dipeptides. The critical micellar concentration (c{sub cmc}) and the degree of counterion dissociation of SDS micelles obtained from electrical conductivity data have been estimated at various concentrations of glutamine dipeptide. Thermodynamic parameters of micellization of SDS in aqueous dipeptide solutions have been determined from c{sub cmc} values and an enthalpy-entropy compensation effect was observed for the ternary systems. The pyrene fluorescence spectra were used to study the change of micropolarity produced by the interaction of SDS with glutamine dipeptide, and the aggregation behavior of SDS. The results have been interpreted in terms of solute-solvent interactions and structural changes in the mixed solutions.

  4. Reaction Intermediate Analogues as Bisubstrate Inhibitors of Pantothenate Synthetase

    OpenAIRE

    Xu, Zhixiang; Yin, Wei; Martinelli, Leonardo K.; Evans, Joanna; Chen, Jinglei; Yu, Yang; Wilson, Daniel J.; Mizrahi, Valerie; Qiao, Chunhua; Aldrich, Courtney C.

    2014-01-01

    The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β–alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the in...

  5. Selective 15N labeling and direct observation by NMR of the active-site glutamine of Fe-containing superoxide dismutase

    International Nuclear Information System (INIS)

    Vance, Carrie K.; Kang, Young M.; Miller, Anne-Frances

    1997-01-01

    The glutamine in position 69 is one of only three conserved active-site amino acid differences between Fe- and Mn-containing superoxide dismutases (SODs). We have refined the conditions for extremely selective labeling of the side chains of glutamine with 15N, and thus obtained dramatically simplified spectra, despite the large size of Fe-SOD. The improved resolution afforded by such highly specific labeling permits the use of direct 15N detection to observe and assign Gln 69, even though its distance to the paramagnetic Fe2+ is only 5A. Selective glutamine side-chain labeling is inexpensive and has general utility for large (and paramagnet-containing) proteins

  6. In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

    International Nuclear Information System (INIS)

    Peters, J.

    1977-01-01

    Some experimental work is described showing that near-U.V. irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-U.V., and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex. (U.K.)

  7. In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Peters, J [California Univ., Irvine (USA)

    1977-06-09

    Some experimental work is described showing that near-uv irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-uv, and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex.

  8. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Zhukhlistova, N. E. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2016-01-15

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  9. Effects of Dietary Glutamine Supplementation on the Body Composition and Protein Status of Early-Weaned Mice Inoculated with Mycobacterium bovis Bacillus Calmette-Guerin

    Science.gov (United States)

    Rogero, Marcelo Macedo; Borges, Maria Carolina; de Castro, Inar Alves; Pires, Ivanir S. O.; Borelli, Primavera; Tirapegui, Julio

    2011-01-01

    Glutamine, one of the most abundant amino acids found in maternal milk, favors protein anabolism. Early-weaned babies are deprived of this source of glutamine, in a period during which endogenous biosynthesis may be insufficient for tissue needs in states of metabolic stress, mainly during infections. The objective of this study was to verify the effects of dietary glutamine supplementation on the body composition and visceral protein status of early-weaned mice inoculated with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Mice were weaned early on their 14th day of life and seperated into two groups, one of which was fed a glutamine-free diet (n = 16) and the other a glutamine-supplemented diet (40 g/kg diet) (n = 16). At 21 days of age, some mice were intraperitoneally injected with BCG. Euthanasia was performed at the 28th day of age. BCG inoculation significantly reduced body weight (P < 0.001), lean mass (P = 0.002), water (P = 0.006), protein (P = 0.007) and lipid content (P = 0.001) in the carcass. Dietary glutamine supplementation resulted in a significant increase in serum IGF-1 (P = 0.019) and albumin (P = 0.025) concentration, muscle protein concentration (P = 0.035) and lipid content (P = 0.002) in the carcass. In conclusion, dietary glutamine supplementation had a positive influence on visceral protein status but did not affect body composition in early-weaned mice inoculated with BCG. PMID:22254124

  10. Effects of Dietary Glutamine Supplementation on the Body Composition and Protein Status of Early-Weaned Mice Inoculated with Mycobacterium bovis Bacillus Calmette-Guerin

    Directory of Open Access Journals (Sweden)

    Ivanir S. O. Pires

    2011-08-01

    Full Text Available Glutamine, one of the most abundant amino acids found in maternal milk, favors protein anabolism. Early-weaned babies are deprived of this source of glutamine, in a period during which endogenous biosynthesis may be insufficient for tissue needs in states of metabolic stress, mainly during infections. The objective of this study was to verify the effects of dietary glutamine supplementation on the body composition and visceral protein status of early-weaned mice inoculated with Mycobacterium bovis Bacillus Calmette-Guérin (BCG. Mice were weaned early on their 14th day of life and seperated into two groups, one of which was fed a glutamine-free diet (n = 16 and the other a glutamine-supplemented diet (40 g/kg diet (n = 16. At 21 days of age, some mice were intraperitoneally injected with BCG. Euthanasia was performed at the 28th day of age. BCG inoculation significantly reduced body weight (P < 0.001, lean mass (P = 0.002, water (P = 0.006, protein (P = 0.007 and lipid content (P = 0.001 in the carcass. Dietary glutamine supplementation resulted in a significant increase in serum IGF-1 (P = 0.019 and albumin (P = 0.025 concentration, muscle protein concentration (P = 0.035 and lipid content (P = 0.002 in the carcass. In conclusion, dietary glutamine supplementation had a positive influence on visceral protein status but did not affect body composition in early-weaned mice inoculated with BCG.

  11. Evidence for Tautomerisation of Glutamine in BLUF Blue Light Receptors by Vibrational Spectroscopy and Computational Chemistry

    Science.gov (United States)

    Domratcheva, Tatiana; Hartmann, Elisabeth; Schlichting, Ilme; Kottke, Tilman

    2016-01-01

    BLUF (blue light sensor using flavin) domains regulate the activity of various enzymatic effector domains in bacteria and euglenids. BLUF features a unique photoactivation through restructuring of the hydrogen-bonding network as opposed to a redox reaction or an isomerization of the chromophore. A conserved glutamine residue close to the flavin chromophore plays a central role in the light response, but the underlying modification is still unclear. We labelled this glutamine with 15N in two representative BLUF domains and performed time-resolved infrared double difference spectroscopy. The assignment of the signals was conducted by extensive quantum chemical calculations on large models with 187 atoms reproducing the UV-vis and infrared signatures of BLUF photoactivation. In the dark state, the comparatively low frequency of 1,667 cm−1 is assigned to the glutamine C=O accepting a hydrogen bond from tyrosine. In the light state, the signature of a tautomerised glutamine was extracted with the C=N stretch at ~1,691 cm−1 exhibiting the characteristic strong downshift by 15N labelling. Moreover, an indirect isotope effect on the flavin C4=O stretch was found. We conclude that photoactivation of the BLUF receptor does not only involve a rearrangement of hydrogen bonds but includes a change in covalent bonds of the protein. PMID:26947391

  12. Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.

    Science.gov (United States)

    Hoepfner, Dominic; McNamara, Case W; Lim, Chek Shik; Studer, Christian; Riedl, Ralph; Aust, Thomas; McCormack, Susan L; Plouffe, David M; Meister, Stephan; Schuierer, Sven; Plikat, Uwe; Hartmann, Nicole; Staedtler, Frank; Cotesta, Simona; Schmitt, Esther K; Petersen, Frank; Supek, Frantisek; Glynne, Richard J; Tallarico, John A; Porter, Jeffrey A; Fishman, Mark C; Bodenreider, Christophe; Diagana, Thierry T; Movva, N Rao; Winzeler, Elizabeth A

    2012-06-14

    With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Alleviation of Drought Stress by Nitrogen Application in Brassica campestris ssp. Chinensis L.

    Directory of Open Access Journals (Sweden)

    Xin Xiong

    2018-05-01

    Full Text Available To assess the influence of drought stress on the growth and nitrogen nutrition status of pakchoi (Brassica campestris ssp. Chinensis L. at different nitrogen (N levels, the changes in N accumulation and enzyme activities involved in N assimilation were investigated. The drought was induced by adding polyethylene glycol (PEG under hydroponic culture conditions. Pakchoi seedlings were exposed to a modified nutrient solution with different nitrogen concentration (N1, N2, and N3 represent 2, 9 and 18 mM NaNO3, respectively and osmotic potential (W1, W2 and W3 represent 0, 60 and 120 g·L−1 PEG 6000 in a full factorial, replicated randomized block design. A short time (seven days of drought stress caused a significant decline in plant water content, transpiration rate, shoot biomass and shoot nitrogen concentration. Increasing N availability considerably alleviate drought stress by increasing the content of total free amino acids in the roots, promoting the acceleration of root biomass accumulation, and improving the activities of nitrate reductase (NR; EC 1.7.1.1 and glutamine synthetase (GS; EC 6.3.1.2 which would reduce moisture limitations. The results suggested that pakchoi supplied with relative higher N had better growth performance under drought stress.

  14. Effect of cadmium toxicity on nitrogen metabolism in leaves of Solanum nigrum L. as a newly found cadmium hyperaccumulator

    International Nuclear Information System (INIS)

    Wang Lin; Zhou Qixing; Ding Lingling; Sun Yuebing

    2008-01-01

    Hyperaccumulators are ideal plant species used for phytoremediation of soils contaminated by heavy metals. A full understanding of metal tolerance mechanisms of hyperaccumulators will facilitate enhancing their phytoremediation efficiency. However, how Cd affects N metabolism and which role plays the response of N metabolism to Cd toxicity in the tolerance of hyperaccumulators are still unknown. To clarify these questions, this study investigated the effects of various soil Cd levels on the concentrations of N forms and the activity of key enzymes involved in N metabolism in leaves of the Cd hyperaccumulator, Solanum nigrum L. The results showed that its growth and all N metabolism indicators were normal at low Cd exposure (≤12 mg kg -1 ). At 24 mg Cd kg -1 soil, nitrate assimilation indicators (nitrate concentration and activity of nitrate reductase) were reduced significantly, whereas most ammonia assimilation indicators (ammonium concentration and activity of glutamine synthetase) remained normal. However, when exposed to a higher Cd level (48 mg kg -1 ), growth and most N metabolism indicators were reduced significantly. Therefore, N metabolism in leaves of S. nigrum could be tolerant of Cd toxicity to a certain extent (soil Cd concentration ≤12 mg kg -1 ), and this might be involved in the Cd-tolerance of this Cd-hyperaccumulator

  15. Novel insights into regulation of asparagine synthetase in conifers

    Directory of Open Access Journals (Sweden)

    Javier eCanales

    2012-05-01

    Full Text Available Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4. In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait., PpAS1 and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1 was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed.

  16. The Bacillus subtilis and Bacillus halodurans Aspartyl-tRNA Synthetases Retain Recognition of tRNA(Asn).

    Science.gov (United States)

    Nair, Nilendra; Raff, Hannah; Islam, Mohammed Tarek; Feen, Melanie; Garofalo, Denise M; Sheppard, Kelly

    2016-02-13

    Synthesis of asparaginyl-tRNA (Asn-tRNA(Asn)) in bacteria can be formed either by directly ligating Asn to tRNA(Asn) using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA(Asn) and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA(Gln) formation. Most bacteria are predicted to use only one route for Asn-tRNA(Asn) formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA(Asn) formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to be specific for tRNA(Asp). However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA(Asp) by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

    Science.gov (United States)

    Hoff-Risseti, Caroline; Dörr, Felipe Augusto; Schaker, Patricia Dayane Carvalho; Pinto, Ernani; Werner, Vera Regina; Fiore, Marli Fatima

    2013-01-01

    The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins. PMID:24015317

  18. Cylindrospermopsin and saxitoxin synthetase genes in Cylindrospermopsis raciborskii strains from Brazilian freshwater.

    Directory of Open Access Journals (Sweden)

    Caroline Hoff-Risseti

    Full Text Available The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX, while cylindrospermopsin (CYN, which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.

  19. Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-α in LPS-stimulated monocytes in human whole-blood.

    Science.gov (United States)

    Raspé, C; Czeslick, E; Weimann, A; Schinke, C; Leimert, A; Kellner, P; Simm, A; Bucher, M; Sablotzki, A

    2013-04-01

    To investigate the effects of the commonly-used immunomodulators l-glutamine, l-alanine, and the combination of both l-alanyl-l-glutamine (Dipeptamin(®)) on intracellular expression of IL-6, IL-8, and TNF-α during endotoxemia, lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system were investigated by flow cytometry. Whole blood of twenty-seven healthy volunteers was stimulated with LPS and incubated with three different amino acid solutions (1. l-glutamine, 2. l-alanine, 3. l-alanyl-l-glutamine, each concentration 2 mM, 5 mM, incubation time 3 h). CD14(+) monocytes were phenotyped in whole-blood and intracellular expression of cytokines was assessed by flow cytometry. Our investigations showed for the first time in whole blood probes, imitating best physiologically present cellular interactions, that l-glutamine caused a dose-independent inhibitory effect on IL-6 and TNF-α production in human monocytes stimulated with LPS. However, l-alanine had contrary effects on IL-6 expression, significantly upregulating expression of IL-6 in LPS-treated monocytes. The impact of l-alanine on the expression of TNF-α was comparable with glutamine. Neither amino acid was able to affect IL-8 production in LPS-stimulated monocytes. The combination of both did not influence significantly IL-6 and IL-8 expression in monocytes during endotoxemia, however strongly reduced TNF-α production. For the regulation of TNF-α, l-glutamine, l-alanine and the combination of both show a congruent and exponentiated downregulating effect during endotoxemia, for the modulation of IL-6, l-glutamine and l-alanine featured opposite regulation leading to a canceling impact of each other when recombining both amino acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Influence of endogenous pyrogen on the cerebral prostaglandin-synthetase system.

    Science.gov (United States)

    Ziel, R; Krupp, P

    1976-11-15

    The biotransformation of arachidonic acid to prostaglandins in vitro is specifically augmented by endogenous pyrogen to a degree depending on the concentration applied, providing that the microsomal fraction of the cerebral cortex is used as prostaglandin-synthetase system. This effect is inhibited by non-steroidal anti-inflammatory agents. These findings are compatible with the hypothesis that prostaglandins might act as mediators of the febrile reaction induced by endogenous pyrogen.

  1. A randomised trial of enteral glutamine supplementation for very preterm children showed no beneficial or adverse long-term neurodevelopmental outcomes

    NARCIS (Netherlands)

    Twilhaar, E. Sabrina; de Kieviet, Jorrit F.; Oosterlaan, Jaap; van Elburg, Ruurd M.

    2017-01-01

    This study evaluated the long-term effects of enteral glutamine supplementation on neurodevelopmental outcomes of a Dutch cohort of very preterm children at 13 years of age. The cohort was enrolled in a randomised placebo-controlled trial between 2001-2003 in which infants received glutamine- or

  2. Analysis of Fungicide Sensitivity and Genetic Diversity among Colletotrichum Species in Sweet Persimmon.

    Science.gov (United States)

    Gang, Geun-Hye; Cho, Hyun Ji; Kim, Hye Sun; Kwack, Yong-Bum; Kwak, Youn-Sig

    2015-06-01

    Anthracnose, caused by Colletotrichum gloeosporioides (C. gloeosporioides; Teleomorph: Glomerella cingulata), is the most destructive disease that affects sweet persimmon production worldwide. However, the biology, ecology, and genetic variations of C. gloeosporioides remain largely unknown. Therefore, in this study, the development of fungicide resistance and genetic diversity among an anthracnose pathogen population with different geographical origins and the exposure of this population to different cultivation strategies were investigated. A total of 150 pathogen isolates were tested in fungicide sensitivity assays. Five of the tested fungicides suppressed mycelial pathogen growth effectively. However, there were significant differences in the sensitivities exhibited by the pathogen isolates examined. Interestingly, the isolates obtained from practical management orchards versus organic cultivation orchards showed no differences in sensitivity to the same fungicide. PCR-restriction fragment length polymorphism (RFLP) analyses were performed to detect internal transcribed spacer regions and the β-tubulin and glutamine synthetase genes of the pathogens examined. Both the glutamine synthetase and β-tubulin genes contained a complex set of polymorphisms. Based on these results, the pathogens isolated from organic cultivation orchards were found to have more diversity than the isolates obtained from the practical management orchards.

  3. Analysis of Fungicide Sensitivity and Genetic Diversity among Colletotrichum Species in Sweet Persimmon

    Directory of Open Access Journals (Sweden)

    Geun-Hye Gang

    2015-06-01

    Full Text Available Anthracnose, caused by Colletotrichum gloeosporioides (C. gloeosporioides; Teleomorph: Glomerella cingulata, is the most destructive disease that affects sweet persimmon production worldwide. However, the biology, ecology, and genetic variations of C. gloeosporioides remain largely unknown. Therefore, in this study, the development of fungicide resistance and genetic diversity among an anthracnose pathogen population with different geographical origins and the exposure of this population to different cultivation strategies were investigated. A total of 150 pathogen isolates were tested in fungicide sensitivity assays. Five of the tested fungicides suppressed mycelial pathogen growth effectively. However, there were significant differences in the sensitivities exhibited by the pathogen isolates examined. Interestingly, the isolates obtained from practical management orchards versus organic cultivation orchards showed no differences in sensitivity to the same fungicide. PCR-restriction fragment length polymorphism (RFLP analyses were performed to detect internal transcribed spacer regions and the β-tubulin and glutamine synthetase genes of the pathogens examined. Both the glutamine synthetase and β-tubulin genes contained a complex set of polymorphisms. Based on these results, the pathogens isolated from organic cultivation orchards were found to have more diversity than the isolates obtained from the practical management orchards.

  4. Impact of dietary glutamine on amino acid digestibility values and ...

    African Journals Online (AJOL)

    Nebonid F. Namroud

    2017-05-22

    May 22, 2017 ... optimum growth during and after the critical period of birds' life. However ... For the first 18 days of incubation, the temperature was set at. 37.8 ºC ... Segments were cut longitudinally and flushed with cold phosphate-buffered saline (PBS). For ...... Muscle glutamine depletion in the intensive care unit. Int. J.

  5. Is long term creatine and glutamine supplementation effective in enhancing physical performance of military police officers?

    Science.gov (United States)

    da Silveira, Celismar Lázaro; de Souza, Thiago Siqueira Paiva; Batista, Gilmário Ricarte; de Araújo, Adenilson Targino; da Silva, Júlio César Gomes; de Sousa, Maria do Socorro Cirilo; Marta, Carlos; Garrido, Nuno Domingo

    2014-09-29

    The objective of this study was to analyze the effect of supplementation with creatine and glutamine on physical fitness of military police officers. Therefore, an experimental double blind study was developed, with the final sample composed by 32 men randomly distributed into three groups: a group supplemented with creatine (n=10), glutamine (n=10) and a placebo group (n=12) and evaluated in three distinct moments, in an interval of three months (T1, T2 and T3). The physical training had a weekly frequency of 5 sessions × 90 min, including strength exercises, local muscular resistance, flexibility and both aerobic and anaerobic capacity. After analyzing the effect of time, group and interaction (group × time) for measures that indicated the physical capabilities of the subjects, a significant effect of time for the entire variable was identified (p0,05). In face of the results it was concluded that supplementation with creatine and glutamine showed no ergogenic effect on physical performance in military police officers.

  6. Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Hove-Jensen, Bjarne; Switzer, Robert L.

    1988-01-01

    in a 416-base-pair 5' untranslated leader in the prsA transcript, which was shown by deletion to be necessary for maximal synthesis of phosphoribosylpyrophosphate synthetase. The S. typhimurium leader contains a 115-base-pair insert relative to the E. coli leader. The insert appears to have no functional...

  7. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

  8. Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

    OpenAIRE

    Sanya Chadha; N. Arjunreddy Mallampudi; Debendra K. Mohapatra; Rentala Madhubala; Ira J. Blader; Greg Matlashewski; Frederick Buckner

    2017-01-01

    ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L.?donovani lysyl-tRNA synthetase (LdLysRS). Two different codin...

  9. Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

    Science.gov (United States)

    González, Alberto; Moenne, Fabiola; Gómez, Melissa; Sáez, Claudio A; Contreras, Rodrigo A; Moenne, Alejandra

    2014-01-01

    In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL(-1), or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5'-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

  10. Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees

    Directory of Open Access Journals (Sweden)

    Alberto eGonzález

    2014-10-01

    Full Text Available In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control, with OC kappa at 1 mg mL-1, or treated with inhibitors of NAD(PH, ascorbate (ASC and glutathione (GSH syntheses and thioredoxin reductase (TRR activity, CHS-828, lycorine, buthionine sulfoximine (BSO and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS, adenosine 5´-phosphosulfate reductase (APR, involved in C, N and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism and growth in Eucalyptus trees.

  11. Differential gene expression in Rhododendron fortunei roots colonized by an ericoid mycorrhizal fungus and increased nitrogen absorption and plant growth

    Directory of Open Access Journals (Sweden)

    Xiangying Wei

    2016-10-01

    Full Text Available Ericoid mycorrhizal (ERM fungi are specifically symbiotic with plants in the family Ericaceae. Little is known thus far about their symbiotic establishment and subsequent nitrogen (N uptake at the molecular level. The present study devised a system for establishing a symbiotic relationship between Rhododendron fortunei Lindl. and an ERM fungus (Oidiodendron maius var. maius strain Om19, quantified seedling growth and N uptake, and compared transcriptome profiling between colonized and uncolonized roots using RNA-Seq. The Om19 colonization induced 16,892 genes that were differentially expressed in plant roots, of which 14,364 were upregulated and 2,528 were downregulated. These genes included those homologous to ATP-binding cassette transporters, calcium/calmodulin-dependent kinases, and symbiosis receptor-like kinases. N metabolism was particularly active in Om19-colonized roots, and 51 genes were upregulated, such as nitrate transporters, nitrate reductase, nitrite reductase, ammonium transporters, glutamine synthetase, and glutamate synthase. Transcriptome analysis also identified a series of genes involving endocytosis, Fc-gamma R-mediated phagocytosis, glycerophospholipid metabolism, and GnRH signal pathway that have not been reported previously. Their roles in the symbiosis require further investigation. The Om19 colonization significantly increased N uptake and seedling growth. Total N content and dry weight of colonized seedlings were 36.6% and 46.6% greater than control seedlings. This is the first transcriptome analysis of a species from the family Ericaceae colonized by an ERM fungus. The findings from this study will shed light on the mechanisms underlying symbiotic relationships of ericaceous species with ERM fungi and the symbiosis-resultant N uptake and plant growth.

  12. Association between methylenetetrahydrofolate reductase (MTHFR ...

    African Journals Online (AJOL)

    Association between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and risk of ischemic stroke in North Indian population: A hospital based case–control study. Amit Kumar, Shubham Misra, Anjali Hazarika, Pradeep Kumar, Ram Sagar, Abhishek Pathak, Kamalesh Chakravarty, Kameshwar ...

  13. Azolla-Anabaena Relationship 1

    Science.gov (United States)

    Meeks, John C.; Steinberg, Nisan A.; Enderlin, Carol S.; Joseph, Cecillia M.; Peters, Gerald A.

    1987-01-01

    The major radioactive products of the fixation of [13N]N2 by Azolla caroliniana Willd.-Anabaena azollae Stras. were ammonium, glutamine, and glutamate, plus a small amount of alanine. Ammonium accounted for 70 and 32% of the total radioactivity recovered after fixation for 1 and 10 minutes, respectively. The presence of a substantial pool of [13N]N2-derived 13NH4+ after longer incubation periods was attributed to the spatial separation between the site of N2-fixation (Anabaena) and a second, major site of assimilation (Azolla). Initially, glutamine was the most highly radioactive organic product formed from [13N]N2, but after 10 minutes of fixation glutamate had 1.5 times more radiolabel than glutamine. These kinetics of radiolabeling, along with the effects of inhibitors of glutamine synthetase and glutamate synthase on assimilation of exogenous and [13N]N2-derived 13NH4+, indicate that ammonium assimilation occurred by the glutamate synthase cycle and that glutamate dehydrogenase played little or no role in the synthesis of glutamate by Azolla-Anabaena. PMID:16665538

  14. Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

    Science.gov (United States)

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-01

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

  15. Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

    Science.gov (United States)

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-09

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The effect of ionic and non-ionic surfactants on the growth, nitrate reductase and nitrite reductase activities of Spirodela polyrrhiza (L. Schleiden

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available Inclusion into the medium of 5 mg•dm-3 of non-ionic (ENF or ionic (DBST surfactant caused 50-60% inhibition of nitrite reductase MR activity in S. polyrrhiza. At the same time, increased accumulation of NO2- in the plant tissues and lowering of the total and soluble protein contents were found. DBST also lowered the nitrate reductase (NR activity and the dry mass of the plants.

  17. Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes.

    Science.gov (United States)

    Armen, Roger S; Schiller, Stefan M; Brooks, Charles L

    2010-06-01

    Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-alpha-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable.

  18. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthal, Cindy [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Mueller, Uwe [Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH, Albert-Einstein-Strasse 15, D-12489 Berlin (Germany); Panjikar, Santosh [European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Sun, Lianli [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Ruppert, Martin [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Zhao, Yu [Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Stöckigt, Joachim [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China)

    2006-12-01

    Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

  19. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

    International Nuclear Information System (INIS)

    Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

    2006-01-01

    Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222 1 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å

  20. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses...

  1. ORF Alignment: NC_000962 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_000962 gi|15609358 >1v4aA 6 435 65 483 2e-72 ... ref|NP_216737.1| GLUTAMATE-AMMONIA...erium tuberculosis H37Rv] ref|NP_855894.1| ... GLUTAMATE-AMMONIA-LIGASE ADENYLYLTRANSFERASE GLNE ... ... AF2122/97] emb|CAA94664.1| ... GLUTAMATE-AMMONIA-LIGASE ADENYLYLTRANSFERASE GLNE ... (Glutami...se] adenylyltransferase) ... (Glutamine-synthetase adenylyltransferase) (ATase) ... emb|CAD97098.1| GLUTAMATE-AMMONIA

  2. ORF Alignment: NC_002755 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002755 gi|15841713 >1v4aA 6 435 65 483 2e-72 ... ref|NP_216737.1| GLUTAMATE-AMMONIA...erium tuberculosis H37Rv] ref|NP_855894.1| ... GLUTAMATE-AMMONIA-LIGASE ADENYLYLTRANSFERASE GLNE ... ... AF2122/97] emb|CAA94664.1| ... GLUTAMATE-AMMONIA-LIGASE ADENYLYLTRANSFERASE GLNE ... (Glutami...se] adenylyltransferase) ... (Glutamine-synthetase adenylyltransferase) (ATase) ... emb|CAD97098.1| GLUTAMATE-AMMONIA

  3. ORF Alignment: NC_002945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002945 gi|31793401 >1v4aA 6 435 65 483 2e-72 ... ref|NP_216737.1| GLUTAMATE-AMMONIA...erium tuberculosis H37Rv] ref|NP_855894.1| ... GLUTAMATE-AMMONIA-LIGASE ADENYLYLTRANSFERASE GLNE ... ... AF2122/97] emb|CAA94664.1| ... GLUTAMATE-AMMONIA-LIGASE ADENYLYLTRANSFERASE GLNE ... (Glutami...se] adenylyltransferase) ... (Glutamine-synthetase adenylyltransferase) (ATase) ... emb|CAD97098.1| GLUTAMATE-AMMONIA

  4. Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis[W

    Science.gov (United States)

    Meguro, Miki; Ito, Hisashi; Takabayashi, Atsushi; Tanaka, Ryouichi; Tanaka, Ayumi

    2011-01-01

    The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation. PMID:21934147

  5. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.

    1986-01-01

    Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...... the UAA translation stop codon, within a Thy-rich region following an inverted repeat sequence, indicative of an rho-independent transcription terminator....

  6. 7T Proton Magnetic Resonance Spectroscopy of Gamma-Aminobutyric Acid, Glutamate, and Glutamine Reveals Altered Concentrations in Patients With Schizophrenia and Healthy Siblings

    DEFF Research Database (Denmark)

    Thakkar, Katharine N; Rösler, Lara; Wijnen, Jannie P

    2017-01-01

    BACKGROUND: The N-methyl-D-aspartate receptor hypofunction model of schizophrenia predicts dysfunction in both glutamatergic and gamma-aminobutyric acidergic (GABAergic) transmission. We addressed this hypothesis by measuring GABA, glutamate, glutamine, and the sum of glutamine plus glutamate...... concentrations in vivo in patients with schizophrenia using proton magnetic resonance spectroscopy at 7T, which allows separation of metabolites that would otherwise overlap at lower field strengths. In addition, we investigated whether altered levels of GABA, glutamate, glutamine, and the sum of glutamine plus...... glutamate reflect genetic vulnerability to schizophrenia by including healthy first-degree relatives. METHODS: Proton magnetic resonance spectroscopy at 7T was performed in 21 patients with chronic schizophrenia who were taking medication, 23 healthy first-degree relatives of patients with schizophrenia...

  7. Glutamine Triggers and Potentiates Glucagon-Like Peptide-1 Secretion by Raising Cytosolic Ca2+ and cAMP

    OpenAIRE

    Tolhurst, Gwen; Zheng, Yue; Parker, Helen E.; Habib, Abdella M.; Reimann, Frank; Gribble, Fiona M.

    2011-01-01

    L-glutamine stimulates glucagon-like peptide 1 (GLP-1) secretion in human subjects and cell lines. As recent advances have enabled the study of primary GLP-1–releasing L cells, this study aimed to characterize glutamine-sensing pathways in native murine L cells. L cells were identified using transgenic mice with cell-specific expression of fluorescent markers. Cells were studied in primary colonic cultures from adult mice, or purified by flow cytometry for expression analysis. Intracellular C...

  8. Glutamine-enriched enteral diet increases splanchnic blood flow in the rat

    NARCIS (Netherlands)

    Houdijk, A. P.; van Leeuwen, P. A.; Boermeester, M. A.; van Lambalgen, T.; Teerlink, T.; FLINKERBUSCH, E. L.; Sauerwein, H. P.; Wesdorp, R. I.

    1994-01-01

    The hemodynamic consequences of glutamine (Gln)-enriched nutrition have not been investigated. This study investigates the effects of a Gln-enriched enteral diet on organ blood flows and systemic hemodynamics. Male Fischer 344 rats (n = 24) were randomized to a group that received a 12.5% (wt/wt)

  9. Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate †

    Science.gov (United States)

    Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D.

    2009-01-01

    O-succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered Bi Uni Uni Bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct-binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first-half reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the ‘F’ form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered Bi Uni Uni Bi Iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogs with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogs tested as none were active except 4-(2-trifluoromethylphenyl)-4-oxobutyric acid which exhibited a 100-fold decrease in kcat/Km. Based on an understanding of OSB-CoA synthetase’s kinetic mechanism and substrate specificity, a reaction intermediate analog of OSB-AMP, 5’-O-(N-(2-trifluoromethylphenyl)-4-oxobutyl) adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of

  10. Glutamine prevents gastric oxidative stress in an animal model of portal hypertension gastropathy.

    Science.gov (United States)

    Marques, Camila; Mauriz, José L; Simonetto, Douglas; Marroni, Claudio A; Tuñon, María J; González-Gallego, Javier; Marrón, Norma P

    2011-01-01

    Portal hypertension (PHI) is a clinical syndrome characterized by increases of the blood flow and/or of the vascular resistance in the portal system. A direct consequence of PHI can appearance different lesions on the gastric mucosa and submucosa, cumulatively termed portal hypertensive gastropathy (PHG). To investigate the effects of glutamine on oxidative stress in an experimental model of PHG induced by partial portal vein ligation (PPVL). Portal pressure, transaminase and alkaline phosphatase activity were quantified. Gastric tissue damage was assessed by histological analysis. Oxidative stress was measured by quantification of cytosolic concentration of thiobarbituric acid reactive substances (TBARS), hydroperoxide-initiated chemiluminescence (QL), and nitric oxide (NO) production. Moreover, activities of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were analyzed. Transaminase and alkaline phosphatase activities were not significantly modified by PPVL, indicating absence of liver injury. Histological analysis of gastric sections showed a lost of normal architecture, with edema and vasodilatation. TBARS, QL, and NO production were significantly increased in PPVL animals. A reduction of SOD activity was found. Glutamine administration markedly alleviated histological abnormalities and oxidative stress, normalized SOD activity, and blocked NO overproduction. Our results confirm that the use of molecules with antioxidant capacity can provide protection of the gastric tissue in portal hypertension. Glutamine treatment can be useful to reduce the oxidative damage induced by PHI on gastric tissue.

  11. Proofreading in vivo: Editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli

    International Nuclear Information System (INIS)

    Jakubowski, H.

    1990-01-01

    Previous in vitro studies have established a pre-transfer proofreading mechanism for editing of homocysteine by bacterial methionyl-, isoleucyl-, and valyl-tRNA synthetases. The unusual feature of the editing is the formation of a distinct compound, homocysteine thiolactone. Now, two-dimensional TLC analysis of 35S-labeled amino acids extracted from cultures of the bacterium Escherichia coli reveals that the thiolactone is also synthesized in vivo. In E. coli, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 109 molecules of methionine incorporated into protein in vivo. These results not only directly demonstrate that the adenylate proofreading pathway for rejection of misactivated homocysteine operates in vivo in E. coli but, in general, establish the importance of error-editing mechanisms in living cells

  12. Fusion of the subunits α and β of succinyl-CoA synthetase as a phylogenetic marker for Pezizomycotina fungi

    Directory of Open Access Journals (Sweden)

    Amanda M. Koire

    2011-01-01

    Full Text Available Gene fusions, yielding the formation of multidomain proteins, are evolutionary events that can be utilized as phylogenetic markers. Here we describe a fusion gene comprising the α and β subunits of succinyl-coA synthetase, an enzyme of the TCA cycle, in Pezizomycotina fungi. This fusion is present in all Pezizomycotina with complete genome sequences and absent from all other organisms. Phylogenetic analysis of the α and β subunits of succinyl-CoA synthetase suggests that both subunits were duplicated and retained in Pezizomycotina while one copy was lost from other fungi. One of the duplicated copies was then fused in Pezizomycotina. Our results suggest that the fusion of the α and β subunits of succinyl-CoA synthetase can be used as a molecular marker for membership in the Pezizomycotina subphylum. If a species has the fusion it can be reliably classified as Pezizomycotina, while the absence of the fusion is suggestive that the species is not a member of this subphylum.

  13. Genome sequence analysis of predicted polyprenol reductase gene from mangrove plant kandelia obovata

    Science.gov (United States)

    Basyuni, M.; Sagami, H.; Baba, S.; Oku, H.

    2018-03-01

    It has been previously reported that dolichols but not polyprenols were predominated in mangrove leaves and roots. Therefore, the occurrence of larger amounts of dolichol in leaves of mangrove plants implies that polyprenol reductase is responsible for the conversion of polyprenol to dolichol may be active in mangrove leaves. Here we report the early assessment of probably polyprenol reductase gene from genome sequence of mangrove plant Kandelia obovata. The functional assignment of the gene was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI using Blastx. The degree of sequence identity between DNA sequence and known polyprenol reductase was confirmed using the Blastx probability E-value, total score, and identity. The genome sequence data resulted in three partial sequences, termed c23157 (700 bp), c23901 (960 bp), and c24171 (531 bp). The c23157 gene showed the highest similarity (61%) to predicted polyprenol reductase 2- like from Gossypium raimondii with E-value 2e-100. The second gene was c23901 to exhibit high similarity (78%) to the steroid 5-alpha-reductase Det2 from J. curcas with E-value 2e-140. Furthermore, the c24171 gene depicted highest similarity (79%) to the polyprenol reductase 2 isoform X1 from Jatropha curcas with E- value 7e-21.The present study suggested that the c23157, c23901, and c24171, genes may encode predicted polyprenol reductase. The c23157, c23901, c24171 are therefore the new type of predicted polyprenol reductase from K. obovata.

  14. Effects of combined pulse electromagnetic field stimulation plus glutamine on the healing of colonic anastomosis in rats.

    Science.gov (United States)

    Girgin, Sadullah; Gedik, Ercan; Ozturk, Hayrettin; Akpolat, Veysi; Akbulut, Veysi; Kale, Ebru; Buyukbayram, Huseyin; Celik, Salih

    2009-04-01

    An experimental study was designed to investigate the effect of combined pulse electromagnetic field (PEMF) stimulation plus glutamine administration on colonic anastomosis. Anastomosis of the left colon was performed in 28 rats, which were divided into four groups; Group 1: normal resection anastomosis plus oral 50 mg/kg/day glutamine; Group 2: normal resection anastomosis plus PEMF stimulation plus oral 50 mg/kg/day glutamine; Group 3: normal resection anastomosis plus PEMF stimulation; Group 4: normal resection anastomosis. On the seventh postoperative day, the animals were killed and the bursting pressure and tissue hydroxyproline concentration of the anastomosis were analyzed and compared. The mean anastomotic bursting pressure in Group 2 was significantly higher than in Groups 1 and 4. On the other hand, the mean anastomotic bursting pressure in Group 1 was significantly higher than in Group 4. The collagen deposition and the fibroblast infiltration were significantly increased on the seventh day in Group 3 compared the other groups. On the other hand, Groups 1 and 2 had higher scores for collagen deposition and fibroblast infiltration than Group 4. In conclusion, burst pressures, hydroxyproline, and histologic features (fibroblast infiltration and collagen deposition) were improved in the PEMF group, and both PEMF and glutamine-enriched nutrition provide a significant gain in the strength of colonic anastomoses in rats.

  15. Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin.

    Science.gov (United States)

    Khan, Sameena; Sharma, Arvind; Belrhali, Hassan; Yogavel, Manickam; Sharma, Amit

    2014-06-01

    Malaria parasites inevitably develop drug resistance to anti-malarials over time. Hence the immediacy for discovering new chemical scaffolds to include in combination malaria drug therapy. The desirable attributes of new chemotherapeutic agents currently include activity against both liver and blood stage malaria parasites. One such recently discovered compound called cladosporin abrogates parasite growth via inhibition of Plasmodium falciparum lysyl-tRNA synthetase (PfKRS), an enzyme central to protein translation. Here, we present crystal structure of ternary PfKRS-lysine-cladosporin (PfKRS-K-C) complex that reveals cladosporin's remarkable ability to mimic the natural substrate adenosine and thereby colonize PfKRS active site. The isocoumarin fragment of cladosporin sandwiches between critical adenine-recognizing residues while its pyran ring fits snugly in the ribose-recognizing cavity. PfKRS-K-C structure highlights ample space within PfKRS active site for further chemical derivatization of cladosporin. Such derivatives may be useful against additional human pathogens that retain high conservation in cladosporin chelating residues within their lysyl-tRNA synthetase.

  16. Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

    DEFF Research Database (Denmark)

    Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

    1992-01-01

    Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression. ...

  17. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

    Science.gov (United States)

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

    2014-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

  18. Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans

    International Nuclear Information System (INIS)

    Rocha, Rita; Barbosa Pereira, Pedro José; Santos, Manuel A. S.; Macedo-Ribeiro, Sandra

    2010-01-01

    The seryl-tRNA synthetase from C. albicans was crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and diffraction data were collected to 2.0 Å resolution at a synchrotron source. The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2–3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Å resolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS

  19. Deamidation Reactions of Asparagine- and Glutamine-Containing Dipeptides Investigated by Ion Spectroscopy

    NARCIS (Netherlands)

    Kempkes, L.J.M.; Martens, J.; Grzetic, J.; Berden, G.; Oomens, J.

    2016-01-01

    Deamidation is a major fragmentation channel upon activation by collision induced dissociation (CID) for protonated peptides containing glutamine (Gln) and asparagine (Asn) residues. Here, we investigate these NH3-loss reactions for four Asn- and Gln-containing protonated peptides in terms of the

  20. effects of standard and/or glutamine dipeptide and/or omega-3 fatty

    African Journals Online (AJOL)

    2013-02-01

    Feb 1, 2013 ... effects of these to neutrophil functions and IL-8 levels are compared. Methods: ... acid solution to S-O group and TPN with omega 3 fatty acids solution and glutamine to S-D-O group ..... exudation involving multiple cell types.

  1. Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

    Directory of Open Access Journals (Sweden)

    Jason M van Rooyen

    Full Text Available In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

  2. Simultaneous infusion of glutamine and branched-chain amino acids (BCAA) to septic rats does not have more favorable effect on protein synthesis in muscle, liver, and small intestine than separate infusions.

    Science.gov (United States)

    Holecek, Milan; Muthny, Tomas; Kovarik, Miroslav; Sispera, Ludek

    2006-01-01

    Glutamine and branched-chain amino acids (BCAA; valine, leucine, and isoleucine) are used as nutrition supplements in the treatment of proteocatabolic illness. We hypothesized that simultaneous administration of BCAA and glutamine affects protein metabolism more significantly than separate administration. In the present study, we evaluated their effect on protein synthesis in skeletal muscle, liver, and jejunum of septic rats. Twenty-four hours after induction of sepsis by subcutaneous injection of turpentine, the rats were infused for 6 hours with 5 mL of 1.75% glutamine, 1.75% BCAA, 1.75% glutamine+BCAA, or saline solution. The control group consisted of intact rats infused with saline. Protein synthesis was measured at the end of infusion by a "flooding method" with [3,4,5-(3)H]phenylalanine. In turpentine-treated animals, we observed a decrease in glutamine concentration in blood plasma and skeletal muscle, a decrease in BCAA concentration in liver and jejunum, and a decrease in protein synthesis in all tissues. Glutamine or glutamine+BCAA infusion increased glutamine concentration in plasma and muscle and stimulated protein synthesis in the liver. The BCAA infusion enhanced concentrations of BCAA in plasma and tissues, but the effect of BCAA on protein synthesis was insignificant. Synergistic effect of simultaneous infusion of glutamine and BCAA on protein synthesis was not observed. We conclude that glutamine infusion to rats with septic injury may significantly improve impaired protein synthesis in the liver and that there is no synergistic effect of glutamine and BCAA infusion on protein synthesis in skeletal muscle, liver, and jejunum.

  3. Supplementation of diets for weaned piglets withL-Valine and L-Glutamine+ L-Glutamic acid

    Directory of Open Access Journals (Sweden)

    Naiara Diedrich Rodrigues

    2015-10-01

    Full Text Available The objective of this study was to evaluate the effects of supplementation of diets for weaned piglets with L-valine and L-glutamine + L-glutamic acid on performance, frequency of diarrhea, organ weight, digesta pH, intestinal morphology, and economic viability. Seventy-two piglets with a live weight of 7.53 ± 0.84 kg and 24 days of age were used. The animals were submitted to the following four treatments from 24 to 46 days of age: diet not supplemented with amino acids (control diet, CD; diet supplemented with glutamine + glutamic acid (GD; diet supplemented with glutamine + glutamic acid + valine (GVD, and diet supplemented with valine (VD. Two sequential phases (pre-initial I and pre-initial II with a duration of 12 and 11 days, respectively, were established. A completely randomized design, consisting of six repetitions and three pigs per experimental unit, was used. Nine days after weaning, at 32 days of age, a piglet per pen was slaughtered for the evaluation of organ weight, digesta pH and intestinal morphology. All animals received a single diet from days 47 to 65. No effects on performance were observed during the pre-initial phases I and II; however, when the whole study period was considered (24 to 65 days of age, piglets fed GVD consumed less feed and exhibited better feed conversion than animals of the VD group. With respect to morphometric parameters, GD provided a greater ileal crypt depth than CD and VD. There was an economic advantage of diets supplemented with L-valine and L-glutamine + L-glutamic acid, validating their use in diets for weaned piglets until 46 days of age.

  4. Bioinformatics analysis of the predicted polyprenol reductase genes in higher plants

    Science.gov (United States)

    Basyuni, M.; Wati, R.

    2018-03-01

    The present study evaluates the bioinformatics methods to analyze twenty-four predicted polyprenol reductase genes from higher plants on GenBank as well as predicted the structure, composition, similarity, subcellular localization, and phylogenetic. The physicochemical properties of plant polyprenol showed diversity among the observed genes. The percentage of the secondary structure of plant polyprenol genes followed the ratio order of α helix > random coil > extended chain structure. The values of chloroplast but not signal peptide were too low, indicated that few chloroplast transit peptide in plant polyprenol reductase genes. The possibility of the potential transit peptide showed variation among the plant polyprenol reductase, suggested the importance of understanding the variety of peptide components of plant polyprenol genes. To clarify this finding, a phylogenetic tree was drawn. The phylogenetic tree shows several branches in the tree, suggested that plant polyprenol reductase genes grouped into divergent clusters in the tree.

  5. Is Long Term Creatine and Glutamine Supplementation Effective in Enhancing Physical Performance of Military Police Officers?

    Directory of Open Access Journals (Sweden)

    Lázaro da Silveira Celismar

    2014-12-01

    Full Text Available The objective of this study was to analyze the effect of supplementation with creatine and glutamine on physical fitness of military police officers. Therefore, an experimental double blind study was developed, with the final sample composed by 32 men randomly distributed into three groups: a group supplemented with creatine (n=10, glutamine (n=10 and a placebo group (n=12 and evaluated in three distinct moments, in an interval of three months (T1, T2 and T3. The physical training had a weekly frequency of 5 sessions x 90 min, including strength exercises, local muscular resistance, flexibility and both aerobic and anaerobic capacity. After analyzing the effect of time, group and interaction (group x time for measures that indicated the physical capabilities of the subjects, a significant effect of time for the entire variable was identified (p0,05. In face of the results it was concluded that supplementation with creatine and glutamine showed no ergogenic effect on physical performance in military police officers.

  6. Metabolic fate of 13N-labeled ammonia in rat brain

    International Nuclear Information System (INIS)

    Cooper, A.J.L.; McDonald, J.M.; Gelbard, A.S.; Gledhill, R.F.; Duffy, T.E.

    1979-01-01

    After infusion of physiological concentrations of [ 13 N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (α-amino), and glutamine (amide) in rat brain were approximately 1:5:400, respectively. Analysis of metabolites, after infusion of [ 13 N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment with methionine sulfoximine led to a decrease in the label present in brain glutamine following carotid artery infusion of [ 13 N]ammonia. 13 N activity in brain glutamate was greater than in the α-amino group of glutamine. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [ 13 N]ammonia via the right common carotid artery, was independent of concentration within the bolus over a 1000-fold range indicating that ammonia enters the brain largely by diffusion. In normal rats approximately 60% of the label recovered in brain was incorporated into glutamine, indicating that the t 1 /sub// 2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered

  7. Association of mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol involves catalytic domain of the synthetase and transframe and integrase domains of Pol

    Directory of Open Access Journals (Sweden)

    Shalak V. F.

    2011-10-01

    Full Text Available Aim. Analyze the interaction between Lysyl-tRNA synthetase (LysRS and HIV-1 GagPol to know whether a particular N-terminal sequence of mitochondrial LysRS triggers a specific recognition with GagPol. Methods. Yeast two-hybrid analysis, immunoprecipitation. Results. We have shown that LysRS associates with the Pol domain of GagPol. Conclusions. A model of the assembly of the LysRS:tRNA3Lys:GagPol packaging complex is proposed.

  8. A meta-analysis of trials using the intention to treat principle for glutamine supplementation in critically ill patients with burn.

    Science.gov (United States)

    Lin, Jiun-Jie; Chung, Xiu-Juan; Yang, Chung-Yih; Lau, Hui-Ling

    2013-06-01

    During critical illness, the demand for glutamine may exceed that which can be mobilized from muscle stores. Infections increase mortality, morbidity, length-of-stay, antibiotic usage and the cost of care. This is a major health care issue. RCTs were identified from the electronic databases: the Cochrane Library, MEDLINE, PubMed web of knowledge and hand searching journals. The trials compared the supplementation with glutamine and non-supplementation in burn. Statistical analysis was performed using RevMan5.1 software, from Cochrane Collaboration. 216 papers showed a match, in the keyword search. Upon screening the title, reading the abstract and the entire article, only four RCTs, involving 155 patients, were included. For both the glutamine group and control group, total burn surface area (TBSA) (MD=2.02, 95% CI -2.17, 6.21, p=0.34) was similar. Glutamine supplementation was associated with a statistically significant decrease in the number of patients with gram-negative bacteremia (OR 0.27, 95% CI 0.08-0.92, p=0.04) and hospital mortality (OR=0.13, 95% CI 0.03, 0.51, p=0.004), however, no statistical difference was noted between groups, for the other results. Glutamine supplemented nutrition can be associated with a reduction in mortality in hospital, complications due to gram-negative bacteremia in burn patients. Further larger and better quality trials are required, in order to determine whether any differences are statistically and clinically important. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

  9. Zinc, vitamin A, and glutamine supplementation in Brazilian shantytown children at risk for diarrhea results in sex-specific improvements in verbal learning

    Directory of Open Access Journals (Sweden)

    Aldo A. M. Lima

    2013-01-01

    Full Text Available OBJECTIVE: To identify the impact of supplemental zinc, vitamin A, and glutamine, alone or in combination, on long-term cognitive outcomes among Brazilian shantytown children with low median height-for-age z-scores. METHODS: A randomized, double-blind, placebo-controlled trial was conducted in children aged three months to nine years old from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for cognitive testing (total of 167 children were: (1 placebo, n = 25; (2 glutamine, n = 23; (3 zinc, n = 18; (4 vitamin A, n = 19; (5 glutamine+zinc, n = 20; (6 glutamine+vitamin A, n = 21; (7 zinc+vitamin A, n = 23; and (8 glutamine+zinc+vitamin A, n = 18. Neuropsychological tests were administered for the cognitive domains of non-verbal intelligence and abstraction, psychomotor speed, verbal memory and recall ability, and semantic and phonetic verbal fluency. Statistical analyses were performed using SPSS, version 16.0. ClinicalTrials.gov: NCT00133406. RESULTS: Girls receiving a combination of glutamine, zinc, and vitamin A had higher mean age-adjusted verbal learning scores than girls receiving only placebo (9.5 versus 6.4, p = 0.007 and girls receiving zinc+vitamin A (9.5 versus 6.5, p = 0.006. Similar group differences were not found between male study children. CONCLUSIONS: The findings suggest that combination therapy offers a sex-specific advantage on tests of verbal learning, similar to that seen among female patients following traumatic brain injury.

  10. Purification and properties of the dihydrofolate synthetase from Serratia indica

    International Nuclear Information System (INIS)

    Ikeda, Masamichi; Iwai, Kazuo

    1976-01-01

    The dihydrofolate synthetase (EC6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg 2+ and K + as cofactors. γ-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of ADP, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP). (auth.)

  11. Disruption of the Glutamate–Glutamine Cycle Involving Astrocytes in an Animal Model of Depression for Males and Females

    Science.gov (United States)

    Rappeneau, Virginie; Blaker, Amanda; Petro, Jeff R.; Yamamoto, Bryan K.; Shimamoto, Akiko

    2016-01-01

    Background: Women are twice as likely as men to develop major depression. The brain mechanisms underlying this sex disparity are not clear. Disruption of the glutamate–glutamine cycle has been implicated in psychiatric disturbances. This study identifies sex-based impairments in the glutamate–glutamine cycle involving astrocytes using an animal model of depression. Methods: Male and female adult Long-Evans rats were exposed to chronic social defeat stress (CSDS) for 21 days, using a modified resident-intruder paradigm. Territorial aggression was used for males and maternal aggression was used for females to induce depressive-like deficits for intruders. The depressive-like phenotype was assessed with intake for saccharin solution, weight gain, estrous cycle, and corticosterone (CORT). Behaviors displayed by the intruders during daily encounters with residents were characterized. Rats with daily handling were used as controls for each sex. Ten days after the last encounter, both the intruders and controls were subjected to a no-net-flux in vivo microdialysis to assess glutamate accumulation and extracellular glutamine in the nucleus accumbens (NAc). The contralateral hemispheres were used for determining changes in astrocytic markers, including glial fibrillary acidic protein (GFAP) and glutamate transporter-1 (GLT-1). Results: Both male and female intruders reduced saccharin intake over the course of CSDS, compared to their pre-stress period and to their respective controls. Male intruders exhibited submissive/defensive behaviors to territorial aggression by receiving sideways threats and bites. These males showed reductions in striatal GLT-1 and spontaneous glutamine in the NAc, compared to controls. Female intruders exhibited isolated behaviors to maternal aggression, including immobility, rearing, and selfgrooming. Their non-reproductive days were extended. Also, they showed reductions in prefrontal and accumbal GFAP+ cells and prefrontal GLT-1, compared to

  12. Conformation-Specific IR and UV Spectroscopy of the Amino Acid Glutamine: Amide-Stacking and Hydrogen Bonding in AN Important Residue in Neurodegenerative Diseases

    Science.gov (United States)

    Walsh, Patrick S.; Dean, Jacob C.; Zwier, Timothy S.

    2014-06-01

    Glutamine plays an important role in several neurodegenerative diseases including Huntington's disease (HD) and Alzheimer's disease (AD). An intriguing aspect of the structure of glutamine is its incorporation of an amide group in its side chain, thereby opening up the possibility of forming amide-amide H-bonds between the peptide backbone and side chain. In this study the conformational preferences of two capped gluatamines Z(carboxybenzyl)-Glutamine-X (X=OH, NHMe) are studied under jet-cooled conditions in the gas phase in order to unlock the intrinsic structural motifs that are favored by this flexible sidechain. Conformational assignments are made by comparing the hydride stretch ( 3100-3700 cm-1) and amide I and II ( 1400-1800 cm-1) resonant ion-dip infrared spectra with predictions from harmonic frequency calculations. Assigned structures will be compared to previously published results on both natural and unnatural residues. Particular emphasis will be placed on the comparison between glutamine and unconstrained γ-peptides due to the similar three-carbon spacing between backbone and side chain in glutamine to the backbone spacing in γ-peptides. The ability of the glutamine side-chain to form amide stacked conformations will be a main focus, along with the prevalence of extended backbone type structures. W. H. James, III, C W. Müller, E. G. Buchanan, M. G. D. Nix, L. Guo, L. Roskop, M. S. Gordon, L. V. Slipchenko, S. H. Gellman, and T. S. Zwier, J. Am. Chem. Soc., 2009, 131(40), 14243-14245.

  13. 7T Proton Magnetic Resonance Spectroscopy of Gamma-Aminobutyric Acid, Glutamate, and Glutamine Reveals Altered Concentrations in Patients With Schizophrenia and Healthy Siblings

    NARCIS (Netherlands)

    Thakkar, Katharine N; Rösler, Lara; Wijnen, Jannie P; Boer, Vincent O.; Klomp, Dennis W J; Cahn, Wiepke; Kahn, René S; Neggers, Sebastiaan F W

    BACKGROUND: The N-methyl-D-aspartate receptor hypofunction model of schizophrenia predicts dysfunction in both glutamatergic and gamma-aminobutyric acidergic (GABAergic) transmission. We addressed this hypothesis by measuring GABA, glutamate, glutamine, and the sum of glutamine plus glutamate

  14. Effect of ethanol on γ-aminobutyric acid in the brain

    International Nuclear Information System (INIS)

    Lassanova, M.; Tursky, T.; Homerova, D.

    1989-01-01

    The effect of acute and chronic ethanol administration on the level of γ-aminobutyric acid (GABA), glutamate, aspartate, and glutamine was investigated using 14 C-labelled compounds. The level of GABA rose after both acute and chronic ethanol administration. In chronic experiments also the levels of glutamate, aspartate and glutamine were increased. In acute experiments the incorporation from glucose into the studied amino acids (neuronal compartment) increased, while in chronic experiments a decreasing trend was observed. In the glial compartment the incorporation increased only into glutamate and glutamine in acute experiments, while in chronic experiments a decreased incorporation into glutamine was recorded. The activities of three enzymes were studied in seven parts of the brain after acute ethanol administration. The activity of glutamic acid decarboxylase increased in the hypothalamus and brain cortex and decreased in the medulla oblongata. The activity of GABA transaminase did not change and the activity of glutamine synthetase decreased only in the hippocampus. In accordance with several other studies, the presented results show that ethanol interferes with the GABA system in the brain. It is suggested that the primary effect of ethanol is exerted on the cell membranes with preference for the regions connected with the GABA system. (author). 3 figs., 6 tabs., 18 refs

  15. AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

    Science.gov (United States)

    Ingram-Smith, Cheryl; Smith, Kerry S.

    2007-01-01

    Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1. PMID:17350930

  16. AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

    Directory of Open Access Journals (Sweden)

    Cheryl Ingram-Smith

    2006-01-01

    Full Text Available Adenosine monophosphate (AMP-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming, EC 6.2.1.1 is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1 and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2, revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.

  17. Time dependent effects of haloperidol on glutamine and GABA homeostasis and astrocyte activity in the rat brain

    Science.gov (United States)

    Konopaske, Glenn T.; Bolo, Nicolas R.; Basu, Alo C.; Renshaw, Perry F.; Coyle, Joseph T.

    2013-01-01

    Rationale Schizophrenia is a severe, persistent, and fairly common mental illness. Haloperidol is widely used and is effective against the symptoms of psychosis seen in schizophrenia. Chronic oral haloperidol administration decreased the number of astrocytes in the parietal cortex of macaque monkeys (Konopaske et al. Biol Psych, 2008). Since astrocytes play a key role in glutamate metabolism, chronic haloperidol administration was hypothesized to modulate astrocyte metabolic function and glutamate homeostasis. Objectives This study investigated the effects of chronic haloperidol administration on astrocyte metabolic activity and glutamate, glutamine, and GABA homeostasis. Methods We used ex vivo 13C magnetic resonance spectroscopy along with high performance liquid chromatography after [1-13C]glucose and [1,2-13C]acetate administration to analyze forebrain tissue from rats administered oral haloperidol for 1 or 6 months. Results Administration of haloperidol for 1 month produced no changes in 13C labeling of glutamate, glutamine, or GABA, or in their total levels. However, a 6 month haloperidol administration increased 13C labeling of glutamine by [1,2-13C]acetate. Moreover, total GABA levels were also increased. Haloperidol administration also increased the acetate/glucose utilization ratio for glutamine in the 6 month cohort. Conclusions Chronic haloperidol administration in rats appears to increase forebrain GABA production along with astrocyte metabolic activity. Studies exploring these processes in subjects with schizophrenia should take into account the potential confounding effects of antipsychotic medication treatment. PMID:23660600

  18. Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallization

    International Nuclear Information System (INIS)

    Anstrom, David M.; Colip, Leslie; Moshofsky, Brian; Hatcher, Eric; Remington, S. James

    2005-01-01

    Alanine and glutamine mutations were made to the same 15 lysine positions on the surface of E. coli malate synthase G and the impact on crystallization observed. The results support lysine replacement for improvement of crystallization and provide insight into site selection and type of amino-acid replacement. Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success

  19. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosin...

  20. Modulation of the nuclear factor-kappa B (NF-κB) signalling pathway by glutamine in peritoneal macrophages of a murine model of protein malnutrition.

    Science.gov (United States)

    da Silva Lima, Fabiana; Rogero, Marcelo Macedo; Ramos, Mayara Caldas; Borelli, Primavera; Fock, Ricardo Ambrósio

    2013-06-01

    Protein malnutrition affects resistance to infection by impairing the inflammatory response, modifying the function of effector cells, such as macrophages. Recent studies have revealed that glutamine-a non-essential amino acid, which could become conditionally essential in some situations like trauma, infection, post-surgery and sepsis-is able to modulate the synthesis of cytokines. The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappa B (NF-κB) signalling pathway of peritoneal macrophages from malnourished mice. Two-month-old male Balb/c mice were submitted to protein-energy malnutrition (n = 10) with a low-protein diet containing 2 % protein, whereas control mice (n = 10) were fed a 12 % protein-containing diet. The haemogram and analysis of plasma glutamine and corticosterone were evaluated. Peritoneal macrophages were pre-treated in vitro with glutamine (0, 0.6, 2 and 10 mmol/L) for 24 h and then stimulated with 1.25 μg LPS for 30 min, and the synthesis of TNF-α and IL-1α and the expression of proteins related to the NF-κB pathway were evaluated. Malnourished animals had anaemia, leucopoenia, lower plasma glutamine and increased corticosterone levels. TNF-α production of macrophages stimulated with LPS was significantly lower in cells from malnourished animals when cultivated in supraphysiological (2 and 10 mmol/L) concentrations of glutamine. Further, glutamine has a dose-dependent effect on the activation of macrophages, in both groups, when stimulated with LPS, inducing a decrease in TNF-α and IL-1α production and negatively modulating the NF-κB signalling pathway. These data lead us to infer that the protein malnutrition state interferes with the activation of macrophages and that higher glutamine concentrations, in vitro, have the capacity to act negatively in the NF-κB signalling pathway.

  1. Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

    Science.gov (United States)

    Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

    2004-04-01

    The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

  2. Identification of 5α-reductase isoenzymes in canine skin.

    Science.gov (United States)

    Bernardi de Souza, Lucilene; Paradis, Manon; Zamberlam, Gustavo; Benoit-Biancamano, Marie-Odile; Price, Christopher

    2015-10-01

    Alopecia X in dogs is a noninflammatory alopecia that may be caused by a hormonal dysfunction. It may be similar to androgenic alopecia in men that is caused by the effect of dihydrotestosterone (DHT). The 5α-reductase isoenzymes, 5αR1 and 5αR2, and a recently described 5αR3, are responsible for the conversion of testosterone into DHT. However, which 5α-reductases are present in canine skin has not yet been described. The main objective of this study was to determine the pattern of expression of 5α-reductase genes in canine skin. Skin biopsies were obtained from healthy, intact young-mature beagles (three males, four females) at three anatomical sites normally affected by alopecia X (dorsal neck, back of thighs and base of tail) and two sites generally unaffected (dorsal head and ventral thorax). Prostate samples (n = 3) were collected as positive controls for 5α-reductase mRNA abundance measurement by real-time PCR. We detected mRNA encoding 5αR1 and 5αR3 but not 5αR2. There were no significant differences in 5αR1 and 5αR3 mRNA levels between the different anatomical sites, irrespective of gender (P > 0.05). Moreover, the mean mRNA abundance in each anatomical site did not differ between males and females (P > 0.05). To the best of the authors' knowledge, this is the first study demonstrating the expression of 5α-reductases in canine skin and the expression of 5αR3 in this tissue. These results may help to elucidate the pathogenesis of alopecia X and to determine more appropriate treatments for this disorder. © 2015 ESVD and ACVD.

  3. N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction

    Directory of Open Access Journals (Sweden)

    Costa Nicola

    2007-12-01

    Full Text Available Abstract Background HIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC. Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood. Here we investigated on the effect of N-acetylcysteine (NAC, on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS in the imbalanced activity of glutamine synthase (GS, the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders. Results Incubation of Lipari human cultured astroglial cells with gp 120 (0.1–10 nM produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS. This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA. In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5–5 mM, dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells. Conclusion In conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered

  4. Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus.

    OpenAIRE

    Pandey, K N; Sabharwal, P S

    1982-01-01

    Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through i...

  5. Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

    DEFF Research Database (Denmark)

    Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J

    2011-01-01

    Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post...... and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25...

  6. Amino acid environment determines expression of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in embryonic rat hepatocytes

    NARCIS (Netherlands)

    Lamers, W. H.; van Roon, M.; Mooren, P. G.; de Graaf, A.; Charles, R.

    1985-01-01

    A completely defined medium (EHM-1), which reflects the amino acid composition of fetal rat serum and contains albumin as the sole proteinaceous compound, allows the accumulation of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in the presence of dexamethasone, dibutyryl cyclic

  7. Transgenic plants with enhanced growth characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2018-01-09

    The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

  8. Transgenic plants with enhanced growth characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2016-09-06

    The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

  9. PDHA1 gene knockout in prostate cancer cells results in metabolic reprogramming towards greater glutamine dependence

    Science.gov (United States)

    Li, Yaqing; Li, Xiaoran; Li, Xiaoli; Zhong, Yali; Ji, Yasai; Yu, Dandan; Zhang, Mingzhi; Wen, Jian-Guo; Zhang, Hongquan; Goscinski, Mariusz Adam; Nesland, Jahn M.; Suo, Zhenhe

    2016-01-01

    Alternative pathways of metabolism endowed cancer cells with metabolic stress. Inhibiting the related compensatory pathways might achieve synergistic anticancer results. This study demonstrated that pyruvate dehydrogenase E1α gene knockout (PDHA1 KO) resulted in alterations in tumor cell metabolism by rendering the cells with increased expression of glutaminase1 (GLS1) and glutamate dehydrogenase1 (GLUD1), leading to an increase in glutamine-dependent cell survival. Deprivation of glutamine induced cell growth inhibition, increased reactive oxygen species and decreased ATP production. Pharmacological blockade of the glutaminolysis pathway resulted in massive tumor cells apoptosis and dysfunction of ROS scavenge in the LNCaP PDHA1 KO cells. Further examination of the key glutaminolysis enzymes in human prostate cancer samples also revealed that higher levels of GLS1 and GLUD1 expression were significantly associated with aggressive clinicopathological features and poor clinical outcome. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon alternative energy metabolism and targeting the glutamine anaplerosis of energy metabolism via GLS1 and GLUD1 in cancer cells may offer a potential novel therapeutic strategy. PMID:27462778

  10. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    Directory of Open Access Journals (Sweden)

    Yeon Bok Kim

    2014-01-01

    Full Text Available Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

  11. Aminoacyl-tRNA synthetases database Y2K.

    Science.gov (United States)

    Szymanski, M; Barciszewski, J

    2000-01-01

    The aminoacyl-tRNA synthetases (AARS) are a diverse group of enzymes that ensure the fidelity of transfer of genetic information from DNA into protein. They catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Currently, 818 AARS primary structures have been reported from archaebacteria, eubacteria, mitochondria, chloro-plasts and eukaryotic cells. The database is a compilation of the amino acid sequences of all AARSs, known to date, which are available as separate entries or alignments of related proteins via the WWW at http://rose.man.poznan.pl/aars/index.html

  12. Comparative modelling and molecular docking of nitrate reductase from Bacillus weihenstephanensis (DS45

    Directory of Open Access Journals (Sweden)

    R. Seenivasagan

    2016-07-01

    Full Text Available Nitrate reductase catalyses the oxidation of NAD(PH and the reduction of nitrate to nitrite. NR serves as a central point for the integration of metabolic pathways by governing the flux of reduced nitrogen through several regulatory mechanisms in plants, algae and fungi. Bacteria express nitrate reductases that convert nitrate to nitrite, but mammals lack these specific enzymes. The microbial nitrate reductase reduces toxic compounds to nontoxic compounds with the help of NAD(PH. In the present study, our results revealed that Bacillus weihenstephanensis expresses a nitrate reductase enzyme, which was made to generate the 3D structure of the enzyme. Six different modelling servers, namely Phyre2, RaptorX, M4T Server, HHpred, SWISS MODEL and Mod Web, were used for comparative modelling of the structure. The model was validated with standard parameters (PROCHECK and Verify 3D. This study will be useful in the functional characterization of the nitrate reductase enzyme and its docking with nitrate molecules, as well as for use with autodocking.

  13. Characterisation of glufosinate resistance mechanisms in Eleusine indica.

    Science.gov (United States)

    Jalaludin, Adam; Yu, Qin; Zoellner, Peter; Beffa, Roland; Powles, Stephen B

    2017-06-01

    An Eleusine indica population has evolved resistance to glufosinate, a major post-emergence herbicide of global agriculture. This population was analysed for target-site (glutamine synthetase) and non-target-site (glufosinate uptake, translocation and metabolism) resistance mechanisms. Glutamine synthetase (GS) activity extracted from susceptible (S) and resistant (R*) plants was equally sensitive to glufosinate inhibition, with IC 50 values of 0.85 mm and 0.99 mm, respectively. The extractable GS activity was also similar in S and R* samples. Foliar uptake of [ 14 C]-glufosinate did not differ in S and R* plants, nor did glufosinate net uptake in leaf discs. Translocation of [ 14 C]-glufosinate into untreated shoots and roots was also similar in both populations, with 44% to 47% of the herbicide translocated out from the treated leaf 24 h after treatment. The HPLC and LC-MS analysis of glufosinate metabolism revealed no major metabolites in S or R* leaf tissue. Glufosinate resistance in this resistant population is not due to an insensitive GS, or increased activity, or altered glufosinate uptake and translocation, or enhanced glufosinate metabolism. Thus, target-site resistance is likely excluded and the exact resistance mechanism(s) remain to be determined. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  14. Addiction to Coupling of the Warburg Effect with Glutamine Catabolism in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Bradley Smith

    2016-10-01

    Full Text Available Metabolic reprogramming is critical to oncogenesis, but the emergence and function of this profound reorganization remain poorly understood. Here we find that cooperating oncogenic mutations drive large-scale metabolic reprogramming, which is both intrinsic to cancer cells and obligatory for the transition to malignancy. This involves synergistic regulation of several genes encoding metabolic enzymes, including the lactate dehydrogenases LDHA and LDHB and mitochondrial glutamic pyruvate transaminase 2 (GPT2. Notably, GPT2 engages activated glycolysis to drive the utilization of glutamine as a carbon source for TCA cycle anaplerosis in colon cancer cells. Our data indicate that the Warburg effect supports oncogenesis via GPT2-mediated coupling of pyruvate production to glutamine catabolism. Although critical to the cancer phenotype, GPT2 activity is dispensable in cells that are not fully transformed, thus pinpointing a metabolic vulnerability specifically associated with cancer cell progression to malignancy.

  15. Cerebral ammonia metabolism in hyperammonemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, A J; Mora, S N; Cruz, N F; Gelbard, A S

    1985-06-01

    The short-term metabolic fate of blood-borne (/sup 13/N)ammonia was determined in the brains of chronically (8- or 14-week portacaval-shunted rats) or acutely (urease-treated) hyperammonemic rats. Using a freeze-blowing technique it was shown that the overwhelming route for metabolism of blood-borne (/sup 13/N)ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of (/sup 13/N)ammonia to L-(amide-/sup 13/N)glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14-week portacaval-shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight-matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.

  16. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

    Science.gov (United States)

    Leke, Renata; Bak, Lasse K; Anker, Malene; Melø, Torun M; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Portela, Luis V; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

    2011-04-01

    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

  17. L-Glutamine Metabolism Is Not A Major Source Of Increased Free ...

    African Journals Online (AJOL)

    10 weeks (2K-1C) and 4 weeks (1K-1C) respectively after renal artery clamping, clipped rats exhibited elevated blood pressures (P<0.001), which was sustained under anaesthesia. No significant difference in plasma glutamine levels were found in hypertensive rats compared to controls (11.3±1.3 mg/l in 2K-1C vs.

  18. Addition of Alanyl-Glutamine to Dialysis Fluid Restores Peritoneal Cellular Stress Responses - A First-In-Man Trial.

    Directory of Open Access Journals (Sweden)

    Klaus Kratochwill

    Full Text Available Peritonitis and ultrafiltration failure remain serious complications of chronic peritoneal dialysis (PD. Dysfunctional cellular stress responses aggravate peritoneal injury associated with PD fluid exposure, potentially due to peritoneal glutamine depletion. In this randomized cross-over phase I/II trial we investigated cytoprotective effects of alanyl-glutamine (AlaGln addition to glucose-based PDF.In a prospective randomized cross-over design, 20 stable PD outpatients underwent paired peritoneal equilibration tests 4 weeks apart, using conventional acidic, single chamber 3.86% glucose PD fluid, with and without 8 mM supplemental AlaGln. Heat-shock protein 72 expression was assessed in peritoneal effluent cells as surrogate parameter of cellular stress responses, complemented by metabolomics and functional immunocompetence assays.AlaGln restored peritoneal glutamine levels and increased the primary outcome heat-shock protein expression (effect 1.51-fold, CI 1.07-2.14; p = 0.022, without changes in peritoneal ultrafiltration, small solute transport, or biomarkers reflecting cell mass and inflammation. Further effects were glutamine-like metabolomic changes and increased ex-vivo LPS-stimulated cytokine release from healthy donor peripheral blood monocytes. In patients with a history of peritonitis (5 of 20, AlaGln supplementation decreased dialysate interleukin-8 levels. Supplemented PD fluid also attenuated inflammation and enhanced stimulated cytokine release in a mouse model of PD-associated peritonitis.We conclude that AlaGln-supplemented, glucose-based PD fluid can restore peritoneal cellular stress responses with attenuation of sterile inflammation, and may improve peritoneal host-defense in the setting of PD.

  19. Glutathione reductase: solvent equilibrium and kinetic isotope effects

    International Nuclear Information System (INIS)

    Wong, K.K.; Vanoni, M.A.; Blanchard, J.S.

    1988-01-01

    Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D 2 O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

  20. Kinetic isotope effect studies of the S-adenosylmethionine synthetase reaction

    International Nuclear Information System (INIS)

    Markham, G.D.; Parkin, D.W.; Schramm, V.L.

    1986-01-01

    S-adenosylmethionine (AdoMet) synthetase catalyzes a unique substitution reaction at the 5' carbon of MgATP. Kinetic isotope effect (V/K) measurements have been used to investigate the mechanism of AdoMet synthetase from E. coli. Changes in 3 H/ 14 C ratios when AdoMet is formed from a mixture of either ([5'- 14 C]ATP and [5'- 12 C,1'- 3 H]ATP) or ([5'- 3 H]ATP and [5'- 1 H,1'- 14 C]ATP) were examined. The effects of varying the concentrations of the co-substrate methionine and the monovalent cation activator K + were investigated. Substitution of 14 C for 12 C at the 5' position of ATP yields a primary V/K kinetic isotope effect ( 12 C/ 14 C) of 1.128 +/- 0.004 at low K + and methionine concentrations. The observed isotope effect diminishes slightly to 1.107 +/- 0.003 when both K + and methionine are present at saturating concentrations, suggesting that MgATP has only a low commitment to catalysis from at conditions near Vmax. No secondary V/K 3 H isotope effect from [5'- 3 H]ATP was detected ( 1 H/ 3 H) = 0.997 +/- 0.003. The magnitude of the primary 14 C isotope effect and the small secondary 3 H effect demonstrate that AdoMet synthesis occurs with a S/sub N/ 2 transition state which is symmetric with respect to the sulfur nucleophile and the departing tripolyphosphate group

  1. Comparison of the eight weeks of supplementation Creatine and Glutamine consumption along with resistance exercise on the level of ALP in female mice

    Directory of Open Access Journals (Sweden)

    A eskandari

    2015-11-01

    Full Text Available Background and purpose: in recent years, in order to improve power, speed, the increase in the volume of the musculature, preventing sports injuries and maintain the muscle performance athletes use from different resistance exercises and food supplements. In this regard, present study has been conducted with the aim of comparison the influence of an 8 week period consumption of creatine (2 gr.kg-1.day-1 in 1st week and 0.48 gr.kg-1.day-1during 2nd to 8th weeks and glutamine (1 gr.kg-1.day-1 from first to eighth weeks along with resistance exercise on level of ALP of female mice. Materials and methods: This experimental study was done on 80 Small adult female mice of Surrey species (28 ± 5 gram. The animals were randomly divided into 8 groups of: resistance exercise, resistance exercise + creatine, resistance exercise + glutamine, resistance exercise + glutamine + creatine, creatine, glutamine, creatine + glutamine and control groups (N= 10. Resistance exercise (5 days a week was including: climbing (4 sets, 5 times repetition with two minutes rest between the sets from a ladder (with the height of one meter and including 26 steps and bearing 30 percent of the weight of the Mouse body (hanging from tail in the first week and the increasing it up to 200 percent of body weight till the last week of the experiment. During 48 hours after the last practice session of resistance exercise, the blood sample was taken and the the level of ALP has been measured. Findings:The results showed that the level of ALP enzyme in creatine + glutamine + resistance exercise groug had been increased in comparison with the control group (144.3 ± 15.86 in comparison with 234.7 ± 25.69 U.L-1 P < 0.05. Conclusion: The results of this research indicate Creatine and Glutamine supplementation consumption along with resistance exercise increases in the level of ALP enzyme in the liver of mice.

  2. Disruption of the glutamate-glutamine cycle involving astrocytes in an animal model of depression for males and females

    Directory of Open Access Journals (Sweden)

    Virginie Rappeneau

    2016-12-01

    Full Text Available Background: Women are twice as likely as men to develop major depression (MD. The brain mechanisms underlying this sex disparity are not clear. Disruption of the glutamate-glutamine cycle has been implicated in psychiatric disturbances. This study identifies sex-based impairments in the glutamate-glutamine cycle involving astrocytes using an animal model of depression. Methods: Male and female adult Long-Evans rats were exposed to chronic social defeat stress (CSDS for 21 days, using a modified resident-intruder paradigm. Territorial aggression was used for males and maternal aggression was used for females to induce depressive-like deficits for intruders. The depressive-like phenotype was assessed with intake for saccharin solution, weight gain, estrous cycle, and corticosterone (CORT. Behaviors displayed by the intruders during daily encounters with residents were characterized. Rats with daily handling were used as controls for each sex. Ten days after the last encounter, both the intruders and controls were subjected to a no-net-flux in vivo microdialysis to assess glutamate accumulation and extracellular glutamine in the nucleus accumbens (NAc. The contralateral hemispheres were used for determining changes in astrocytic markers, including glial fibrillary acidic protein (GFAP and glutamate transporter-1 (GLT-1. Results: Both male and female intruders reduced saccharin intake over the course of CSDS, compared to their pre-stress period and to their respective controls. Male intruders exhibited submissive/defensive behaviors to territorial aggression by receiving sideways threats and bites. These males showed reductions in striatal GLT-1 and spontaneous glutamine in the NAc, compared to controls. Female intruders exhibited isolated behaviors to maternal aggression, including immobility, rearing, and self-grooming. Their non-reproductive days were extended. Also, they showed reductions in prefrontal and accumbal GFAP+ cells and prefrontal GLT

  3. Aging Reduces an ERRalpha-Directed Mitochondrial Glutaminase Expression Suppressing Glutamine Anaplerosis and Osteogenic Differentiation of Mesenchymal Stem Cells.

    Science.gov (United States)

    Huang, Tongling; Liu, Renzhong; Fu, Xuekun; Yao, Dongsheng; Yang, Meng; Liu, Qingli; Lu, William W; Wu, Chuanyue; Guan, Min

    2017-02-01

    Aging deteriorates osteogenic capacity of mesenchymal stem/stromal cells (MSCs), contributing to imbalanced bone remodeling and osteoporosis. Glutaminase (Gls) catabolizes glutamine into glutamate at the first step of mitochondrial glutamine (Gln)-dependent anaplerosis which is essential for MSCs upon osteogenic differentiation. Estrogen-related receptor α (ERRα) regulates genes required for mitochondrial function. Here, we found that ERRα and Gls are upregulated by osteogenic induction in human MSCs (hMSCs). In contrast, osteogenic differentiation capacity and glutamine consumption of MSCs, as well as ERRα, Gls and osteogenic marker genes are significantly reduced with age. We demonstrated that ERRα binds to response elements on Gls promoter and affects glutamine anaplerosis through transcriptional induction of Gls. Conversely, mTOR inhibitor rapamycin, ERRα inverse agonist compound 29 or Gls inhibitor BPTES leads to reduced Gln anaplerosis and deteriorated osteogenic differentiation of hMSCs. Importantly, overexpression of ERRα or Gls restored impairment by these inhibitors. Finally, we proved that compensated ERRα or Gls expression indeed potentiated Gln anaplerosis and osteogenic capability of elderly mice MSCs in vitro. Together, we establish that Gls is a novel ERRα target gene and ERRα/Gls signaling pathway plays an important role in osteogenic differentiation of MSCs, providing new sights into novel regenerative therapeutics development. Our findings suggest that restoring age-related mitochondrial Gln-dependent anaplerosis may be beneficial for degenerative bone disorders such as osteoporosis. Stem Cells 2017;35:411-424. © 2016 AlphaMed Press.

  4. Combined effects of Lanthanum(III) and elevated Ultraviolet-B radiation on root nitrogen nutrient in soybean seedlings.

    Science.gov (United States)

    Huang, Guangrong; Wang, Lihong; Sun, Zhaoguo; Li, Xiaodong; Zhou, Qing; Huang, Xiaohua

    2015-02-01

    Rare earth element pollution and elevated ultraviolet-B (UV-B) radiation occur simultaneously in some regions, but the combined effects of these two factors on plants have not attracted enough attention. Nitrogen nutrient is vital to plant growth. In this study, the combined effects of lanthanum(III) and elevated UV-B radiation on nitrate reduction and ammonia assimilation in soybean (Glycine max L.) roots were investigated. Treatment with 0.08 mmol L(-1) La(III) did not change the effects of elevated UV-B radiation on nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), nitrate, ammonium, amino acids, or soluble protein in the roots. Treatment with 0.24 mmol L(-1) La(III) and elevated UV-B radiation synergistically decreased the NR, NiR, GS, and GOGAT activities as well as the nitrate, amino acid, and soluble protein levels, except for the GDH activity and ammonium content. Combined treatment with 1.20 mmol L(-1) La(III) and elevated UV-B radiation produced severely deleterious effects on all test indices, and these effects were stronger than those induced by La(III) or elevated UV-B radiation treatment alone. Following the withdrawal of La(III) and elevated UV-B radiation, all test indices for the combined treatments with 0.08/0.24 mmol L(-1) La(III) and elevated UV-B radiation recovered to a certain extent, but they could not recover for treatments with 1.20 mmol L(-1) La(III) and elevated UV-B radiation. In summary, combined treatment with La(III) and elevated UV-B radiation seriously affected nitrogen nutrition in soybean roots through the inhibition of nitrate reduction and ammonia assimilation.

  5. Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound

    Directory of Open Access Journals (Sweden)

    Das Pralay

    2011-05-01

    Full Text Available Abstract Background Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. Results When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide, respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. Conclusions We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed.

  6. Effects of L-glutamine on rectal temperature and some markers of oxidative stress in Red Sokoto goats during the hot-dry season.

    Science.gov (United States)

    Ocheja, Ohiemi Benjamin; Ayo, Joseph Olusegun; Aluwong, Tagang; Minka, Ndazo Salka

    2017-08-01

    The experiment investigated the ameliorative effects of L-glutamine administration on rectal temperature (RT), erythrocyte osmotic fragility (EOF), serum antioxidant enzyme activities and malondialdehyde (MDA) concentration in Red Sokoto goats during the hot-dry season. Twenty eight healthy Red Sokoto goats, comprising 14 experimental (administered 0.2 g/kg of L-glutamine dissolved in 10 mL of distilled water, once daily for 21 days) and 14 control (administered equivalent of distilled water) goats served as subjects. Rectal temperature (measured at 6:00, 13:00 and 18:00 h) and blood samples (taken at 8:00 h) were obtained from all subjects weekly, before, during and after L-glutamine administration. Data obtained were compared using one-way repeated-measures ANOVA, followed by Tukey's post-hoc test. The dry-bulb temperature, relative humidity and temperature-humidity index for the study period ranged between 24.0 and 37.5 °C, 26.0 and 84.0% and 73.0 and 86.3, respectively. L-glutamine administration decreased (P heat-stressed goats during the hot-dry season.

  7. Growth hormone enhances fat-free mass and glutamine availability in patients with short-bowel syndrome: an ancillary double-blind, randomized crossover study.

    Science.gov (United States)

    Seguy, David; Darmaun, Dominique; Duhamel, Alain; Thuillier, François; Cynober, Luc; Cortot, Antoine; Gottrand, Frédéric; Messing, Bernard

    2014-09-01

    Benefits of recombinant human growth hormone (rhGH) alone or combined with glutamine in patients with intestinal failure because of short-bowel syndrome remain controversial. We explored effects of rhGH on whole-body protein metabolism in patients with short-bowel syndrome with intestinal failure (SBS-IF) to gain insight into its mechanism of action. Eight stable hyperphagic patients with severe SBS-IF received, in a double-blind, randomized crossover study, low-dose rhGH (0.05 mg · kg⁻¹ · d⁻¹) and a placebo for two 3-wk periods. Leucine and glutamine kinetics under fasting and fed conditions, fat-free mass (FFM), and serum insulin were determined on the final day of each treatment. rhGH increased FFM and nonoxidative leucine disposal (NOLD; an index of protein synthesis) (P de novo synthesis (P de novo synthesis and intestinal absorption increase glutamine availability over the physiologic range, suggesting that beneficial effects of rhGH in hyperphagic patients might be achieved without glutamine supplementation. © 2014 American Society for Nutrition.

  8. Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

    DEFF Research Database (Denmark)

    Walls, Anne Byriel; Eyjolfsson, Elvar M.; Smeland, Olav B.

    2011-01-01

    γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of G...... glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice.......65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-(13)C]glucose and the astrocyte-specific substrate [1,2-(13)C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses...

  9. The Disappearance of a Hepatic Mass in Anti-Synthetase Syndrome

    Directory of Open Access Journals (Sweden)

    Christopher J Mesa

    2017-06-01

    Full Text Available Anti-Synthetase Syndrome (ASyS is a rare chronic autoimmune disorder characterized by myositis, interstitial lung disease (ILD, polyarthralgia, “mechanic’s hands” and Raynaud’s phenomenon. Liver lesions are quite rare in ASyS. In our ASyS case, we will discuss a 58-year-old man presenting with muscle weakness, arthralgia, and interstitial lung disease (ILD. He was positive for anti-Jo-1 antibodies, substantiating the diagnosis, and was started on treatment. This was followed by the appearance of a liver mass that disappeared when the patient achieved remission.

  10. 7T Proton Magnetic Resonance Spectroscopy of Gamma-Aminobutyric Acid, Glutamate, and Glutamine Reveals Altered Concentrations in Patients With Schizophrenia and Healthy Siblings.

    Science.gov (United States)

    Thakkar, Katharine N; Rösler, Lara; Wijnen, Jannie P; Boer, Vincent O; Klomp, Dennis W J; Cahn, Wiepke; Kahn, René S; Neggers, Sebastiaan F W

    2017-03-15

    The N-methyl-D-aspartate receptor hypofunction model of schizophrenia predicts dysfunction in both glutamatergic and gamma-aminobutyric acidergic (GABAergic) transmission. We addressed this hypothesis by measuring GABA, glutamate, glutamine, and the sum of glutamine plus glutamate concentrations in vivo in patients with schizophrenia using proton magnetic resonance spectroscopy at 7T, which allows separation of metabolites that would otherwise overlap at lower field strengths. In addition, we investigated whether altered levels of GABA, glutamate, glutamine, and the sum of glutamine plus glutamate reflect genetic vulnerability to schizophrenia by including healthy first-degree relatives. Proton magnetic resonance spectroscopy at 7T was performed in 21 patients with chronic schizophrenia who were taking medication, 23 healthy first-degree relatives of patients with schizophrenia, and 24 healthy nonrelatives. Glutamate, glutamine, and GABA were measured cortically and subcortically in bilateral basal ganglia and occipital cortex. Patients with schizophrenia had reduced cortical GABA compared with healthy relatives and the combined sample of healthy relatives and healthy nonrelatives, suggesting that altered GABAergic systems in schizophrenia are associated with either disease state or medication effects. Reduced cortical glutamine relative to healthy control subjects was observed in patients with schizophrenia and the combined sample of healthy relatives and patients with schizophrenia, suggesting that altered glutamatergic metabolite levels are associated with illness liability. No group differences were found in the basal ganglia. Taken together, these findings are consistent with alterations in GABAergic and glutamatergic systems in patients with schizophrenia and provide novel insights into these systems in healthy relatives. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  11. Mapping hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase, in Escherichia coli.

    Science.gov (United States)

    Parker, J; Fishman, S E

    1979-04-01

    The structural gene for histidyl-tRNA synthetase was localized to 53.8 min on the Escherichia coli genome. The gene order in this region was determined to be dapE-purC-upp-purG-(guaA, guaB)-hisS-glyA.

  12. Substrate and cofactor binding to nitrile reductase : A mass spectrometry based study

    NARCIS (Netherlands)

    Gjonaj, L.; Pinkse, M.W.H.; Fernandez Fueyo, E.; Hollmann, F.; Hanefeld, U.

    2016-01-01

    Nitrile reductases catalyse a two-step reduction of nitriles to amines. This requires the binding of two NADPH molecules during one catalytic cycle. For the nitrile reductase from E. coli (EcoNR) mass spectrometry studies of the catalytic mechanism were performed. EcoNR is dimeric and has no Rossman

  13. Cell Death-Autophagy Loop and Glutamate-Glutamine Cycle in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Shu Yuan

    2017-07-01

    Full Text Available Although we know that amyotrophic lateral sclerosis (ALS is correlated with the glutamate-mediated corticomotor neuronal hyperexcitability, detailed ALS pathology remains largely unexplained. While a number of drugs have been developed, no cure exists so far. Here, we propose a hypothesis of neuronal cell death—incomplete autophagy positive-feedback loop—and summarize the role of the neuron-astrocyte glutamate-glutamine cycle in ALS. The disruption of these two cycles might ideally retard ALS progression. Cerebrovascular injuries (such as multiple embolization sessions and strokes induce neuronal cell death and the subsequent autophagy. ALS impairs autophagosome-lysosome fusion and leads to magnified cell death. Trehalose rescues this impaired fusion step, significantly delaying the onset of the disease, although it does not affect the duration of the disease. Therefore, trehalose might be a prophylactic drug for ALS. Given that a major part of neuronal glutamate is converted from glutamine through neuronal glutaminase (GA, GA inhibitors may decrease the neuronal glutamate accumulation, and, therefore, might be therapeutic ALS drugs. Of these, Ebselen is the most promising one with strong antioxidant properties.

  14. Double-blinded, placebo-controlled trial on intravenous L-alanyl-L-glutamine in the incidence of oral mucositis following chemoradiotherapy in patients with head-and-neck cancer

    International Nuclear Information System (INIS)

    Cerchietti, Leandro C.A.; Navigante, Alfredo H.; Lutteral, Maribel A.; Castro, Monica A.; Kirchuk, Ricardo; Bonomi, Marcelo; Cabalar, Maria Esther; Roth, Berta; Negretti, Graciela; Sheinker, Beatriz; Uchima, Patricia

    2006-01-01

    Purpose: We performed this double-blinded, placebo-controlled study to determine the safety and efficacy of L-alanyl-L-glutamine in the prevention of mucositis in patients with head-and-neck cancer. Methods and Materials: Thirty-two patients with head-and-neck cancer were treated with chemoradiotherapy (CRT) (radiotherapy daily up to 70 Gy plus cisplatin/5-fluoruracil once a week) and were asked to participate. Twenty-nine patients received the CRT schedule and were double-blindly assigned to receive either intravenous L-alanyl-L-glutamine 0.4 g/kg weight/day or an equal volume of saline (placebo) during chemotherapy days. Results: Fourteen patients received L-alanyl-L-glutamine and 15 received placebo. Mucositis was assessed by the Objective Mucositis Score (OMS) and the World Health Organization (WHO) grading system. There was a significant difference in incidence of mucositis developed in patients receiving placebo compared with those who received L-alanyl-L-glutamine (p = 0.035). The number of patients with severe objective mucositis (OMS >1.49) was higher in the placebo group compared with the L-alanyl-L-glutamine group (67% vs. 14%, p 0.007). L-alanyl-L-glutamine patients experienced less pain (three highest Numeric Rating Scale scores of 1.3/10 vs. 6.3/10 respectively, p = 0.008) and need for feeding tubes (14% vs. 60% respectively, p = 0.020) compared with placebo patients. No adverse effects related to the drug or the infusions were noted in either group. Conclusion: For patients with head-and-neck cancer receiving CRT, intravenous L-alanyl-L-glutamine may be an effective preventive measure to decrease the severity of mucositis

  15. Emersion induces nitrogen release and alteration of nitrogen metabolism in the intertidal genus Porphyra.

    Directory of Open Access Journals (Sweden)

    Jang K Kim

    Full Text Available We investigated emersion-induced nitrogen (N release from Porphyra umbilicalis Kütz. Thallus N concentration decreased during 4 h of emersion. Tissue N and soluble protein contents of P. umbilicalis were positively correlated and decreased during emersion. Growth of P. umbilicalis did not simply dilute the pre-emersion tissue N concentration. Rather, N was lost from tissues during emersion. We hypothesize that emersion-induced N release occurs when proteins are catabolized. While the δ(15N value of tissues exposed to emersion was higher than that of continuously submerged tissues, further discrimination of stable N isotopes did not occur during the 4 h emersion. We conclude that N release from Porphyra during emersion did not result from bacterial denitrification, but possibly as a consequence of photorespiration. The release of N by P. umbilicalis into the environment during emersion suggests a novel role of intertidal seaweeds in the global N cycle. Emersion also altered the physiological function (nitrate uptake, nitrate reductase and glutamine synthetase activity, growth rate of P. umbilicalis and the co-occurring upper intertidal species P. linearis Grev., though in a seasonally influenced manner. Individuals of the year round perennial species P. umbilicalis were more tolerant of emersion than ephemeral, cold temperate P. linearis in early winter. However, the mid-winter populations of both P. linearis and P. umbilicalis, had similar temporal physiological patterns during emersion.

  16. Relationship of Nitrogen Use Efficiency with the Activities of Enzymes Involved in Nitrogen Uptake and Assimilation of Finger Millet Genotypes Grown under Different Nitrogen Inputs

    Directory of Open Access Journals (Sweden)

    Nidhi Gupta

    2012-01-01

    Full Text Available Nitrogen responsiveness of three-finger millet genotypes (differing in their seed coat colour PRM-1 (brown, PRM-701 (golden, and PRM-801 (white grown under different nitrogen doses was determined by analyzing the growth, yield parameters and activities of nitrate reductase (NR, glutamine synthetase (GS, glutamate synthase; GOGAT, and glutamate dehydrogenase (GDH at different developmental stages. High nitrogen use efficiency and nitrogen utilization efficiency were observed in PRM-1 genotype, whereas high nitrogen uptake efficiency was observed in PRM-801 genotype. At grain filling nitrogen uptake efficiency in PRM-1 negatively correlated with NR, GS, GOGAT activities whereas it was positively correlated in PRM-701 and PRM-801, however, GDH showed a negative correlation. Growth and yield parameters indicated that PRM-1 responds well at high nitrogen conditions while PRM-701 and PRM-801 respond well at normal and low nitrogen conditions respectively. The study indicates that PRM-1 is high nitrogen responsive and has high nitrogen use efficiency, whereas golden PRM-701 and white PRM-801 are low nitrogen responsive genotypes and have low nitrogen use efficiency. However, the crude grain protein content was higher in PRM-801 genotype followed by PRM-701 and PRM-1, indicating negative correlation of nitrogen use efficiency with source to sink relationship in terms of seed protein content.

  17. Mitochondrial Sirt3 supports cell proliferation by regulating glutamine-dependent oxidation in renal cell carcinoma

    International Nuclear Information System (INIS)

    Choi, Jieun; Koh, Eunjin; Lee, Yu Shin; Lee, Hyun-Woo; Kang, Hyeok Gu; Yoon, Young Eun; Han, Woong Kyu; Choi, Kyung Hwa; Kim, Kyung-Sup

    2016-01-01

    Clear cell renal carcinoma (RCC), the most common malignancy arising in the adult kidney, exhibits increased aerobic glycolysis and low mitochondrial respiration due to von Hippel-Lindau gene defects and constitutive hypoxia-inducible factor-α expression. Sirt3 is a major mitochondrial deacetylase that mediates various types of energy metabolism. However, the role of Sirt3 as a tumor suppressor or oncogene in cancer depends on cell types. We show increased Sirt3 expression in the mitochondrial fraction of human RCC tissues. Sirt3 depletion by lentiviral short-hairpin RNA, as well as the stable expression of the inactive mutant of Sirt3, inhibited cell proliferation and tumor growth in xenograft nude mice, respectively. Furthermore, mitochondrial pyruvate, which was used for oxidation in RCC, might be derived from glutamine, but not from glucose and cytosolic pyruvate, due to depletion of mitochondrial pyruvate carrier and the relatively high expression of malic enzyme 2. Depletion of Sirt3 suppressed glutamate dehydrogenase activity, leading to impaired mitochondrial oxygen consumption. Our findings suggest that Sirt3 plays a tumor-progressive role in human RCC by regulating glutamine-derived mitochondrial respiration, particularly in cells where mitochondrial usage of cytosolic pyruvate is severely compromised. -- Highlights: •Sirt3 is required for the maintenance of RCC cell proliferation. •Mitochondrial usage of cytosolic pyruvate is severely compromised in RCC. •Sirt3 supports glutamine-dependent oxidation in RCC.

  18. Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

    Science.gov (United States)

    Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

    2014-01-01

    Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

  19. Mitochondrial Sirt3 supports cell proliferation by regulating glutamine-dependent oxidation in renal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jieun; Koh, Eunjin; Lee, Yu Shin; Lee, Hyun-Woo; Kang, Hyeok Gu [Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Yoon, Young Eun; Han, Woong Kyu [Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Choi, Kyung Hwa [Department of Urology, CHA Bundang Medical Center, CHA University, Seongnam 463-712 (Korea, Republic of); Kim, Kyung-Sup, E-mail: KYUNGSUP59@yuhs.ac [Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2016-06-03

    Clear cell renal carcinoma (RCC), the most common malignancy arising in the adult kidney, exhibits increased aerobic glycolysis and low mitochondrial respiration due to von Hippel-Lindau gene defects and constitutive hypoxia-inducible factor-α expression. Sirt3 is a major mitochondrial deacetylase that mediates various types of energy metabolism. However, the role of Sirt3 as a tumor suppressor or oncogene in cancer depends on cell types. We show increased Sirt3 expression in the mitochondrial fraction of human RCC tissues. Sirt3 depletion by lentiviral short-hairpin RNA, as well as the stable expression of the inactive mutant of Sirt3, inhibited cell proliferation and tumor growth in xenograft nude mice, respectively. Furthermore, mitochondrial pyruvate, which was used for oxidation in RCC, might be derived from glutamine, but not from glucose and cytosolic pyruvate, due to depletion of mitochondrial pyruvate carrier and the relatively high expression of malic enzyme 2. Depletion of Sirt3 suppressed glutamate dehydrogenase activity, leading to impaired mitochondrial oxygen consumption. Our findings suggest that Sirt3 plays a tumor-progressive role in human RCC by regulating glutamine-derived mitochondrial respiration, particularly in cells where mitochondrial usage of cytosolic pyruvate is severely compromised. -- Highlights: •Sirt3 is required for the maintenance of RCC cell proliferation. •Mitochondrial usage of cytosolic pyruvate is severely compromised in RCC. •Sirt3 supports glutamine-dependent oxidation in RCC.

  20. N-terminus determines activity and specificity of styrene monooxygenase reductases.

    Science.gov (United States)

    Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

    2017-12-01

    Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.