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

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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

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...

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

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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

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

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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.

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

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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.

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

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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.

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

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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.

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

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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.

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

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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

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

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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.

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

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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

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

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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.

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

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

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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.

Nitrogen metabolism in a grapevine in vitro system

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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.

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

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

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

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

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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+.

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

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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.

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

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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.

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

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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

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

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.

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

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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.

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

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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.

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.

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

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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

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...

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

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...

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)

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

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.

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...

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.

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.

[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.

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.

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

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

ASN1-encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds.

Science.gov (United States)

Gaufichon, Laure; Marmagne, Anne; Belcram, Katia; Yoneyama, Tadakatsu; Sakakibara, Yukiko; Hase, Toshiharu; Grandjean, Olivier; Clément, Gilles; Citerne, Sylvie; Boutet-Mercey, Stéphanie; Masclaux-Daubresse, Céline; Chardon, Fabien; Soulay, Fabienne; Xu, Xiaole; Trassaert, Marion; Shakiebaei, Maryam; Najihi, Amina; Suzuki, Akira

2017-08-01

Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoter Ca MV 35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter Napin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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)

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

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.

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...

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...

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.

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

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

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

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

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van Heeswijk, Wally C.; Westerhoff, Hans V.

2013-01-01

SUMMARY We present a comprehensive overview of the hierarchical network of intracellular processes revolving around central nitrogen metabolism in Escherichia coli. The hierarchy intertwines transport, metabolism, signaling leading to posttranslational modification, and transcription. The protein components of the network include an ammonium transporter (AmtB), a glutamine transporter (GlnHPQ), two ammonium assimilation pathways (glutamine synthetase [GS]-glutamate synthase [glutamine 2-oxoglutarate amidotransferase {GOGAT}] and glutamate dehydrogenase [GDH]), the two bifunctional enzymes adenylyl transferase/adenylyl-removing enzyme (ATase) and uridylyl transferase/uridylyl-removing enzyme (UTase), the two trimeric signal transduction proteins (GlnB and GlnK), the two-component regulatory system composed of the histidine protein kinase nitrogen regulator II (NRII) and the response nitrogen regulator I (NRI), three global transcriptional regulators called nitrogen assimilation control (Nac) protein, leucine-responsive regulatory protein (Lrp), and cyclic AMP (cAMP) receptor protein (Crp), the glutaminases, and the nitrogen-phosphotransferase system. First, the structural and molecular knowledge on these proteins is reviewed. Thereafter, the activities of the components as they engage together in transport, metabolism, signal transduction, and transcription and their regulation are discussed. Next, old and new molecular data and physiological data are put into a common perspective on integral cellular functioning, especially with the aim of resolving counterintuitive or paradoxical processes featured in nitrogen assimilation. Finally, we articulate what still remains to be discovered and what general lessons can be learned from the vast amounts of data that are available now. PMID:24296575

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

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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.

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

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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.

[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.

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

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.

Functions of Glutamine Synthetase Isoforms in the Nitrogen Metabolism of Plants

DEFF Research Database (Denmark)

Guan, Miao

Nitrogen is one of the major plant nutrients limiting crop production worldwide. In many parts of the world the availability of N fertilizers is limited, whereas in other parts of the world too much N fertilizer is applied, leading to serious negative environmental consequences. The use of N...... fertilizers accordingly needs to be optimized in order to make agriculture more sustainable. One pathway to achieve such optimization is to improve plant N use efficiency (NUE) by developing new crop genotypes with improved yield per unit of N fertilizer applied. For this purpose, more and better knowledge...... about bottlenecks in plant N assimilation is needed. Based on a reverse genetics strategy embracing characterization of knockout mutants in the model plant species Arabidopsis, the results obtained in this PhD study have provided new information about the specific roles of two genes Gln1;1 and Gln1...

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

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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.

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

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

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.

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

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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.

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

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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.

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.

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.

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.

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

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.

Glutamine nitrogen and ammonium nitrogen supplied as a nitrogen source is not converted into nitrate nitrogen of plant tissues of hydroponically grown pak-choi (Brassica chinensis L.).

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Wang, H-J; Wu, L-H; Tao, Q-N; Miller, D D; Welch, R M

2009-03-01

Many vegetables, especially leafy vegetables, accumulate NO(-) (3)-N in their edible portions. High nitrate levels in vegetables constitute a health hazard, such as cancers and blue baby syndrome. The aim of this study was to determine if (1) ammonium nitrogen (NH(+) (4)-N) and glutamine-nitrogen (Gln-N) absorbed by plant roots is converted into nitrate-nitrogen of pak-choi (Brassica chinensis L.) tissues, and (2) if nitrate-nitrogen (NO(-) (3)-N) accumulation and concentration of pak-choi tissues linearly increase with increasing NO(-) (3)-N supply when grown in nutrient solution. In experiment 1, 4 different nitrogen treatments (no nitrogen, NH(+) (4)-N, Gln-N, and NO(-) (3)-N) with equal total N concentrations in treatments with added N were applied under sterile nutrient medium culture conditions. In experiment 2, 5 concentrations of N (from 0 to 48 mM), supplied as NO(-) (3)-N in the nutrient solution, were tested. The results showed that Gln-N and NH(+) (4)-N added to the nutrient media were not converted into nitrate-nitrogen of plant tissues. Also, NO(-) (3)-N accumulation in the pak-choi tissues was the highest when plants were supplied 24 mM NO(-) (3)-N in the media. The NO(-) (3)-N concentration in plant tissues was quadratically correlated to the NO(-) (3)-N concentration supplied in the nutrient solution.

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

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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.

Glutamine supplementation of parenteral nutrition does not improve intestinal permeability, nitrogen balance, or outcome in newborns and infants undergoing digestive-tract surgery: results from a double-blind, randomized, controlled trial

NARCIS (Netherlands)

E.W. Steyerberg (Ewout); F.W.J. Hazebroek (Frans); M. Mourik; G.J.J.M. Borsboom (Gerard); T. Rietveld (Trinet); J.G.M. Huijmans (Jan); D. Tibboel (Dick); M.J.I.J. Albers (Marcel)

2005-01-01

textabstractOBJECTIVE: To assess the effect of isocaloric isonitrogenous parenteral glutamine supplementation on intestinal permeability and nitrogen loss in newborns and infants after major digestive-tract surgery. SUMMARY BACKGROUND DATA: Glutamine supplementation in critically

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

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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.

Novel insights into regulation of asparagine synthetase in conifers

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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.

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

Nitrogen nutrition in cotton and control strategies for greenhouse gas emissions: a review.

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Khan, Aziz; Tan, Daniel Kean Yuen; Munsif, Fazal; Afridi, Muhammad Zahir; Shah, Farooq; Wei, Fan; Fahad, Shah; Zhou, Ruiyang

2017-10-01

Cotton (Gossypium hirustum L.) is grown globally as a major source of natural fiber. Nitrogen (N) management is cumbersome in cotton production systems; it has more impacts on yield, maturity, and lint quality of a cotton crop than other primary plant nutrient. Application and production of N fertilizers consume large amounts of energy, and excess application can cause environmental concerns, i.e., nitrate in ground water, and the production of nitrous oxide a highly potent greenhouse gas (GHG) to the atmosphere, which is a global concern. Therefore, improving nitrogen use efficiency (NUE) of cotton plant is critical in this context. Slow-release fertilizers (e.g., polymer-coated urea) have the potential to increase cotton yield and reduce environmental pollution due to more efficient use of nutrients. Limited literature is available on the mitigation of GHG emissions for cotton production. Therefore, this review focuses on the role of N fertilization, in cotton growth and GHG emission management strategies, and will assess, justify, and organize the researchable priorities. Nitrate and ammonium nitrogen are essential nutrients for successful crop production. Ammonia (NH 3 ) is a central intermediate in plant N metabolism. NH 3 is assimilated in cotton by the mediation of glutamine synthetase, glutamine (z-) oxoglutarate amino-transferase enzyme systems in two steps: the first step requires adenosine triphosphate (ATP) to add NH 3 to glutamate to form glutamine (Gln), and the second step transfers the NH 3 from glutamine (Gln) to α-ketoglutarate to form two glutamates. Once NH 3 has been incorporated into glutamate, it can be transferred to other carbon skeletons by various transaminases to form additional amino acids. The glutamate and glutamine formed can rapidly be used for the synthesis of low-molecular-weight organic N compounds (LMWONCs) such as amides, amino acids, ureides, amines, and peptides that are further synthesized into high-molecular-weight organic

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

Resistance to Phosphinothricin (Glufosinate) and Its Utilization as a Nitrogen Source by Chlamydomonas reinhardtii.

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Franco, A R; Lopez-Siles, F J; Cardenas, J

1996-10-01

Wild-type strain 21gr of the green alga Chlamydomonas reinhardtii was resistant to the ammonium salt of l-phosphinothricin (PPT, also called glufosinate), an irreversible inhibitor of glutamine synthetase activity and the main active component of the herbicide BASTA (AgrEvo, Frankfurt am Main, Germany). Under the same conditions, however, this strain was highly sensitive to l-methionine-S-sulfoximine, a structural analog of PPT which has been reported to be 5 to 10 times less effective than PPT as an inhibitor in plants. Moreover, this alga was able to grow with PPT as the sole nitrogen source when this compound was provided at low concentrations. This utilization of PPT was dependent upon the addition of acetate and light and did not take place in the presence of ammonium. Resistance was due neither to the presence of N-acetyltransferase or transaminase activity nor to the presence of glutamine synthetase isoforms resistant to PPT. By using l-[methyl-(sup14)C]PPT, we demonstrated that resistance is due to lack of PPT transport into the cells. This strongly suggests that PPT and l-methionine-S-sulfoximine enter the cells through different systems. Growth with PPT is supported by its deamination by an l-amino acid oxidase activity which has been previously described to be located at the periplasm.

Insecticidal activity of glufosinate through glutamine depletion in a caterpillar.

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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.

Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels.

Science.gov (United States)

Muro-Pastor, M I; Reyes, J C; Florencio, F J

2001-10-12

The regulatory circuits that control nitrogen metabolism are relatively well known in several bacterial model groups. However, much less is understood about how the nitrogen status of the cell is perceived in vivo. In cyanobacteria, the transcription factor NtcA is required for regulation (activation or repression) of an extensive number of genes involved in nitrogen metabolism. In contrast, how NtcA activity is regulated is largely unknown. Assimilation of ammonium by most microorganisms occurs through the sequential action of two enzymes: glutamine synthetase (GS) and glutamate synthase. Interestingly, regulation of the expression of NtcA-dependent genes in the cyanobacterium Synechocystis sp. PCC 6803 is altered in mutants with modified levels of GS activity. Two types of mutants were analyzed: glnA null mutants that lack GS type I and gif mutants unable to inactivate GS in the presence of ammonium. Changes in the intracellular pools of 19 different amino acids and the keto acid 2-oxoglutarate were recorded in wild-type and mutant strains under different nitrogen conditions. Our data strongly indicate that the nitrogen status in cyanobacteria is perceived as changes in the intracellular 2-oxoglutarate pool.

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

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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.

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.

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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.

Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

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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.

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.

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

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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.

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.

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

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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

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...

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

[Research advance in nitrogen metabolism of plant and its environmental regulation].

Science.gov (United States)

Xu, Zhenzhu; Zhou, Guangsheng

2004-03-01

Nitrogen metabolism is not only one of the basic processes of plant physiology, but also one of the important parts of global chemical cycle. Plant nitrogen assimilation directly takes part in the synthesis and conversion of amino acid through the reduction of nitrate. During this stage, some key enzymes, e.g., nitrate reductase (NR), glutamine synthetase (GS), glutamate dehydrogenase (GDH), glutamine synthase (GOGAT), aspargine synthetase (AS), and asparate aminotransferase (AspAT) participate these processes. The protein is assimilated in plant cell through amino acid, and becomes a part of plant organism through modifying, classifying, transporting and storing processes, etc. The nitrogen metabolism is associated with carbonic metabolism through key enzyme regulations and the conversion of products, which consists of basic life process. Among these amino acids in plant cell, glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp) and asparagines (Asn), etc., play a key role, which regulates their conversion each other and their contents in the plant cell through regulating formation and activity of those key enzymes. Environmental factors also affect the conversion and recycle of the key amino acids through regulating gene expression of the key enzymes and their activities. Nitrate and light intensity positively regulate the gene transcription of NR, but ammonium ions and Glu, Gln do the negative way. Water deficit is a very serious constraint on N2 fixation rate and soybean (Glycine max Merr.) grain yield, in which, ureide accumulation and degradation under water deficit appear to be the key issues of feedback mechanism on nitrogen fixation. Water stress decreases NR activity, but increases proteinase activity, and thus, they regulate plant nitrogen metabolism, although there are some different effects among species and cultivars. Water stress also decreases plant tissue protein content, ratio of protein and amino acid, and reduces the absorption of amino

Nitrogen-responsive Regulation of GATA Protein Family Activators Gln3 and Gat1 Occurs by Two Distinct Pathways, One Inhibited by Rapamycin and the Other by Methionine Sulfoximine*

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Georis, Isabelle; Tate, Jennifer J.; Cooper, Terrance G.; Dubois, Evelyne

2011-01-01

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin. PMID:22039046

Nitrogen-responsive regulation of GATA protein family activators Gln3 and Gat1 occurs by two distinct pathways, one inhibited by rapamycin and the other by methionine sulfoximine.

Science.gov (United States)

Georis, Isabelle; Tate, Jennifer J; Cooper, Terrance G; Dubois, Evelyne

2011-12-30

Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

Nitrogen Control in Pseudomonas aeruginosa : A Role for Glutamine in the Regulation of the Synthesis of NADP-Dependent Glutamate Dehydrogenase, Urease and Histidase

NARCIS (Netherlands)

Janssen, Dick B.; Herst, Patricia M.; Joosten, Han M.L.J.; Drift, Chris van der

1981-01-01

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress

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.

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

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....

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.

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...

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.

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.

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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.

Quantitative proteomic analyses of the microbial degradation of estrone under various background nitrogen and carbon conditions.

Science.gov (United States)

Du, Zhe; Chen, Yinguang; Li, Xu

2017-10-15

Microbial degradation of estrogenic compounds can be affected by the nitrogen source and background carbon in the environment. However, the underlying mechanisms are not well understood. The objective of this study was to elucidate the molecular mechanisms of estrone (E1) biodegradation at the protein level under various background nitrogen (nitrate or ammonium) and carbon conditions (no background carbon, acetic acid, or humic acid as background carbon) by a newly isolated bacterial strain. The E1 degrading bacterial strain, Hydrogenophaga atypica ZD1, was isolated from river sediments and its proteome was characterized under various experimental conditions using quantitative proteomics. Results show that the E1 degradation rate was faster when ammonium was used as the nitrogen source than with nitrate. The degradation rate was also faster when either acetic acid or humic acid was present in the background. Proteomics analyses suggested that the E1 biodegradation products enter the tyrosine metabolism pathway. Compared to nitrate, ammonium likely promoted E1 degradation by increasing the activities of the branched-chain-amino-acid aminotransferase (IlvE) and enzymes involved in the glutamine synthetase-glutamine oxoglutarate aminotransferase (GS-GOGAT) pathway. The increased E1 degradation rate with acetic acid or humic acid in the background can also be attributed to the up-regulation of IlvE. Results from this study can help predict and explain E1 biodegradation kinetics under various environmental conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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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.

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

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...

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)

Dynamic optimal control of homeostasis: an integrative system approach for modeling of the central nitrogen metabolism in Saccharomyces cerevisiae.

Science.gov (United States)

van Riel, N A; Giuseppin, M L; Verrips, C T

2000-01-01

The theory of dynamic optimal metabolic control (DOMC), as developed by Giuseppin and Van Riel (Metab. Eng., 2000), is applied to model the central nitrogen metabolism (CNM) in Saccharomyces cerevisiae. The CNM represents a typical system encountered in advanced metabolic engineering. The CNM is the source of the cellular amino acids and proteins, including flavors and potentially valuable biomolecules; therefore, it is also of industrial interest. In the DOMC approach the cell is regarded as an optimally controlled system. Given the metabolic genotype, the cell faces a control problem to maintain an optimal flux distribution in a changing environment. The regulation is based on strategies and balances feedback control of homeostasis and feedforward regulation for adaptation. The DOMC approach is an integrative, holistic approach, not based on mechanistic descriptions and (therefore) not biased by the variation present in biochemical and molecular biological data. It is an effective tool to structure the rapidly increasing amount of data on the function of genes and pathways. The DOMC model is used successfully to predict the responses of pulses of ammonia and glutamine to nitrogen-limited continuous cultures of a wild-type strain and a glutamine synthetase-negative mutant. The simulation results are validated with experimental data.

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...

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...

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

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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.

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

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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

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

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

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|

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.

Alleviation of Drought Stress by Nitrogen Application in Brassica campestris ssp. Chinensis L.

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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.

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

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

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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

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

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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.

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.

ORF Alignment: NC_002755 [GENIUS II[Archive

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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

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

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

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

Mid-Season Leaf Glutamine Predicts End-Season Maize Grain Yield and Nitrogen Content in Response to Nitrogen Fertilization under Field Conditions

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Travis Goron

2017-06-01

Full Text Available After uptake in cereal crops, nitrogen (N is rapidly assimilated into glutamine (Gln and other amino acids for transport to sinks. Therefore Gln has potential as an improved indicator of soil N availability compared to plant N demand. Gln has primarily been assayed to understand basic plant physiology, rather than to measure plant/soil-N under field conditions. It was hypothesized that leaf Gln at early-to-mid season could report the N application rate and predict end-season grain yield in field-grown maize. A three-year maize field experiment was conducted with N application rates ranging from 30 to 218 kg ha−1. Relative leaf Gln was assayed from leaf disk tissue using a whole-cell biosensor for Gln (GlnLux at the V3-V14 growth stages. SPAD (Soil Plant Analysis Development and NDVI (Normalized Difference Vegetation Index measurements were also performed. When sampled at V6 or later, GlnLux glutamine output consistently correlated with the N application rate, end-season yield, and grain N content. Yield correlation outperformed GreenSeekerTM NDVI, and was equivalent to SPAD chlorophyll, indicating the potential for yield prediction. Additionally, depleting soil N via overplanting increased GlnLux resolution to the earlier V5 stage. The results of the study are discussed in the context of luxury N consumption, leaf N remobilization, senescence, and grain fill. The potential and challenges of leaf Gln and GlnLux for the study of crop N physiology, and future N management are also discussed.

Construction of hybrid peptide synthetases by module and domain fusions.

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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.

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

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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.

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

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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.

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

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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.

Delaying chloroplast turnover increases water-deficit stress tolerance through the enhancement of nitrogen assimilation in rice.

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Sade, Nir; Umnajkitikorn, Kamolchanok; Rubio Wilhelmi, Maria Del Mar; Wright, Matthew; Wang, Songhu; Blumwald, Eduardo

2018-02-12

Abiotic stress-induced senescence in crops is a process particularly affecting the photosynthetic apparatus, decreasing photosynthetic activity and inducing chloroplast degradation. A pathway for stress-induced chloroplast degradation that involves the CHLOROPLAST VESICULATION (CV) gene was characterized in rice (Oryza sativa) plants. OsCV expression was up-regulated with the age of the plants and when plants were exposed to water-deficit conditions. The down-regulation of OsCV expression contributed to the maintenance of the chloroplast integrity under stress. OsCV-silenced plants displayed enhanced source fitness (i.e. carbon and nitrogen assimilation) and photorespiration, leading to water-deficit stress tolerance. Co-immunoprecipitation, intracellular co-localization, and bimolecular fluorescence demonstrated the in vivo interaction between OsCV and chloroplastic glutamine synthetase (OsGS2), affecting source-sink relationships of the plants under stress. Our results would indicate that the OsCV-mediated chloroplast degradation pathway is involved in the regulation of nitrogen assimilation during stress-induced plant senescence. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Is there in vivo evidence for amino acid shuttles carrying ammonia from neurons to astrocytes?

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Rothman, Douglas L; De Feyter, Henk M; Maciejewski, Paul K; Behar, Kevin L

2012-11-01

The high in vivo flux of the glutamate/glutamine cycle puts a strong demand on the return of ammonia released by phosphate activated glutaminase from the neurons to the astrocytes in order to maintain nitrogen balance. In this paper we review several amino acid shuttles that have been proposed for balancing the nitrogen flows between neurons and astrocytes in the glutamate/glutamine cycle. All of these cycles depend on the directionality of glutamate dehydrogenase, catalyzing reductive glutamate synthesis (forward reaction) in the neuron in order to capture the ammonia released by phosphate activated glutaminase, while catalyzing oxidative deamination of glutamate (reverse reaction) in the astrocytes to release ammonia for glutamine synthesis. Reanalysis of results from in vivo experiments using (13)N and (15)N labeled ammonia and (15)N leucine in rats suggests that the maximum flux of the alanine/lactate or branched chain amino acid/branched chain amino acid transaminase shuttles between neurons and astrocytes are approximately 3-5 times lower than would be required to account for the ammonia transfer from neurons to astrocytes needed for glutamine synthesis (amide nitrogen) to sustain the glutamate/glutamine cycle. However, in the rat brain both the total ammonia fixation rate by glutamate dehydrogenase and the total branched chain amino acid transaminase activity are sufficient to support a branched chain amino acid/branched chain keto acid shuttle, as proposed by Hutson and coworkers, which would support the de novo synthesis of glutamine in the astrocyte to replace the ~20 % of neurotransmitter glutamate that is oxidized. A higher fraction of the nitrogen needs of total glutamate neurotransmitter cycling could be supported by hybrid cycles in which glutamate and tricarboxylic acid cycle intermediates act as a nitrogen shuttle. A limitation of all in vivo studies in animals conducted to date is that none have shown transfer of nitrogen for glutamine amide

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

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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

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.

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

Science.gov (United States)

Kajimura, Makiko; Walsh, Patrick J; Mommsen, Thomas P; Wood, Chris M

2006-01-01

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.

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

Functional specialization of one copy of glutamine phosphoribosyl pyrophosphate amidotransferase in ureide production from symbiotically fixed nitrogen in Phaseolus vulgaris.

Science.gov (United States)

Coleto, Inmaculada; Trenas, Almudena T; Erban, Alexander; Kopka, Joachim; Pineda, Manuel; Alamillo, Josefa M

2016-08-01

Purines are essential molecules formed in a highly regulated pathway in all organisms. In tropical legumes, the nitrogen fixed in the nodules is used to generate ureides through the oxidation of de novo synthesized purines. Glutamine phosphoribosyl pyrophosphate amidotransferase (PRAT) catalyses the first committed step of de novo purine synthesis. In Phaseolus vulgaris there are three genes coding for PRAT. The three full-length sequences, which are intron-less genes, were cloned, and their expression levels were determined under conditions that affect the synthesis of purines. One of the three genes, PvPRAT3, is highly expressed in nodules and protein amount and enzymatic activity in these tissues correlate with nitrogen fixation activity. Inhibition of PvPRAT3 gene expression by RNAi-silencing and subsequent metabolomic analysis of the transformed roots shows that PvPRAT3 is essential for the synthesis of ureides in P. vulgaris nodules. © 2016 John Wiley & Sons Ltd.

The better growth phenotype of DvGS1-transgenic arabidopsis thaliana is attributed to the improved efficiency of nitrogen assimilation

Directory of Open Access Journals (Sweden)

Zhu Chenguang

2015-01-01

Full Text Available The overexpression of the algal glutamine synthetase (GS gene DvGS1 in Arabidopsis thaliana resulted in higher plant biomass and better growth phenotype. The purpose of this study was to recognize the biological mechanism for the growth improvement of DvGS1-transgenic Arabidopsis. A series of molecular and biochemical investigations related to nitrogen and carbon metabolism in the DvGS1-transgenic line was conducted. Analysis of nitrogen use efficiency (NUE-related gene transcription and enzymatic activity revealed that the transcriptional level and enzymatic activity of the genes encoding GS, glutamate synthase, glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, were significantly upregulated, especially from leaf tissues of the DvGS1-transgenic line under two nitrate conditions. The DvGS1-transgenic line showed increased total nitrogen content and decreased carbon: nitrogen ratio compared to wild-type plants. Significant reduced concentrations of free nitrate, ammonium, sucrose, glucose and starch, together with higher concentrations of total amino acids, individual amino acids (glutamate, aspartate, asparagine, methionine, soluble proteins and fructose in leaf tissues confirmed that the DvGS1-transgenic line demonstrated a higher efficiency of nitrogen assimilation, which subsequently affected carbon metabolism. These improved metabolisms of nitrogen and carbon conferred the DvGS1-transgenic Arabidopsis higher NUE, more biomass and better growth phenotype compared with the wild-type plants.

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.

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

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

The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites

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Vivek A. Hariharan

2017-03-01

Full Text Available Many enzymes make “mistakes”. Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH and mitochondrial malate dehydrogenase (mMDH slowly catalyze the reduction of 2-oxoglutarate (2-OG to the oncometabolite l-2-hydroxyglutarate (l-2-HG. l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine. We show here that human glutamine synthetase (GS catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM. We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain “mistakes” of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.

The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites.

Science.gov (United States)

Hariharan, Vivek A; Denton, Travis T; Paraszcszak, Sarah; McEvoy, Kyle; Jeitner, Thomas M; Krasnikov, Boris F; Cooper, Arthur J L

2017-03-30

Many enzymes make "mistakes". Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH) and mitochondrial malate dehydrogenase (mMDH) slowly catalyze the reduction of 2-oxoglutarate (2-OG) to the oncometabolite l-2-hydroxyglutarate (l-2-HG). l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine). We show here that human glutamine synthetase (GS) catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM). We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG) can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine) is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM) to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain "mistakes" of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.

Glutamine alimentation in catabolic state.

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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.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

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da Silva Neto José F

2010-08-01

Full Text Available Abstract Background Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase, was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. Conclusions Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.

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

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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.

Modifications of proteins by polyunsaturated fatty acid peroxidation products

DEFF Research Database (Denmark)

Refsgaard, Hanne; Tsai, Lin; Stadtman, Earl

2000-01-01

The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(lll)/O-2] depends on the degree of unsaturation of the fatty acid. The fatty acid......-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate fatty acids were oxidized in the presence...... in the formation of protein carbonyls, These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (alpha,beta-unsaturated aldehydes) with lysine residues...

Metabolic features involved in drought stress tolerance mechanisms in peanut nodules and their contribution to biological nitrogen fixation.

Science.gov (United States)

Furlan, Ana Laura; Bianucci, Eliana; Castro, Stella; Dietz, Karl-Josef

2017-10-01

Legumes belong to the most important crops worldwide. They increase soil fertility due their ability to establish symbiotic associations with soil microorganisms, known as rhizobia, capable of fixing nitrogen from the atmosphere. However, they are frequently exposed to abiotic stress conditions in particular drought. Such adverse conditions impair the biological nitrogen fixation (BNF) and depend largely on the legume. Therefore, two peanut cultivars with contrasting tolerance to drought, namely the more tolerant EC-98 and the sensitive Granoleico, were investigated to elucidate the relative contribution of BNF to the tolerance to drought. The tolerant cultivar EC-98 sustained growth and BNF similar to the control condition despite the reduced water potential and photosynthesis, suggesting the functioning of distinct metabolic pathways that contributed to enhance the tolerance. The biochemical and metabolomics approaches revealed that nodules from the tolerant cultivar accumulated trehalose, proline and gamma-aminobutyric acid (GABA), metabolites with known function in protecting against drought stress. The amide metabolism was severely affected in nodules from the sensitive cultivar Granoleico as revealed by the low content of asparagine and glutamine in the drought stressed plants. The sensitive cultivar upon rehydration was unable to re-establish a metabolism similar to well-watered plants. This was evidenced by the low level of metabolites and, transcripts and specific activities of enzymes from the carbon (sucrose synthase) and nitrogen (glutamine synthetase) metabolism which decreased below the values of control plants. Therefore, the increased content of metabolites with protective functions under drought stress likely is crucial for the full restoration upon rehydration. Smaller changes of drought stress-related metabolites in nodule are another trait that contributes to the effective control of BNF in the tolerant peanut cultivar (EC-98). Copyright © 2017

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.

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...

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.

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

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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.

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

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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

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...

Emersion induces nitrogen release and alteration of nitrogen metabolism in the intertidal genus Porphyra.

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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.

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

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.

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...

Nitrogen-13-labeled ammonia for myocardial imaging

Energy Technology Data Exchange (ETDEWEB)

Walsh, W.F.; Fill, H.R.; Harper, P.V.

1977-01-01

Cyclotron-produced nitrogen-13 (half-life 10 min), as labeled ammonia (/sup 13/NH/sub 4//sup +/), has been evaluated as a myocardial perfusion imaging agent. The regional myocardial uptake of /sup 13/NH/sub 4//sup +/ has been shown to be proportional to regional tissue perfusion in animal studies. Intravenously administered /sup 13/NH/sub 4//sup +/ is rapidly cleared from the circulation, being extracted by the liver (15 percent), lungs, myocardium (2 percent--4 percent), brain, kidney, and bladder. Myocardial ammonia is metabolized mainly to glutamine via the glutamine synthetase pathway. Pulmonary uptake is substantial, but usually transient, except in smokers where clearance may be delayed. The positron annihilation irradiation (511 keV) of /sup 13/N may be imaged with a scintillation camera, using either a specially designed tungsten collimator or a pinhole collimator. After early technical problems with collimation and the production method of /sup 13/NH/sub 4//sup +/ were overcome, reproducible high quality myocardial images were consistently obtained. The normal myocardial image was established to be of a homogeneous ''doughnut'' configuration. Imaging studies performed in patients with varying manifestations of ischemic and valvular heart disease showed a high incidence of localized perfusion defects, especially in patients with acute myocardial infarction. Sequential studies at short intervals in patients with acute infarction showed correlation between alterations in regional perfusion and the clinical course of the patient. It is concluded that myocardial imaging with /sup 13/NH/sub 4//sup +/ and a scintillation camera provides a valid and noninvasive means of assessing regional myocardial perfusion. This method is especially suitable for sequential studies of acute cardiac patients at short intervals. Coincidence imaging of the 511 keV annihilation irradiation provides a tomographic and potentially quantitative assessment of the

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...

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.

Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

International Nuclear Information System (INIS)

Levin, Inna; Kessler, Naama; Moor, Nina; Klipcan, Liron; Koc, Emine; Templeton, Paul; Spremulli, Linda; Safro, Mark

2007-01-01

The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 55, b = 90, c = 96 Å

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...

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...

Exogenous glutamine increases lipid accumulation in developing seeds of castor bean (Ricinus communis L. cultured in vitro

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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.

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

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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.

Ammonium and methylammonium transport in Rhodobacter sphaeroides

International Nuclear Information System (INIS)

Cordts, M.L.; Gibson, J.

1987-01-01

Rhodobacter spheroides maintained intracellular ammonium pools of 1.1 to 2.6 mM during growth in several fixed nitrogen sources as well as during diazotrophic growth. Addition of 0.15 mM NH 4 + to washed, nitrogen-free cell suspensions was followed by linear uptake of NH 4 + from the medium and transient formation of intracellular pools of 0.9 to 1.5 mM NH 4 + . Transport of NH 4 + was shown to be independent of assimilation by glutamine synthetase because intracellular pools of over 1 mM represented NH 4 + concentration gradients of at least 100-fold across the cytoplasmic membrane. Ammonium pools of over 1 mM were also found in non-growing cell suspensions in nitrogen-free medium after glutamine synthetase was inhibited with methionine sulfoximine. In NH 4 + -free cell suspensions, methylammonium ( 14 CH 3 NH 3 + ) was taken up rapidly, and intracellular concentrations of 0.4 to 0.5 mM were maintained. The 14 CN 3 NH 3 + pool was not affected by methionine sulfoximine. Unlike NH 4 + uptake, 14 CH 3 NH 3 + uptake in nitrogen-free cell suspensions was repressed by growth in NH 4 + . These results suggest that R. sphaeroides may produce an NH 4 + -specific transport system in addition to the NH 4 + / 14 CH 3 NH 3 + transporter. This second transporter is able to produce normal-size NH 4 + pools but has very little affinity for 14 CH 3 NH 3 + and is not repressed by growth in high concentrations of NH 4 +

AMPK Activation Affects Glutamate Metabolism in Astrocytes

DEFF Research Database (Denmark)

Voss, Caroline Marie; Pajęcka, Kamilla; Stridh, Malin H

2015-01-01

acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon...... on glutamate metabolism in astrocytes was studied using primary cultures of these cells from mouse cerebral cortex during incubation in media containing 2.5 mM glucose and 100 µM [U-(13)C]glutamate. The metabolism of glutamate including a detailed analysis of its metabolic pathways involving the tricarboxylic...... skeleton into the TCA cycle was reduced. On the other hand, glutamate uptake into the astrocytes as well as its conversion to glutamine catalyzed by glutamine synthetase was not affected by AMPK activation. Interestingly, synthesis and release of citrate, which are hallmarks of astrocytic function, were...

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

Nitrogen assimilation in soybean nodules, 1

International Nuclear Information System (INIS)

Ohyama, Takuji; Kumazawa, Kikuo

1980-01-01

In order to elucidate the pathways to assimilate the ammonia produced by N 2 -fixation in soybean nodules, 15 N-labeled compounds were administered to intact nodules or nodule slices pretreated with various inhibitors of nitrogen assimilation. After exposure to 15 N 2 , 15 N-incorporation into various nitrogenous compounds was investigated in attached nodules injected with methionine sulfoximine (MSX) or azaserine (AS). MSX treatment increased the 15 N content of ammonia more than 6 times, however, depressed 15 N content of most of amides and amino acids. AS treatment enhanced 15 N content of amido-N of glutamine as well as ammonia, but decreased amino-N of glutamine and most of amino acids. Experiments with nodule slices pretreated with MSX or AS solution and then fed with 15 N-labeled ammonia or amido- 15 N of glutamine showed the same trends. Aminooxyacetate inhibited nitrogen flow from glutamic acid to other amino acids. These results strongly indicate that the ammonia produced by N 2 -fixation is assimilated by GS/GOGAT system to glutamic acid and then transaminated to various amino acids in situ. 15 N-incorporation patterns in nodule slices fed with 15 N-labeled ammonia, hydroxylamine, nitrite were similar, but nitrate seemed to be reduced in a definite compartment and assimilated similarly as in intact nodules fed with 15 N 2 (author)

Essential nontranslational functions of tRNA synthetases.

Science.gov (United States)

Guo, Min; Schimmel, Paul

2013-03-01

Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. Although these new functions were thought to be 'moonlighting activities', many are as critical for cellular homeostasis as their activity in translation. New roles have been associated with their cytoplasmic forms as well as with nuclear and secreted extracellular forms that affect pathways for cardiovascular development and the immune response and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. New architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. Although a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid-binding site for another purpose.

Direct Functionalization of Nitrogen Heterocycles via Rh-Catalyzed C-H Bond Activation

Energy Technology Data Exchange (ETDEWEB)

Lewis, Jared; Bergman, Robert; Ellman, Jonathan

2008-02-04

Nitrogen heterocycles are present in many compounds of enormous practical importance, ranging from pharmaceutical agents and biological probes to electroactive materials. Direct funtionalization of nitrogen heterocycles through C-H bond activation constitutes a powerful means of regioselectively introducing a variety of substituents with diverse functional groups onto the heterocycle scaffold. Working together, our two groups have developed a family of Rh-catalyzed heterocycle alkylation and arylation reactions that are notable for their high level of functional-group compatibility. This Account describes their work in this area, emphasizing the relevant mechanistic insights that enabled synthetic advances and distinguished the resulting transformations from other methods. They initially discovered an intramolecular Rh-catalyzed C-2-alkylation of azoles by alkenyl groups. That reaction provided access to a number of di-, tri-, and tetracyclic azole derivatives. They then developed conditions that exploited microwave heating to expedite these reactions. While investigating the mechanism of this transformation, they discovered that a novel substrate-derived Rh-N-heterocyclic carbene (NHC) complex was involved as an intermediate. They then synthesized analogous Rh-NHC complexes directly by treating precursors to the intermediate [RhCl(PCy{sub 3}){sub 2}] with N-methylbenzimidazole, 3-methyl-3,4-dihydroquinazolein, and 1-methyl-1,4-benzodiazepine-2-one. Extensive kinetic analysis and DFT calculations supported a mechanism for carbene formation in which the catalytically active RhCl(PCy{sub 3}){sub 2} fragment coordinates to the heterocycle before intramolecular activation of the C-H bond occurs. The resulting Rh-H intermediate ultimately tautomerizes to the observed carbene complex. With this mechanistic information and the discovery that acid co-catalysts accelerate the alkylation, they developed conditions that efficiently and intermolecularly alkylate a variety of

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

Mutations in nif genes that cause Klebsiella pneumoniae to be derepressed for nitrogenase synthesis in the presence of ammonium.

OpenAIRE

MacNeil, D; Brill, W J

1980-01-01

Four Nif+ revertants from strains with polar insertions in nifL, were insensitive to ammonium and amino acid repression of nitrogenase synthesis. These strains have mutations located in or near the nifL region. The derepressed phenotype was dominant in a merodiploid containing a nif+ plasmid. These nif regulatory mutations also suppressed the Nif- phenotype of Gln- strains. Thus, regulation by fixed nitrogen (possible via glutamine synthetase) occurs on the nifLA operon but not on the other s...

Hyperhydricity Phenomena Problem in Embryonic Callus of Date Palm, Solving by Glutamine and NH4+: No3- Ratio in Basal Nutrient Medium

International Nuclear Information System (INIS)

El-Dawayaty, M.M.; Zayed, Z.E.; Abdel-Gelil, L.M.

2012-01-01

Hyperhydricity is a serious problem faced in vitro date palm propagation which directly effects on the commercial production. So we try to solve this problem by studying, the effect of glutamine as the organic source of nitrogen and NH 4 + :NO 3- ratio as the inorganic source of nitrogen to decrease hyperhydricity phenomena and to produce normal somatic embryos of date palm cv. Gondila.Vitrified embryonic callus were inoculated on MS basal nutrient medium modified with glutamine levels and NH 4 + : NO 3 - ratio. Five concentration ratios of NH 4 : NO 3 (10:15, 15:10, 0:20, 20:0, 0:0 ml/l) were used with 0.1 mg/l NAA for 8 weeks throughout 2 recultures. There were gradually increasing in the percentage of vitrified embryonic callus differentiation to normal somatic embryos by increasing glutamine levels from 0.0 to 400 mg/l. Glutamine at the lowest level (50 mg/l) increased significantly the number of vitrified somatic embryos. Ammonium as the sole source of N resulted in depression in somatic embryos differentiation and escalated the frequency of hyperhydricity whereas,if nitrate was used as the sole N source, somatic embryos good quality were produced and hyperhydricity was eliminated

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.)

Impact of the Disruption of ASN3-Encoding Asparagine Synthetase on Arabidopsis Development

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Laure Gaufichon

2016-02-01

Full Text Available The aim of this study was to investigate the role of ASN3-encoded asparagine synthetase (AS, EC 6.3.5.4 during vegetative growth, seed development and germination of Arabidopsis thaliana. Phenotypic analysis of knockout (asn3-1 and knockdown (asn3-2 T-DNA insertion mutants for the ASN3 gene (At5g10240 demonstrated wild-type contents of asparagine synthetase protein, chlorophyll and ammonium in green leaves at 35 days after sowing. In situ hybridization localized ASN3 mRNA to phloem companion cells of vasculature. Young siliques of the asn3-1 knockout line showed a decrease in asparagine but an increase in glutamate. The seeds of asn3-1 and asn3-2 displayed a wild-type nitrogen status expressed as total nitrogen content, indicating that the repression of ASN3 expression had only a limited effect on mature seeds. An analysis of amino acid labeling of seeds imbibed with (15N ammonium for 24 h revealed that asn3-1 seeds contained 20% less total asparagine while 15N-labeled asparagine ((2-15Nasparagine, (4-15Nasparagine and (2,4-15Nasparagine increased by 12% compared to wild-type seeds. The data indicate a fine regulation of asparagine synthesis and hydrolysis in Arabidopsis seeds.

Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

Energy Technology Data Exchange (ETDEWEB)

Levin, Inna; Kessler, Naama [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Moor, Nina [Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk (Russian Federation); Klipcan, Liron [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Koc, Emine [Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802 (United States); Templeton, Paul [Department Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215 (United States); Spremulli, Linda [Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290 (United States); Safro, Mark, E-mail: mark.safro@weizmann.ac.il [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

2007-09-01

The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55, b = 90, c = 96 Å.

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

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

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.

Mitochondrial citrulline synthesis from ammonia and glutamine in the liver of ureogenic air-breathing catfish, Clarias batrachus (Linnaeus).

Science.gov (United States)

Kharbuli, Zaiba Y; Biswas, Kuheli; Saha, Nirmalendu

2007-12-01

The possible synthesis of citrulline, a rate limiting step for urea synthesis via the ornithine-urea cycle (OUC) in teleosts was tested both in the presence of ammonia and glutamine as nitrogen-donating substrates by the isolated liver mitochondria of ureogenic air-breathing walking catfish, C. batrachus. Both ammonia and glutamine could be used as nitrogen-donating substrates for the synthesis of citrulline by the isolated liver mitochondria, since the rate of citrulline synthesis was almost equal in presence of both the substrates. The citrulline synthesis by the isolated liver mitochondria requires succinate at a concentration of 0.1 mM as an energy source, and also requires the involvement of intramitochondrial carbonic anhydrase activity for supplying HCO3 as another substrate for citrulline synthesis. The rate of citrulline synthesis was further stimulated significantly by the isolated liver mitochondria of the fish after pre-exposure to 25 mM NH4Cl for 7 days. Due to possessing this biochemical adaptational strategy leading to the amelioration of ammonia toxicity mainly by channeling ammonia directly and/or via the formation of glutamine to the OUC, this air-breathing catfish could succeed in surviving in high external ammonia, which it faces in its natural habitat in certain seasons of the year.

Plasma Glutamine Concentrations in Liver Failure.

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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.

Essential Non-Translational Functions of tRNA Synthetases

Science.gov (United States)

Guo, Min; Schimmel, Paul

2013-01-01

Nontranslational functions of vertebrate aminoacyl tRNA synthetases (aaRSs), which catalyze the production of aminoacyl-tRNAs for protein synthesis, have recently been discovered. While these new functions were thought to be ‘moonlighting activities’, many are as critical for cellular homeostasis as the activity in translation. New roles have been associated with cytoplasmic forms as well as with nuclear and secreted extracellular forms that impact pathways for cardiovascular development, the immune response, and mTOR, IFN-γ and p53 signaling. The associations of aaRSs with autoimmune disorders, cancers and neurological disorders further highlight nontranslational functions of these proteins. Novel architecture elaborations of the aaRSs accompany their functional expansion in higher organisms and have been associated with the nontranslational functions for several aaRSs. While a general understanding of how these functions developed is limited, the expropriation of aaRSs for essential nontranslational functions may have been initiated by co-opting the amino acid binding site for another purpose. PMID:23416400

Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

International Nuclear Information System (INIS)

Fujita, Akiko; Sato, Chihiro; Kitajima, Ken.

2007-01-01

The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences

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.

The action of Saraca asoca Roxb. de Wilde bark on the PGH2 synthetase enzyme complex of the sheep vesicular gland.

Science.gov (United States)

Middelkoop, T B; Labadie, R P

1985-01-01

Extracts of S. asoca bark and pure compounds isolated from the bark were tested for properties that might inhibit the conversion of arachidonic acid by the PGH2 synthetase. They were assayed spectrophotometrically with adrenaline as cofactor. Methanol- and ethyl acetate extracts inhibited the conversion. The observed inhibition was confirmed in an oxygraphic assay. Two procyanidin dimers from the ethyl acetate extract showed enzyme catalyzed oxidation in our assay. The ether extract of the bark was also found to contain yet unknown substances which were capable of being oxidised by the PGH2 synthetase. The combined action of the components of the bark may explain the mode of action of the drug Asoka Aristha, the main ingredient of which is the bark of S. asoca. The drug is traditionally used in Sri Lanka to treat menorrhagia.

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.

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.

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.

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)

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

Nitrogen metabolism of the intestine during digestion in a teleost fish, the plainfin midshipman (Porichthys notatus).

Science.gov (United States)

Bucking, Carol; LeMoine, Christophe M R; Craig, Paul M; Walsh, Patrick J

2013-08-01

Digestion affects nitrogen metabolism in fish, as both exogenous and endogenous proteins and amino acids are catabolized, liberating ammonia in the process. Here we present a model of local detoxification of ammonia by the intestinal tissue of the plainfin midshipman (Porichthys notatus) during digestion, resulting in an increase in urea excretion of gastrointestinal origin. Corroborating evidence indicated whole-animal ammonia and urea excretion increased following feeding, and ammonia levels within the lumen of the midshipman intestine increased to high levels (1.8±0.4 μmol N g(-1)). We propose that this ammonia entered the enterocytes and was detoxified to urea via the ornithine-urea cycle (O-UC) enzymes, as evidenced by a 1.5- to 2.9-fold post-prandial increase in glutamine synthetase activity (0.14±0.05 and 0.28±0.02 μmol min(-1) g(-1) versus 0.41±0.03 μmol min(-1) g(-1)) and an 8.7-fold increase in carbamoyl phosphate synthetase III activity (0.3±1.2 versus 2.6±0.4 nmol min(-1) g(-1)). Furthermore, digestion increased urea production by isolated gastrointestinal tissue 1.7-fold, supporting our hypothesis that intestinal tissue synthesizes urea in response to feeding. We further propose that the intestinal urea may have been excreted into the intestinal lumen via an apical urea transporter as visualized using immunohistochemistry. A portion of the urea was then excreted to the environment along with the feces, resulting in the observed increase in urea excretion, while another portion may have been used by intestinal ureolytic bacteria. Overall, we propose that P. notatus produces urea within the enterocytes via a functional O-UC, which is then excreted into the intestinal lumen. Our model of intestinal nitrogen metabolism does not appear to be universal as we were unab le to activate the O-UC in the intestine of fed rainbow trout. However, literature values suggest that multiple fish species could follow this model.

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

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.)

Physiological regulation of isocitrate dehydrogenase and the role of 2-oxoglutarate in Prochlorococcus sp. strain PCC 9511.

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María Agustina Domínguez-Martín

Full Text Available The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42 catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.

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.

Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of 'Honeycrisp' apple (Malus domestica Borkh) with excessive accumulation of carbohydrates.

Science.gov (United States)

Wang, Huicong; Ma, Fangfang; Cheng, Lailiang

2010-07-01

Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of 'Honeycrisp' apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO(2) assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NAD-malate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADH-dependent glutamate synthase, and glutamate pyruvate transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to "consume" the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis.

Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion

Science.gov (United States)

Mitch, William E.; Sands, Jeff M.

2015-01-01

Renal nitrogen metabolism primarily involves urea and ammonia metabolism, and is essential to normal health. Urea is the largest circulating pool of nitrogen, excluding nitrogen in circulating proteins, and its production changes in parallel to the degradation of dietary and endogenous proteins. In addition to serving as a way to excrete nitrogen, urea transport, mediated through specific urea transport proteins, mediates a central role in the urine concentrating mechanism. Renal ammonia excretion, although often considered only in the context of acid-base homeostasis, accounts for approximately 10% of total renal nitrogen excretion under basal conditions, but can increase substantially in a variety of clinical conditions. Because renal ammonia metabolism requires intrarenal ammoniagenesis from glutamine, changes in factors regulating renal ammonia metabolism can have important effects on glutamine in addition to nitrogen balance. This review covers aspects of protein metabolism and the control of the two major molecules involved in renal nitrogen excretion: urea and ammonia. Both urea and ammonia transport can be altered by glucocorticoids and hypokalemia, two conditions that also affect protein metabolism. Clinical conditions associated with altered urine concentrating ability or water homeostasis can result in changes in urea excretion and urea transporters. Clinical conditions associated with altered ammonia excretion can have important effects on nitrogen balance. PMID:25078422

Zn-biofortification enhanced nitrogen metabolism and photorespiration process in green leafy vegetable Lactuca sativa L.

Science.gov (United States)

Barrameda-Medina, Yurena; Lentini, Marco; Esposito, Sergio; Ruiz, Juan M; Blasco, Begoña

2017-04-01

Excessive rates of nitrogen (N) fertilizers may result in elevated concentrations of nitrate (NO 3 - ) in plants. Considering that many programs of biofortification with trace elements are being performed, it has become important to study how the application of these elements affects plant physiology and, particularly, N utilization in leaf crops. The main objective of the present study was to determine whether the NO 3 - accumulation and the nitrogen use efficiency was affected by the application of different doses of Zn in Lactuca sativa plants. Zn doses in the range 80-100 µmol L -1 produced an increase in Zn concentration provoking a decrease of NO 3 - concentration and increase of the nitrate reductase, glutamine synthetase and aspartate aminotransferase activities, as well as the photorespiration processes. As result, we observed an increase in reduced N, total N concentration and N utilization efficiency. Consequently, at a dose of 80 µmol L -1 of Zn, the amino acid concentration increased significantly. Adequate Zn fertilization is an important critical player in lettuce, especially at a dose of 80 µmol L -1 of Zn, because it could result in an increase in the Zn concentration, a reduction of NO 3 - levels and an increase the concentration of essential amino acids, with all of them having beneficial properties for the human diet. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Homogeneous dihydroxylation of olefins catalyzed by OsO(4)(2-) immobilized on a dendritic backbone with a tertiary nitrogen at its core position.

Science.gov (United States)

Fujita, Ken-Ichi; Inoue, Kensuke; Tsuchimoto, Teruhisa; Yasuda, Hiroyuki

2012-01-01

OsO(4)(2-) immobilized on a poly(benzyl ether) dendrimer with a tertiary nitrogen at its core position efficiently catalyzed the homogeneous dihydroxylation of olefins with a low level of osmium leaching. The dendritic osmium catalyst could be applied to the wide range of olefins. Furthermore, the dendritic osmium catalyst was recovered by reprecipitation and then reused up to five times.

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.

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

Ammonium and methylammonium transport in Rhodobacter sphaeroides

Energy Technology Data Exchange (ETDEWEB)

Cordts, M.L.; Gibson, J.

1987-04-01

Rhodobacter spheroides maintained intracellular ammonium pools of 1.1 to 2.6 mM during growth in several fixed nitrogen sources as well as during diazotrophic growth. Addition of 0.15 mM NH/sub 4//sup +/ to washed, nitrogen-free cell suspensions was followed by linear uptake of NH/sub 4//sup +/ from the medium and transient formation of intracellular pools of 0.9 to 1.5 mM NH/sub 4//sup +/. Transport of NH/sub 4//sup +/ was shown to be independent of assimilation by glutamine synthetase because intracellular pools of over 1 mM represented NH/sub 4//sup +/ concentration gradients of at least 100-fold across the cytoplasmic membrane. Ammonium pools of over 1 mM were also found in non-growing cell suspensions in nitrogen-free medium after glutamine synthetase was inhibited with methionine sulfoximine. In NH/sub 4//sup +/-free cell suspensions, methylammonium (/sup 14/CH/sub 3/NH/sub 3//sup +/) was taken up rapidly, and intracellular concentrations of 0.4 to 0.5 mM were maintained. The /sup 14/CN/sub 3/NH/sub 3//sup +/ pool was not affected by methionine sulfoximine. Unlike NH/sub 4//sup +/ uptake, /sup 14/CH/sub 3/NH/sub 3//sup +/ uptake in nitrogen-free cell suspensions was repressed by growth in NH/sub 4//sup +/. These results suggest that R. sphaeroides may produce an NH/sub 4//sup +/-specific transport system in addition to the NH/sub 4//sup +///sup 14/CH/sub 3/NH/sub 3//sup +/ transporter. This second transporter is able to produce normal-size NH/sub 4//sup +/ pools but has very little affinity for /sup 14/CH/sub 3/NH/sub 3//sup +/ and is not repressed by growth in high concentrations of NH/sub 4//sup +/.

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.

Coupled effects of light and nitrogen source on the urea cycle and nitrogen metabolism over a diel cycle in the marine diatom Thalassiosira pseudonana.

Science.gov (United States)

Bender, Sara J; Parker, Micaela S; Armbrust, E Virginia

2012-03-01

Diatoms are photoautotrophic organisms capable of growing on a variety of inorganic and organic nitrogen sources. Discovery of a complete urea cycle in diatoms was surprising, as this pathway commonly functions in heterotrophic organisms to rid cells of waste nitrogen. To determine how the urea cycle is integrated into cellular nitrogen metabolism and energy management, the centric diatom Thalassiosira pseudonana was maintained in semi-continuous batch cultures on nitrate, ammonium, or urea as the sole nitrogen source, under a 16: 8 light: dark cycle and at light intensities that were low, saturating, or high for growth. Steady-state transcript levels were determined for genes encoding enzymes linked to the urea cycle, urea hydrolysis, glutamine synthesis, pyrimidine synthesis, photorespiration, and energy storage. Transcript abundances were significantly affected by nitrogen source, light intensity and a diel cycle. The impact of N source on differential transcript accumulation was most apparent under the highest light intensity. Models of cellular metabolism under high light were developed based on changes in transcript abundance and predicted enzyme localizations. We hypothesize that the urea cycle is integrated into nitrogen metabolism through its connection to glutamine and in the eventual production of urea. These findings have important implications for nitrogen flow in the cell over diel cycles at surface ocean irradiances. Copyright © 2011 Elsevier GmbH. All rights reserved.

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

In situ autoradiographic detection of folylpolyglutamate synthetase activity

International Nuclear Information System (INIS)

Sussman, D.J.; Milman, G.; Osborne, C.; Shane, B.

1986-01-01

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6- 3 H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6- 3 H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which functions in mammalian cells

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...

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...

Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

OpenAIRE

Matussek, Karl; Moritz, Patrick; Brunner, Nina; Eckerskorn, Christoph; Hensel, Reinhard

1998-01-01

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and...

Glutamine and glutamate: Nonessential or essential amino acids?

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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

Enzymology of asparagine metabolism

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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.

Endogenous glutamine decrease is associated with pancreatic cancer progression.

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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.

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

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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)

Introduction to the Glutamate-Glutamine Cycle.

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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.

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

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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.

Mammalian folylpoly-γ-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

International Nuclear Information System (INIS)

Cichowicz, D.J.; Shane, B.

1987-01-01

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of M/sub r/ 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K + was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the K/sub m/ value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied

Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

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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.

Characterisation of a major enzyme of bovine nitrogen metabolism

CSIR Research Space (South Africa)

Mathomu, LM

2010-09-01

Full Text Available of cellular protein metabolism (Curthoys & Watford, 1995; Meister, 1974). Glutamine functions as a major inter-organ transport form of nitrogen, carbon and serves as a source of energy between tissues such as brain, liver, kidney and even muscles...

GLUTAMINE AND HYPERAMMONEMIC CRISES IN PATIENTS WITH UREA CYCLE DISORDERS

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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

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

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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

Thalli Growth, Propagule Survival, and Integrated Physiological Response to Nitrogen Stress of Ramalina calicaris var. japonica in Shennongjia Mountain (China

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Chuan-Hua Wang

2018-05-01

Full Text Available In this study, effects of nitrogen (N availability on growth, survival of Ramalina calicaris var. japonica, and whether it respond nitrogen stress in an integrated physiological way was evaluated. Thalli growth and propagule survival, thalli N and phosphorus (P content, and activity of phosphomonoesterase (PME of R. calicaris var. japonica were determined in a field experiment. Its differentiate adsorption in ammonia and nitrate, the activity of glutamine synthetase (GSA and nitrate reductase (NRA also were investigated in a series of indoor experiments. The results showed that N deposition significantly decreased the growth and survival of this lichen, and the N sensitivity threshold was suggested at 6.0 kg N⋅ha-1⋅y-1. When the N deposition increased from 8.59 kg N⋅ha-1⋅y-1 to 14.24, 20.49, 32.99 and 57.99 kg N⋅ha-1⋅y-1, the growth rates of lichen thalli decreased by 26.47, 39.01, 52.18 and 60.3%, respectively; Whereas the survival rate of the lichen propagules decreased from 92.8% of control (0.0 kg N⋅ha-1⋅y-1 to 10.7% of 50.0 kg N⋅ha-1⋅y-1, when they were treated with 0.00, 6.25, 12.5, 25.0, and 50.0 kg N⋅ha-1⋅y-1 deposition. Compared with an adequate adsorption of ammonium N, no nitrate adsorption occurred when thalli was submerged in solution lower than 0.4 mM. Our results also suggested that thalli total nitrogen, N:P ratio increased with N availability, and the activity of PME was significantly correlated with thalli total nitrogen. These all indicated that phosphorus limitation occurred when R. calicaris var. japonica treated with higher nitrogen deposition. Compared with slightly effects of NRA, GSA of R. calicaris var. japonica responded nitrogen availability significantly; In addition, GSA and NRA negatively correlated with thalli growth rate and propagule survival significantly. These results indicated that nitrogen stress do decrease growth and survival of R. calicaris var. japonica, and lichen would be

Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1.

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Gutiérrez, Rodrigo A; Stokes, Trevor L; Thum, Karen; Xu, Xiaodong; Obertello, Mariana; Katari, Manpreet S; Tanurdzic, Milos; Dean, Alexis; Nero, Damion C; McClung, C Robertson; Coruzzi, Gloria M

2008-03-25

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae.

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Ambrosio, Rafael; Ortiz-Marquez, Juan Cesar Federico; Curatti, Leonardo

2017-03-01

The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N 2 and CO 2 from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N 2 from the air into crops. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All

Nitrogen and carbon relationships between the parasitic weed Orobanche foetida and susceptible and tolerant faba bean lines.

Science.gov (United States)

Abbes, Zouhaier; Kharrat, Mohamed; Delavault, Philippe; Chaïbi, Wided; Simier, Philippe

2009-02-01

The parasitic weed Orobanche foetida (Poiret) is an emergent agronomical problem on faba bean in Tunisia. The Tunisian breeding programs for faba bean resistance to O. foetida have produced several tolerant lines including the line XBJ90.03-16-1-1-1, which limits both parasite attachments to the host roots and growth of the attached parasites. The present study aims to provide a better understanding of the nutritional relationships between the parasite and this tolerant line in comparison with the susceptible Bachaar genotype. Phloem saps of faba bean were harvested using phloem exudation experiments. The major organic compounds potentially transferred from both faba bean genotypes to the parasite were identified as sucrose, raffinose, stachyose, citrate, malate, asparagine (ASN), aspartate (ASP), glutamine, glutamate, serine, alanine and GABA. However, the phloem exudates of the tolerant line were highly deficient in nitrogen when compared to that of the susceptible line. When attached to roots of the tolerant line, the parasite displayed limited activities of soluble invertases in tubercles, and especially in shoots, suggesting that the low performance of the broomrapes attached to the tolerant line resulted from a reduced capacity to utilize the host-derived carbohydrates. On the other hand, the mechanisms involved in the osmotic adjustment and primary metabolism of the parasite did not differ significantly according to the host genotype: mineral cations, especially potassium and calcium, predominated as the major osmotically-active compounds in both tubercles and shoots; shoots accumulated preferentially hexoses as organic solutes although tubercles accumulated preferentially starch and soluble amino acids, especially ASP and ASN. This suggests an important role for a glutamine-dependent asparagine synthetase (EC 6.3.5.4) in the N metabolism of the parasite.

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.)

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.)

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

Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.

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Yoko Kimata-Ariga

Full Text Available Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT, nitrite reductase (NiR and glutamine synthetase (GS, separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE. GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa and multiple sizes (>120 kDa, respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

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

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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.

Title: Potassium application regulates nitrogen metabolism and osmotic adjustment in cotton (Gossypium hirsutum L.) functional leaf under drought stress.

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Zahoor, Rizwan; Zhao, Wenqing; Abid, Muhammad; Dong, Haoran; Zhou, Zhiguo

2017-08-01

To evaluate the role of potassium (K) in maintaining nitrogen metabolism and osmotic adjustment development of cotton functional leaves to sustain growth under soil drought and rewatering conditions, the plants of two cotton cultivars Siza 3 (low-K sensitive) and Simian 3 (low-K tolerant), were grown under three different K rates (K0, K1, and K2; 0, 150, and 300kgK 2 Oha -1 , respectively) and exposed to drought stress with 40±5% soil relative water content (SRWC). The drought stress was applied at flowering stage by withholding water for eight days followed by rewatering to a well-watered level (75±5% SRWC). The results showed that drought-stressed plants of both cultivars showed a decrease in leaf relative water content (RWC) and osmotic potential in the functional leaves and developed osmotic adjustment with an increase in the contents of free amino acids, soluble sugars, inorganic K, and nitrate as compared to well-watered plants. In drought-stressed plants, nitrogen-metabolizing enzyme activities of nitrogen reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT) were diminished significantly (P≤0.05) along with decreased chlorophyll content and soluble proteins. However, drought-stressed plants under K application not only exhibited higher osmotic adjustment with greater accumulation of osmolytes but also regulated nitrogen metabolism by maintaining higher enzyme activities, soluble proteins, and chlorophyll content in functional leaves as compared to the plants without K application. Siza 3 showed better stability in enzyme activities and resulted in 89% higher seed cotton yield under K2 as compared to K0 in drought-stressed plants, whereas this increase was 53% in the case of Simian 3. The results of the study suggested that K application enhances cotton plants' potential for sustaining high nitrogen-metabolizing enzyme activities and related components to supplement osmotic adjustment under soil drought conditions. Copyright © 2017

Effects of CO2 enrichment on photosynthesis, growth, and nitrogen metabolism of the seagrass Zostera noltii

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Alexandre, Ana; Silva, João; Buapet, Pimchanok; Björk, Mats; Santos, Rui

2012-01-01

Seagrass ecosystems are expected to benefit from the global increase in CO2 in the ocean because the photosynthetic rate of these plants may be Ci-limited at the current CO2 level. As well, it is expected that lower external pH will facilitate the nitrate uptake of seagrasses if nitrate is cotransported with H+ across the membrane as in terrestrial plants. Here, we investigate the effects of CO2 enrichment on both carbon and nitrogen metabolism of the seagrass Zostera noltii in a mesocosm experiment where plants were exposed for 5 months to two experimental CO2 concentrations (360 and 700 ppm). Both the maximum photosynthetic rate (Pm) and photosynthetic efficiency (α) were higher (1.3- and 4.1-fold, respectively) in plants exposed to CO2-enriched conditions. On the other hand, no significant effects of CO2 enrichment on leaf growth rates were observed, probably due to nitrogen limitation as revealed by the low nitrogen content of leaves. The leaf ammonium uptake rate and glutamine synthetase activity were not significantly affected by increased CO2 concentrations. On the other hand, the leaf nitrate uptake rate of plants exposed to CO2-enriched conditions was fourfold lower than the uptake of plants exposed to current CO2 level, suggesting that in the seagrass Z. noltii nitrate is not cotransported with H+ as in terrestrial plants. In contrast, the activity of nitrate reductase was threefold higher in plant leaves grown at high-CO2 concentrations. Our results suggest that the global effects of CO2 on seagrass production may be spatially heterogeneous and depend on the specific nitrogen availability of each system. Under a CO2 increase scenario, the natural levels of nutrients will probably become limiting for Z. noltii. This potential limitation becomes more relevant because the expected positive effect of CO2 increase on nitrate uptake rate was not confirmed. PMID:23145346

Effects of CO(2) enrichment on photosynthesis, growth, and nitrogen metabolism of the seagrass Zostera noltii.

Science.gov (United States)

Alexandre, Ana; Silva, João; Buapet, Pimchanok; Björk, Mats; Santos, Rui

2012-10-01

Seagrass ecosystems are expected to benefit from the global increase in CO(2) in the ocean because the photosynthetic rate of these plants may be C(i)-limited at the current CO(2) level. As well, it is expected that lower external pH will facilitate the nitrate uptake of seagrasses if nitrate is cotransported with H(+) across the membrane as in terrestrial plants. Here, we investigate the effects of CO(2) enrichment on both carbon and nitrogen metabolism of the seagrass Zostera noltii in a mesocosm experiment where plants were exposed for 5 months to two experimental CO(2) concentrations (360 and 700 ppm). Both the maximum photosynthetic rate (P(m)) and photosynthetic efficiency (α) were higher (1.3- and 4.1-fold, respectively) in plants exposed to CO(2)-enriched conditions. On the other hand, no significant effects of CO(2) enrichment on leaf growth rates were observed, probably due to nitrogen limitation as revealed by the low nitrogen content of leaves. The leaf ammonium uptake rate and glutamine synthetase activity were not significantly affected by increased CO(2) concentrations. On the other hand, the leaf nitrate uptake rate of plants exposed to CO(2)-enriched conditions was fourfold lower than the uptake of plants exposed to current CO(2) level, suggesting that in the seagrass Z. noltii nitrate is not cotransported with H(+) as in terrestrial plants. In contrast, the activity of nitrate reductase was threefold higher in plant leaves grown at high-CO(2) concentrations. Our results suggest that the global effects of CO(2) on seagrass production may be spatially heterogeneous and depend on the specific nitrogen availability of each system. Under a CO(2) increase scenario, the natural levels of nutrients will probably become limiting for Z. noltii. This potential limitation becomes more relevant because the expected positive effect of CO(2) increase on nitrate uptake rate was not confirmed.

Beauvericin synthetase contains a calmodulin binding motif in the entomopathogenic fungus Beauveria bassiana.

Science.gov (United States)

Kim, Jiyoung; Sung, Gi-Ho

2018-03-19

Beauvericin is a mycotoxin which has insecticidal, anti-microbial, anti-viral and anti-cancer activities. Beauvericin biosynthesis is rapidly catalyzed by the beauvericin synthetase (BEAS) in Beauveria bassiana. Ca 2+ plays crucial roles in multiple signaling pathways in eukaryotic cells. These Ca 2+ signals are partially decoded by Ca 2+ sensor calmodulin (CaM). In this report, we describe that B. bassiana BEAS (BbBEAS) can interact with CaM in a Ca 2+ -dependent manner. A synthetic BbBEAS peptide, corresponding to the putative CaM-binding motif, formed a stable complex with CaM in the presence of Ca 2+ . In addition, in vitro CaM-binding assay revealed that the His-tagged BbBEAS (amino acids 2421-2538) binds to CaM in a Ca 2+ -dependent manner. Therefore, this work suggests that BbBEAS is a novel CaM-binding protein in B. bassiana.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

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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

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.

Ecology of nitrogen fixation in soils and rhizospheres. Pt. 5

Energy Technology Data Exchange (ETDEWEB)

Werner, D.; Stripf, R.; Abramowski, R.; Fiedler, U.

1980-12-01

The effects of reduced oxygen concentration on root growth and activities of enzymes of N-metabolism of wheat (Triticum aestivum var. Kolibri) have been studied, for low O/sub 2/ tensions are required for N/sub 2/ fixation by microaerophilic bacteria (e.g. Azospirillum) associated with root systems of grasses. In hydrocultures with oxygen concentrations in the range of 0.2 to 1 mg O/sub 2/ x 1/sup -1/ compared to aerated cultures (8-9 mg O/sub 2/ x 1/sup -1/) root growth was reduced from 10 mg fresh weight x day/sup -1/ x plant/sup -1/ to one tenth 15 to 30 d after sowing. Specific activity of NADH and NADPH dependent glutamate dehydrogenase (E.C. 1.4.1.2 and 1.4.1.4) is reduced by 50% in the cultures with low oxygen concentrations 20 to 30 days after sowing, whereas specific activity of aspartate aminotransferase (E.C. 2.6.1.1) and alanine amino transferase (E.C. 2.6.1.2) is enhanced by a factor of two to three. Specific activity of glutamine synthetase is almost unaffected. Specific activity of glutamate dehydrogenase is lowest in the root tips, medium in young root hair zone and highest in the old root hair zone, glutamine synthetase activity is reverse in the three zones with differences by a factor of 3-5; aspartate aminotransferase is similarly active in the three zones. Nitrate concentration used (100 ..mu..M) for cultivation of the wheat plants was tested with Azospirillum brasilense in pure culture on agar surfaces exposed to air at the same pH (5.8), used for cultivation of the wheat plants. Activiy after a 14 day period (peak activity 70 mmol C/sub 2/H/sub 4/ x mg protein/sup -1/ x h/sup -1/) was not affected, however 1 mM and 5 mM nitrate added reduced the total activity to 50% and 10% respectively.

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

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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.

Synthesis of pyrrolo(2,3-b)quinolines by palladium-catalyzed heteroannulation

International Nuclear Information System (INIS)

Gee, Moon Bae; Lee, Won Jung; Yum, Eul Kgun

2003-01-01

Palladium-catalyzed heteroannulation of 2-amino-3-iodoquinoline derivatives and 1-trimethylsilyl internal alkynes provided highly regioselective pyrrolo(2,3-b)quinolines with trimethylsilyl group next to the nitrogen atom in the pyrrole ring

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)

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.

Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

International Nuclear Information System (INIS)

Bao, Baolong; Wijeratne, Subhashinee S.K.; Rodriguez-Melendez, Rocio; Zempleni, Janos

2011-01-01

Highlights: → Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). → Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. → HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

BLOOD AMMONIA AND GLUTAMINE AS PREDICTORS OF HYPERAMMONEMIC CRISES IN UREA CYCLE DISORDER PATIENTS

Science.gov (United States)

Lee, Brendan; Diaz, George A.; Rhead, William; Lichter-Konecki, U.; Feigenbaum, Annette; Berry, Susan A.; Le Mons, C.; Bartley, James A; Longo, Nicola; Nagamani, Sandesh C.; Berquist, William; Gallagher, Renata; Bartholomew, Dennis; Harding, Cary O.; Korson, Mark S.; McCandless, Shawn E.; Smith, Wendy; Cederbaum, Stephen; Wong, Derek; Merritt, J. Lawrence; Schulze, A.; Vockley, Gerard.; Kronn, David; Zori, Roberto; Summar, Marshall; Milikien, D.A.; Marino, M.; Coakley, D.F.; Mokhtarani, M.; Scharschmidt, B.F.

2014-01-01

Purpose To examine predictors of ammonia exposure and hyperammonemic crises (HAC) in patients with urea cycle disorders (UCDs). Methods The relationships between fasting ammonia, daily ammonia exposure, and HACs were analyzed in >100 UCD patients. Results Fasting ammonia correlated strongly with daily ammonia exposure (r=0.764, p200% (purea nitrogen. Fasting glutamine correlated weakly with AUC0-24 and was not a significant predictor of HACs. Conclusions Fasting ammonia correlates strongly and positively with daily ammonia exposure and with the risk and rate of HACs, suggesting that UCD patients may benefit from tight ammonia control. PMID:25503497

Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

KAUST Repository

Hohl, Adrian

2017-12-06

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.

Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

KAUST Repository

Hohl, Adrian; Karan, Ram; Akal, Anstassja; Renn, Dominik; Liu, Xuechao; Dharamarajnadar, Alaguraj; Ghoprade, Seema Arun; Groll, Michael; Rueping, Magnus; Eppinger, Jö rg

2017-01-01

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.

Identification and in vitro analysis of the GatD/MurT enzyme-complex catalyzing lipid II amidation in Staphylococcus aureus.

Directory of Open Access Journals (Sweden)

Daniela Münch

2012-01-01

Full Text Available The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly₅. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly₅ may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.

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.

A proteomic network for symbiotic nitrogen fixation efficiency in Bradyrhizobium elkanii

Science.gov (United States)

Rhizobia bacteroids colonize legumes and reduce N2 to NH3 in root nodules. The current model is that bacteroids avoid assimilating this NH3. Instead, the legume forms glutamine from it, the nitrogen of which is returned to the bacteroid as leucine, isoleucine, valine, dicarboxylates, and peptides. I...

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.)

The Nitrogen Moieties of Dietary Nonessential Amino Acids Are Distinctively Metabolized in the Gut and Distributed to the Circulation in Rats.

Science.gov (United States)

Nakamura, Hidehiro; Kawamata, Yasuko; Kuwahara, Tomomi; Sakai, Ryosei

2017-08-01

Background: Although previous growth studies in rodents have indicated the importance of dietary nonessential amino acids (NEAAs) as nitrogen sources, individual NEAAs have different growth-promoting activities. This phenomenon might be attributable to differences in the nitrogen metabolism of individual NEAAs. Objective: The aim of this study was to compare nitrogen metabolism across dietary NEAAs with the use of their 15 N isotopologues. Methods: Male Fischer rats (8 wk old) were given 1.0 g amino acid-defined diets containing either 15 N-labeled glutamate, glutamine (amino or amide), aspartate, alanine, proline, glycine, or serine hourly for 5-6 h. Then, steady-state amino acid concentrations and their 15 N enrichments in the gut and in portal and arterial plasma were measured by an amino acid analyzer and LC tandem mass spectrometry, respectively. Results: The intestinal 15 N distribution and portal-arterial balance of 15 N metabolites indicated that most dietary glutamate nitrogen (>90% of dietary input) was incorporated into various amino acids, including alanine, proline, and citrulline, in the gut. Dietary aspartate nitrogen, alanine nitrogen, and amino nitrogen of glutamine were distributed similarly to other amino acids both in the gut and in the circulation. In contrast, incorporation of the nitrogen moieties of dietary proline, serine, and glycine into other amino acids was less than that of other NEAAs, although interconversion between serine and glycine was very active. Cluster analysis of 15 N enrichment data also indicated that dietary glutamate nitrogen, aspartate nitrogen, alanine nitrogen, and the amino nitrogen of glutamine were distributed similarly to intestinal and circulating amino acids. Further, the analysis revealed close relations between intestinal and arterial 15 N enrichment for each amino acid. The steady-state 15 N enrichment of arterial amino acids indicated that substantial amounts of circulating amino acid nitrogen are derived

Carrier gas effects on aluminum-catalyzed nanowire growth

International Nuclear Information System (INIS)

Ke, Yue; Hainey, Mel Jr; Won, Dongjin; Weng, Xiaojun; Eichfeld, Sarah M; Redwing, Joan M

2016-01-01

Aluminum-catalyzed silicon nanowire growth under low-pressure chemical vapor deposition conditions requires higher reactor pressures than gold-catalyzed growth, but the reasons for this difference are not well understood. In this study, the effects of reactor pressure and hydrogen partial pressure on silicon nanowire growth using an aluminum catalyst were studied by growing nanowires in hydrogen and hydrogen/nitrogen carrier gas mixtures at different total reactor pressures. Nanowires grown in the nitrogen/hydrogen mixture have faceted catalyst droplet tips, minimal evidence of aluminum diffusion from the tip down the nanowire sidewalls, and significant vapor–solid deposition of silicon on the sidewalls. In comparison, wires grown in pure hydrogen show less well-defined tips, evidence of aluminum diffusion down the nanowire sidewalls at increasing reactor pressures and reduced vapor–solid deposition of silicon on the sidewalls. The results are explained in terms of a model wherein the hydrogen partial pressure plays a critical role in aluminum-catalyzed nanowire growth by controlling hydrogen termination of the silicon nanowire sidewalls. For a given reactor pressure, increased hydrogen partial pressures increase the extent of hydrogen termination of the sidewalls which suppresses SiH_4 adsorption thereby reducing vapor–solid deposition of silicon but increases the surface diffusion length of aluminum. Conversely, lower hydrogen partial pressures reduce the hydrogen termination and also increase the extent of SiH_4 gas phase decomposition, shifting the nanowire growth window to lower growth temperatures and silane partial pressures. (paper)

Inactivation of the glutamine/amino acid transporter ASCT2 by 1,2,3-dithiazoles: proteoliposomes as a tool to gain insights in the molecular mechanism of action and of antitumor activity

International Nuclear Information System (INIS)

Oppedisano, Francesca; Catto, Marco; Koutentis, Panayiotis A.; Nicolotti, Orazio; Pochini, Lorena; Koyioni, Maria; Introcaso, Antonellina; Michaelidou, Sophia S.; Carotti, Angelo; Indiveri, Cesare

2012-01-01

The ASCT2 transport system catalyses a sodium-dependent antiport of glutamine and other neutral amino acids which is involved in amino acid metabolism. A library of 1,2,3-dithiazoles was designed, synthesized and evaluated as inhibitors of the glutamine/amino acid ASCT2 transporter in the model system of proteoliposomes reconstituted with the rat liver transporter. Fifteen of the tested compounds at concentration of 20 μM or below, inhibited more than 50% the glutamine/glutamine antiport catalysed by the reconstituted transporter. These good inhibitors bear a phenyl ring with electron withdrawing substituents. The inhibition was reversed by 1,4-dithioerythritol indicating that the effect was likely owed to the formation of mixed sulfides with the protein's Cys residue(s). A dose–response analysis of the most active compounds gave IC 50 values in the range of 3–30 μM. Kinetic inhibition studies indicated a non-competitive inhibition, presumably because of a potential covalent interaction of the dithiazoles with cysteine thiol groups that are not located at the substrate binding site. Indeed, computational studies using a homology structural model of ASCT2 transporter, suggested as possible binding targets, Cys-207 or Cys-210, that belong to the CXXC motif of the protein. -- Highlights: ► Non‐competitive inhibition of ASCT2 by 1,2,3-dithiazoles was studied in proteoliposomes. ► Different 1,2,3-dithiazoles were synthesized and evaluated as transporter inhibitors. ► Many compounds potently inhibited the glutamine/glutamine antiport catalyzed by ASCT2. ► The inhibition was reversed by DTE indicating reaction with protein Cys. ► The most active compounds gave IC 50 in the range of 3–30 μM.

Inactivation of the glutamine/amino acid transporter ASCT2 by 1,2,3-dithiazoles: proteoliposomes as a tool to gain insights in the molecular mechanism of action and of antitumor activity

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Oppedisano, Francesca [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy); Catto, Marco [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Koutentis, Panayiotis A. [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Nicolotti, Orazio [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Pochini, Lorena [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy); Koyioni, Maria [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Introcaso, Antonellina [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Michaelidou, Sophia S. [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Carotti, Angelo, E-mail: carotti@farmchim.uniba.it [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Indiveri, Cesare, E-mail: indiveri@unical.it [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy)

2012-11-15

The ASCT2 transport system catalyses a sodium-dependent antiport of glutamine and other neutral amino acids which is involved in amino acid metabolism. A library of 1,2,3-dithiazoles was designed, synthesized and evaluated as inhibitors of the glutamine/amino acid ASCT2 transporter in the model system of proteoliposomes reconstituted with the rat liver transporter. Fifteen of the tested compounds at concentration of 20 μM or below, inhibited more than 50% the glutamine/glutamine antiport catalysed by the reconstituted transporter. These good inhibitors bear a phenyl ring with electron withdrawing substituents. The inhibition was reversed by 1,4-dithioerythritol indicating that the effect was likely owed to the formation of mixed sulfides with the protein's Cys residue(s). A dose–response analysis of the most active compounds gave IC{sub 50} values in the range of 3–30 μM. Kinetic inhibition studies indicated a non-competitive inhibition, presumably because of a potential covalent interaction of the dithiazoles with cysteine thiol groups that are not located at the substrate binding site. Indeed, computational studies using a homology structural model of ASCT2 transporter, suggested as possible binding targets, Cys-207 or Cys-210, that belong to the CXXC motif of the protein. -- Highlights: ► Non‐competitive inhibition of ASCT2 by 1,2,3-dithiazoles was studied in proteoliposomes. ► Different 1,2,3-dithiazoles were synthesized and evaluated as transporter inhibitors. ► Many compounds potently inhibited the glutamine/glutamine antiport catalyzed by ASCT2. ► The inhibition was reversed by DTE indicating reaction with protein Cys. ► The most active compounds gave IC{sub 50} in the range of 3–30 μM.

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.).

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Mihaljević, Snježana; Radić, Sandra; Bauer, Nataša; Garić, Rade; Mihaljević, Branka; Horvat, Gordana; Leljak-Levanić, Dunja; Jelaska, Sibila

2011-11-01

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin. Copyright © 2011 Elsevier GmbH. All rights reserved.

Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor.

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Su, Fei; Takaya, Naoki; Shoun, Hirofumi

2004-02-01

Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.

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

Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3

International Nuclear Information System (INIS)

Wang, Xiaoying; Akasaka, Ryogo; Takemoto, Chie; Morita, Satoshi; Yamaguchi, Machiko; Terada, Takaho; Shirozu, Mikako; Yokoyama, Shigeyuki; Chen, Shilin; Si, Shuyi; Xie, Yong

2011-01-01

A hyperthermophilic adenylosuccinate synthetase from P. horikoshii OT3, which is 90–120 amino acids shorter than those from the vast majority of organisms, was expressed, purified and crystallized and X-ray diffraction data were collected to 2.5 Å resolution. Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430–457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90–120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3 2 12, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å 3 Da −1 and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution

Effects of lanthanum(III) on nitrogen metabolism of soybean seedlings under elevated UV-B radiation.

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Cao, Rui; Huang, Xiao-hua; Zhou, Qing; Cheng, Xiao-ying

2007-01-01

The hydroponic culture experiments of soybean bean seedlings were conducted to investigate the effect of lanthanum (La) on nitrogen metabolism under two different levels of elevated UV-B radiation (UV-B, 280-320 nm). The whole process of nitrogen metabolism involves uptake and transport of nitrate, nitrate assimilation, ammonium assimilation, amino acid biosynthesis, and protein synthesis. Compared with the control, UV-B radiation with the intensity of low level 0.15 W/m2 and high level 0.45 W/m2 significantly affected the whole nitrogen metabolism in soybean seedlings (p nitrogen-metabolism-related enzymes, such as: nitrate reductase (NR) to the nitrate reduction, glutamine systhetase (GS) and glutamine synthase (GOGAT) to the ammonia assimilation, while it increased the content of free amino acids and decreased that of soluble protein as well. The damage effect of high level of UV-B radiation on nitrogen metabolism was greater than that of low level. And UV-B radiation promoted the activity of the anti-adversity enzyme glutamate dehydrogenase (GDH), which reduced the toxicity of excess ammonia in plant. After pretreatment with the optimum concentration of La (20 mg/L), La could increase the activity of NR, GS, GOGAT, and GDH, and ammonia assimilation, but decrease nitrate and ammonia accumulation. In conclusion, La could relieve the damage effect of UV-B radiation on plant by regulating nitrogen metabolism process, and its alleviating effect under low level was better than that under the high one.

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

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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.

[Effects of different NO3--N/NH4+-N ratios on cucumber seedlings growth, nitrogen absorption and metabolism under suboptimal temperature and light intensity].

Science.gov (United States)

Zhang, Xiao Cui; Liu, Yu Mei; Bai, Long Qiang; He, Chao Xing; Yu, Xian Chang; Li, Yan Su

2016-08-01

Cucumber (cv. Zhongnong 26) was used as material, the effects of NO 3 - -N/NH 4 + -N ratios on growth and physiological characteristics of cucumber seedlings under suboptimal temperature and light intensity (18 ℃/10 ℃,180 ± 20 μmol·m -2 ·s -1 ) were studied. Total nitrogen in the nutrient solution was equal and three NO 3 - -N/NH 4 + -N ratios, 26:2, 21:7 and 14:14, were applied as treatments. The results showed that cucumber treated by NO 3 - -N/NH 4 + -N=21:7 had the longest total root length, the biggest root volume and root surface area, and the maximum number of root tips. H + -ATPase activity and relative expression of genes encoding nitrate transporter (NRT) and ammonium transporter (AMT) in cucumber roots were increased significantly by the treatment of NO 3 - -N/NH 4 + -N=21:7. In addition, nitrate reductase (NR), glutamine synthetase (GS) and glutamate synthase (GOGAT) in cucumber leaves under the treatment of NO 3 - -N/NH 4 + -N=21:7 were higher. As a result, the nitrogen content and biomass of cucumber were significantly increased. Compared with the plants under the treatment of NO 3 - -N/NH 4 + -N=26:2 or 14:14, cucumber seedlings under the treatment of NO 3 - -N/NH 4 + -N=21:7 had the highest biomass and total dry mass (DM) which were increased by 14.0% and 19.3% respectively under suboptimal temperature and light intensity. In conclusion, under suboptimal environmental conditions, NO 3 - -N/NH 4 + -N ratio could be adjusted to increase nitrogen absorption and metabolism of cucumber and alleviate the de-trimental effects caused by suboptimal conditions and promoted the cucumber growth.

Flux balance analysis of ammonia assimilation network in E. coli predicts preferred regulation point.

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Wang, Lu; Lai, Luhua; Ouyang, Qi; Tang, Chao

2011-01-25

Nitrogen assimilation is a critical biological process for the synthesis of biomolecules in Escherichia coli. The central ammonium assimilation network in E. coli converts carbon skeleton α-ketoglutarate and ammonium into glutamate and glutamine, which further serve as nitrogen donors for nitrogen metabolism in the cell. This reaction network involves three enzymes: glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT). In minimal media, E. coli tries to maintain an optimal growth rate by regulating the activity of the enzymes to match the availability of the external ammonia. The molecular mechanism and the strategy of the regulation in this network have been the research topics for many investigators. In this paper, we develop a flux balance model for the nitrogen metabolism, taking into account of the cellular composition and biosynthetic requirements for nitrogen. The model agrees well with known experimental results. Specifically, it reproduces all the (15)N isotope labeling experiments in the wild type and the two mutant (ΔGDH and ΔGOGAT) strains of E. coli. Furthermore, the predicted catalytic activities of GDH, GS and GOGAT in different ammonium concentrations and growth rates for the wild type, ΔGDH and ΔGOGAT strains agree well with the enzyme concentrations obtained from western blots. Based on this flux balance model, we show that GS is the preferred regulation point among the three enzymes in the nitrogen assimilation network. Our analysis reveals the pattern of regulation in this central and highly regulated network, thus providing insights into the regulation strategy adopted by the bacteria. Our model and methods may also be useful in future investigations in this and other networks.

Flux balance analysis of ammonia assimilation network in E. coli predicts preferred regulation point.

Directory of Open Access Journals (Sweden)

Lu Wang

Full Text Available Nitrogen assimilation is a critical biological process for the synthesis of biomolecules in Escherichia coli. The central ammonium assimilation network in E. coli converts carbon skeleton α-ketoglutarate and ammonium into glutamate and glutamine, which further serve as nitrogen donors for nitrogen metabolism in the cell. This reaction network involves three enzymes: glutamate dehydrogenase (GDH, glutamine synthetase (GS and glutamate synthase (GOGAT. In minimal media, E. coli tries to maintain an optimal growth rate by regulating the activity of the enzymes to match the availability of the external ammonia. The molecular mechanism and the strategy of the regulation in this network have been the research topics for many investigators. In this paper, we develop a flux balance model for the nitrogen metabolism, taking into account of the cellular composition and biosynthetic requirements for nitrogen. The model agrees well with known experimental results. Specifically, it reproduces all the (15N isotope labeling experiments in the wild type and the two mutant (ΔGDH and ΔGOGAT strains of E. coli. Furthermore, the predicted catalytic activities of GDH, GS and GOGAT in different ammonium concentrations and growth rates for the wild type, ΔGDH and ΔGOGAT strains agree well with the enzyme concentrations obtained from western blots. Based on this flux balance model, we show that GS is the preferred regulation point among the three enzymes in the nitrogen assimilation network. Our analysis reveals the pattern of regulation in this central and highly regulated network, thus providing insights into the regulation strategy adopted by the bacteria. Our model and methods may also be useful in future investigations in this and other networks.

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes*

Science.gov (United States)

Tate, Jennifer J.; Cooper, Terrance G.

2013-01-01

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. PMID:23935103

GlnR-mediated regulation of nitrogen metabolism in the actinomycete Saccharopolyspora erythraea.

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Yao, Li-Li; Liao, Cheng-Heng; Huang, Gang; Zhou, Ying; Rigali, Sebastien; Zhang, Buchang; Ye, Bang-Ce

2014-09-01

Nitrogen source sensing, uptake, and assimilation are central for growth and development of microorganisms which requires the participation of a global control of nitrogen metabolism-associated genes at the transcriptional level. In soil-dwelling antibiotic-producing actinomycetes, this role is played by GlnR, an OmpR family regulator. In this work, we demonstrate that SACE_7101 is the ortholog of actinomycetes' GlnR global regulators in the erythromycin producer Saccharopolyspora erythraea. Indeed, the chromosomal deletion of SACE_7101 severely affects the viability of S. erythraea when inoculated in minimal media supplemented with NaNO3, NaNO2, NH4Cl, glutamine, or glutamate as sole nitrogen source. Combination of in silico prediction of cis-acting elements, subsequent in vitro (through gel shift assays) and in vivo (real-time reverse transcription polymerase chain reaction) validations of the predicted target genes revealed a very large GlnR regulon aimed at adapting the nitrogen metabolism of S. erythraea. Indeed, enzymes/proteins involved in (i) uptake and assimilation of ammonium, (ii) transport and utilization of urea, (iii) nitrite/nitrate, (iv) glutamate/glutamine, (v) arginine metabolism, (vi) nitric oxide biosynthesis, and (vii) signal transduction associated with the nitrogen source supplied have at least one paralog gene which expression is controlled by GlnR. Our work highlights a GlnR-binding site consensus sequence (t/gna/cAC-n6-GaAAc) which is similar although not identical to the consensus sequences proposed for other actinomycetes. Finally, we discuss the distinct and common features of the GlnR-mediated transcriptional control of nitrogen metabolism between S. erythraea and the model organism Streptomyces coelicolor.

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.

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

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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.

Transcriptome of Proteus mirabilis in the Murine Urinary Tract: Virulence and Nitrogen Assimilation Gene Expression▿†

Science.gov (United States)

Pearson, Melanie M.; Yep, Alejandra; Smith, Sara N.; Mobley, Harry L. T.

2011-01-01

The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In this study, microarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across nine microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded mannose-resistant Proteus-like (MR/P) fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo, while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract. PMID:21505083

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

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.

Applications of Palladium-Catalyzed C-N Cross-Coupling Reactions.

Science.gov (United States)

Ruiz-Castillo, Paula; Buchwald, Stephen L

2016-10-12

Pd-catalyzed cross-coupling reactions that form C-N bonds have become useful methods to synthesize anilines and aniline derivatives, an important class of compounds throughout chemical research. A key factor in the widespread adoption of these methods has been the continued development of reliable and versatile catalysts that function under operationally simple, user-friendly conditions. This review provides an overview of Pd-catalyzed N-arylation reactions found in both basic and applied chemical research from 2008 to the present. Selected examples of C-N cross-coupling reactions between nine classes of nitrogen-based coupling partners and (pseudo)aryl halides are described for the synthesis of heterocycles, medicinally relevant compounds, natural products, organic materials, and catalysts.

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

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

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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.

Steady-state and dynamic gene expression programs in Saccharomyces cerevisiae in response to variation in environmental nitrogen

Science.gov (United States)

Airoldi, Edoardo M.; Miller, Darach; Athanasiadou, Rodoniki; Brandt, Nathan; Abdul-Rahman, Farah; Neymotin, Benjamin; Hashimoto, Tatsu; Bahmani, Tayebeh; Gresham, David

2016-01-01

Cell growth rate is regulated in response to the abundance and molecular form of essential nutrients. In Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen is a major determinant of cell growth rate, supporting growth rates that vary at least threefold. Transcriptional control of nitrogen use is mediated in large part by nitrogen catabolite repression (NCR), which results in the repression of specific transcripts in the presence of a preferred nitrogen source that supports a fast growth rate, such as glutamine, that are otherwise expressed in the presence of a nonpreferred nitrogen source, such as proline, which supports a slower growth rate. Differential expression of the NCR regulon and additional nitrogen-responsive genes results in >500 transcripts that are differentially expressed in cells growing in the presence of different nitrogen sources in batch cultures. Here we find that in growth rate–controlled cultures using nitrogen-limited chemostats, gene expression programs are strikingly similar regardless of nitrogen source. NCR expression is derepressed in all nitrogen-limiting chemostat conditions regardless of nitrogen source, and in these conditions, only 34 transcripts exhibit nitrogen source–specific differential gene expression. Addition of either the preferred nitrogen source, glutamine, or the nonpreferred nitrogen source, proline, to cells growing in nitrogen-limited chemostats results in rapid, dose-dependent repression of the NCR regulon. Using a novel means of computational normalization to compare global gene expression programs in steady-state and dynamic conditions, we find evidence that the addition of nitrogen to nitrogen-limited cells results in the transient overproduction of transcripts required for protein translation. Simultaneously, we find that that accelerated mRNA degradation underlies the rapid clearing of a subset of transcripts, which is most pronounced for the highly expressed NCR

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

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

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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.

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

Glutamine prevents oxidative stress in a model of portal hypertension.

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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

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

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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.

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

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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.

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...

The Nitrate-Inducible NAC Transcription Factor TaNAC2-5A Controls Nitrate Response and Increases Wheat Yield1[OPEN

Science.gov (United States)

He, Xue; Qu, Baoyuan; Li, Wenjing; Zhao, Xueqiang; Teng, Wan; Ma, Wenying; Ren, Yongzhe; Li, Bin; Li, Zhensheng; Tong, Yiping

2015-01-01

Nitrate is a major nitrogen resource for cereal crops; thus, understanding nitrate signaling in cereal crops is valuable for engineering crops with improved nitrogen use efficiency. Although several regulators have been identified in nitrate sensing and signaling in Arabidopsis (Arabidopsis thaliana), the equivalent information in cereals is missing. Here, we isolated a nitrate-inducible and cereal-specific NAM, ATAF, and CUC (NAC) transcription factor, TaNAC2-5A, from wheat (Triticum aestivum). A chromatin immunoprecipitation assay showed that TaNAC2-5A could directly bind to the promoter regions of the genes encoding nitrate transporter and glutamine synthetase. Overexpression of TaNAC2-5A in wheat enhanced root growth and nitrate influx rate and, hence, increased the root’s ability to acquire nitrogen. Furthermore, we found that TaNAC2-5A-overexpressing transgenic wheat lines had higher grain yield and higher nitrogen accumulation in aerial parts and allocated more nitrogen in grains in a field experiment. These results suggest that TaNAC2-5A is involved in nitrate signaling and show that it is an exciting gene resource for breeding crops with more efficient use of fertilizer. PMID:26371233

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

The Cell Wall Teichuronic Acid Synthetase (TUAS Is an Enzyme Complex Located in the Cytoplasmic Membrane of Micrococcus luteus

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Lingyi Lynn Deng

2010-01-01

composed of disaccharide repeating units [-4-β-D-ManNAcAp-(1→6α-D-Glcp−1-]n, which is covalently anchored to the peptidoglycan on the inner cell wall and extended to the outer surface of the cell envelope. An enzyme complex responsible for the TUA chain biosynthesis was purified and characterized. The 440 kDa enzyme complex, named teichuronic acid synthetase (TUAS, is an octomer composed of two kinds of glycosyltransferases, Glucosyltransferase, and ManNAcA-transferase, which is capable of catalyzing the transfer of disaccharide glycosyl residues containing both glucose and the N-acetylmannosaminuronic acid residues. TUAS displays hydrophobic properties and is found primarily associated with the cytoplasmic membrane. The purified TUAS contains carotinoids and lipids. TUAS activity is diminished by phospholipase digestion. We propose that TUAS serves as a multitasking polysaccharide assembling station on the bacterial membrane.

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

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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.

Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

Science.gov (United States)

Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

2014-11-07

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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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

Rhodium(III)-Catalyzed Amidation of Unactivated C(sp(3) )-H Bonds.

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Wang, He; Tang, Guodong; Li, Xingwei

2015-10-26

Nitrogenation by direct functionalization of C-H bonds represents an important strategy for constructing C-N bonds. Rhodium(III)-catalyzed direct amidation of unactivated C(sp(3) )-H bonds is rare, especially under mild reaction conditions. Herein, a broad scope of C(sp(3) )-H bonds are amidated under rhodium catalysis in high efficiency using 3-substituted 1,4,2-dioxazol-5-ones as the amide source. The protocol broadens the scope of rhodium(III)-catalyzed C(sp(3) )-H activation chemistry, and is applicable to the late-stage functionalization of natural products. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination

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Aravind L

2008-11-01

Full Text Available Abstract Recently Mycobacterium tuberculosis was shown to possess a novel protein modification, in which a small protein Pup is conjugated to the epsilon-amino groups of lysines in target proteins. Analogous to ubiquitin modification in eukaryotes, this remarkable modification recruits proteins for degradation via archaeal-type proteasomes found in mycobacteria and allied actinobacteria. While a mycobacterial protein named PafA was found to be required for this conjugation reaction, its biochemical mechanism has not been elucidated. Using sensitive sequence profile comparison methods we establish that the PafA family proteins are related to the γ-glutamyl-cysteine synthetase and glutamine synthetase. Hence, we predict that PafA is the Pup ligase, which catalyzes the ATP-dependent ligation of the terminal γ-carboxylate of glutamate to lysines, similar to the above enzymes. We further discovered that an ortholog of the eukaryotic PAC2 (e.g. cg2106 is often present in the vicinity of the actinobacterial Pup-proteasome gene neighborhoods and is likely to represent the ancestral proteasomal chaperone. Pup-conjugation is sporadically present outside the actinobacteria in certain lineages, such as verrucomicrobia, nitrospirae, deltaproteobacteria and planctomycetes, and in the latter two lineages it might modify membrane proteins. Reviewers This article was reviewed by M. Madan Babu and Andrei Osterman

Nitrogen Addition Exacerbates the Negative Effects of Low Temperature Stress on Carbon and Nitrogen Metabolism in Moss

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Bin-Yang Liu

2017-08-01

Full Text Available Global environmental changes are leading to an increase in localized abnormally low temperatures and increasing nitrogen (N deposition is a phenomenon recognized worldwide. Both low temperature stress (LTS and excess N induce oxidative stress in plants, and excess N also reduces their resistance to LTS. Mosses are primitive plants that are generally more sensitive to alterations in environmental factors than vascular species. To study the combined effects of N deposition and LTS on carbon (C and N metabolism in moss, two moss species, Pogonatum cirratum subsp. fuscatum, and Hypnum plumaeforme, exposed to various concentrations of nitrate (KNO3 or ammonium (NH4Cl, were treated with or without LTS. C/N metabolism indices were then monitored, both immediately after the stress and after a short recovery period (10 days. LTS decreased the photosystem II (PSII performance index and inhibited non-cyclic photophosphorylation, ribulose-1,5-bisphosphate carboxylase, and glutamine synthetase activities, indicating damage to PSII and reductions in C/N assimilation in these mosses. LTS did not affect cyclic photophosphorylation, sucrose synthase, sucrose-phosphate synthase, and NADP-isocitrate dehydrogenase activities, suggesting a certain level of energy and C skeleton generation were maintained in the mosses to combat LTS; however, LTS inhibited the activity of glycolate oxidase. As predicted, N supply increased the sensitivity of the mosses to LTS, resulting in greater damage to PSII and a sharper decrease in C/N assimilation. After the recovery period, the performance of PSII and C/N metabolism, which were inhibited by LTS increased significantly, and were generally higher than those of control samples not exposed to LTS, suggesting overcompensation effects; however, N application reduced the extent of compensation effects. Both C and N metabolism exhibited stronger compensation effects in H. plumaeforme than in P. cirratum subsp. fuscatum. The

Palladium-catalyzed aerobic oxidative cross-coupling of arylhydrazines with terminal alkynes.

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Zhao, Yingwei; Song, Qiuling

2015-09-04

The palladium-catalyzed Sonogashira-type aerobic oxidative coupling of arylhydrazines with terminal alkynes via C-N bond cleavage has been developed; internal alkynes were afforded with a broad substrate scope. This reaction proceeds under copper- and base-free conditions with molecular oxygen as the sole oxidant and nitrogen and water as the only by-products.

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

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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.

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

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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.

Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro

International Nuclear Information System (INIS)

Zhao Guohui; Liu Jun; Liu Xiang; Chen Min; Zhang Houcheng; Wang, Peng George

2007-01-01

GDP-perosamine synthetase (Per, E.C. not yet classified) is important to the synthesis of Escherichia coli O157:H7 O-antigen. The mutant in per gene can disrupt the synthesis of O157 O-antigen. In this study, GDP-perosamine synthetase was cloned from E. coli O157:H7 and over-expressed in E. coli BL21 (DE3). The recombinant His-tagged Per fusion protein was a decamer with molecular weight of 431 kDa. The optimal pH value of this recombinant protein was 7.5. The divalent ions had no significant effect on Per-catalyzed reaction. The K m and K cat /K m for GDP-4-keto-6-deoxy-D-mannose were 0.09 mM and 2.1 x 10 5 M -1 S -1 , and those for L-glutamate were 2 mM and 0.52 x 10 5 M -1 S -1 , respectively. Per was used to synthesize GDP-perosamine from GDP-mannose together with recombinant GDP-mannose dehydratase (GMD, E.C. 4.2.1.47). The purified GDP-perosamine was identified by MS and NMR. In summary, this work provided a feasible approach for the synthesis of GDP-perosamine which can lead to the study of LPS biosynthesis of pathogenic E. coli O157:H7

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

Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

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Pengfei Fang

2015-12-01

Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories.

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

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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.

Urea application promotes amino acid metabolism and membrane lipid peroxidation in Azolla.

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Chen, Jiana; Huang, Min; Cao, Fangbo; Pardha-Saradhi, P; Zou, Yingbin

2017-01-01

A pot experiment was conducted to evaluate the effect of urea on nitrogen metabolism and membrane lipid peroxidation in Azolla pinnata. Compared to controls, the application of urea to A. pinnata resulted in a 44% decrease in nitrogenase activity, no significant change in glutamine synthetase activity, 660% higher glutamic-pyruvic transaminase, 39% increase in free amino acid levels, 22% increase in malondialdehyde levels, 21% increase in Na+/K+- levels, 16% increase in Ca2+/Mg2+-ATPase levels, and 11% decrease in superoxide dismutase activity. In terms of H2O2 detoxifying enzymes, peroxidase activity did not change and catalase activity increased by 64% in urea-treated A. pinnata. These findings suggest that urea application promotes amino acid metabolism and membrane lipid peroxidation in A. pinnata.

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

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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.

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

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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.

Blood ammonia and glutamine as predictors of hyperammonemic crises in patients with urea cycle disorder.

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Lee, Brendan; Diaz, George A; Rhead, William; Lichter-Konecki, Uta; Feigenbaum, Annette; Berry, Susan A; Le Mons, Cindy; Bartley, James A; Longo, Nicola; Nagamani, Sandesh C; Berquist, William; Gallagher, Renata; Bartholomew, Dennis; Harding, Cary O; Korson, Mark S; McCandless, Shawn E; Smith, Wendy; Cederbaum, Stephen; Wong, Derek; Merritt, J Lawrence; Schulze, Andreas; Vockley, Jerry; Vockley, Gerard; Kronn, David; Zori, Roberto; Summar, Marshall; Milikien, Douglas A; Marino, Miguel; Coakley, Dion F; Mokhtarani, Masoud; Scharschmidt, Bruce F

2015-07-01

The aim of this study was to examine predictors of ammonia exposure and hyperammonemic crises in patients with urea cycle disorders. The relationships between fasting ammonia, daily ammonia exposure, and hyperammonemic crises were analyzed in >100 patients with urea cycle disorders. Fasting ammonia correlated strongly with daily ammonia exposure (r = 0.764; P 200% (P urea cycle disorder subtype, dietary protein intake, or blood urea nitrogen. Fasting glutamine correlated weakly with daily ammonia exposure assessed as 24-hour area under the curve and was not a significant predictor of hyperammonemic crisis. Fasting ammonia correlates strongly and positively with daily ammonia exposure and with the risk and rate of hyperammonemic crises, suggesting that patients with urea cycle disorder may benefit from tight ammonia control.

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.

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

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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.

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

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

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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.

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

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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.

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

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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.

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

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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

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

Prevention of gastrointestinal injury by using glutamine in cardiac surgery

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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.

Synthesis of Rhodamines from Fluoresceins Using Pd-Catalyzed C–N Cross-Coupling

Science.gov (United States)

2011-01-01

A unified, convenient, and efficient strategy for the preparation of rhodamines and N,N′-diacylated rhodamines has been developed. Fluorescein ditriflates were found to undergo palladium-catalyzed C–N cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores. PMID:22091952

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

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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.

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.

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

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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.

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

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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.

In vivo urea cycle flux distinguishes and correlates with phenotypic severity in disorders of the urea cycle

Science.gov (United States)

Lee, Brendan; Yu, Hong; Jahoor, Farook; O'Brien, William; Beaudet, Arthur L.; Reeds, Peter

2000-01-01

Urea cycle disorders are a group of inborn errors of hepatic metabolism that result in often life-threatening hyperammonemia and hyperglutaminemia. Clinical and laboratory diagnosis of partial deficiencies during asymptomatic periods is difficult, and correlation of phenotypic severity with either genotype and/or in vitro enzyme activity is often imprecise. We hypothesized that stable isotopically determined in vivo rates of total body urea synthesis and urea cycle-specific nitrogen flux would correlate with both phenotypic severity and carrier status in patients with a variety of different enzymatic deficiencies of the urea cycle. We studied control subjects, patients, and their relatives with different enzymatic deficiencies affecting the urea cycle while consuming a low protein diet. On a separate occasion the subjects either received a higher protein intake or were treated with an alternative route medication sodium phenylacetate/benzoate (Ucephan), or oral arginine supplementation. Total urea synthesis from all nitrogen sources was determined from [18O]urea labeling, and the utilization of peripheral nitrogen was estimated from the relative isotopic enrichments of [15N]urea and [15N]glutamine during i.v. co-infusions of [5-(amide)15N]glutamine and [18O]urea. The ratio of the isotopic enrichments of 15N-urea/15N-glutamine distinguished normal control subjects (ratio = 0.42 ± 0.06) from urea cycle patients with late (0.17 ± 0.03) and neonatal (0.003 ± 0.007) presentations irrespective of enzymatic deficiency. This index of urea cycle activity also distinguished asymptomatic heterozygous carriers of argininosuccinate synthetase deficiency (0.22 ± 0.03), argininosuccinate lyase deficiency (0.35 ± 0.11), and partial ornithine transcarbamylase deficiency (0.26 ± 0.06) from normal controls. Administration of Ucephan lowered, and arginine increased, urea synthesis to the degree predicted from their respective rates of metabolism. The 15N-urea/15N-glutamine ratio

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.

A New Oidiodendron maius Strain Isolated from Rhododendron fortunei and Its Effects on Nitrogen Uptake and Plant Growth

Directory of Open Access Journals (Sweden)

Xiangying Wei

2016-08-01

Full Text Available A new mycorrhizal fungal strain was isolated from hair roots of Rhododendron fortunei Lindl. grown in Huading Forest Park, Zhejiang Province, China. Morphological characterization and internal transcribed spacer (ITS rDNA analysis suggested that it belongs to Oidiodendron maius Barron, and we designated it as strain Om19. Methods for culturing Om19 were established, and the ability of Om19 to form mycorrhizae on R. fortunei was evaluated in a peat-based substrate. Microscopic observations showed hyaline hyphae on the surface of hair roots and crowded hyphal complexes (hyphal coils inside root cortical cells of R. fortunei after inoculation, indicating that the roots were well colonized by Om19. In a second experiment, fresh and dry weight of R. fortunei two months after Om19 inoculation were greater than uninoculated plants, and the total nitrogen (N absorbed by plants inoculated with Om19 was greater than the uninoculated controls. qRT-PCR analysis of five genes related to N uptake and metabolism (two nitrate transporters, an ammonium transporter, glutamine synthetase, and glutamate synthase showed that these genes were highly upregulated with 2 to 9 fold greater expression in plants inoculated with Om19 compared to uninoculated plants. In the third experiment, Om19 was inoculated into the peat-based substrate for growing Formosa azalea (R. indica ‘Formosa’. ‘Formosa’ azalea plants grown in the inoculated substrate had larger canopies and root systems compared to uninoculated plants. Our results show that Om19 could be an important microbial tool for improving production of Rhododendron plants.

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...

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.

A Proteomic Network for Symbiotic Nitrogen Fixation Efficiency in Bradyrhizobium elkanii.

Science.gov (United States)

Cooper, Bret; Campbell, Kimberly B; Beard, Hunter S; Garrett, Wesley M; Mowery, Joseph; Bauchan, Gary R; Elia, Patrick

2018-03-01

Rhizobia colonize legumes and reduce N 2 to NH 3 in root nodules. The current model is that symbiotic rhizobia bacteroids avoid assimilating this NH 3 . Instead, host legume cells form glutamine from NH 3 , and the nitrogen is returned to the bacteroid as dicarboxylates, peptides, and amino acids. In soybean cells surrounding bacteroids, glutamine also is converted to ureides. One problem for soybean cultivation is inefficiency in symbiotic N 2 fixation, the biochemical basis of which is unknown. Here, the proteomes of bacteroids of Bradyrhizobium elkanii USDA76 isolated from N 2 fixation-efficient Peking and -inefficient Williams 82 soybean nodules were analyzed by mass spectrometry. Nearly half of the encoded bacterial proteins were quantified. Efficient bacteroids produced greater amounts of enzymes to form Nod factors and had increased amounts of signaling proteins, transporters, and enzymes needed to generate ATP to power nitrogenase and to acquire resources. Parallel investigation of nodule proteins revealed that Peking had no significantly greater accumulation of enzymes needed to assimilate NH 3 than Williams 82. Instead, efficient bacteroids had increased amounts of enzymes to produce amino acids, including glutamine, and to form ureide precursors. These results support a model for efficient symbiotic N 2 fixation in soybean where the bacteroid assimilates NH 3 for itself.

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 ...

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.

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.

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.

Urea application promotes amino acid metabolism and membrane lipid peroxidation in Azolla.

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Jiana Chen

Full Text Available A pot experiment was conducted to evaluate the effect of urea on nitrogen metabolism and membrane lipid peroxidation in Azolla pinnata. Compared to controls, the application of urea to A. pinnata resulted in a 44% decrease in nitrogenase activity, no significant change in glutamine synthetase activity, 660% higher glutamic-pyruvic transaminase, 39% increase in free amino acid levels, 22% increase in malondialdehyde levels, 21% increase in Na+/K+- levels, 16% increase in Ca2+/Mg2+-ATPase levels, and 11% decrease in superoxide dismutase activity. In terms of H2O2 detoxifying enzymes, peroxidase activity did not change and catalase activity increased by 64% in urea-treated A. pinnata. These findings suggest that urea application promotes amino acid metabolism and membrane lipid peroxidation in A. pinnata.

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.

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)

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

Drought tolerance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene.

Science.gov (United States)

Qin, Na; Xu, Weigang; Hu, Lin; Li, Yan; Wang, Huiwei; Qi, Xueli; Fang, Yuhui; Hua, Xia

2016-11-01

Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.

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....

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.

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.

Increased production of free fatty acids in Aspergillus oryzae by disruption of a predicted acyl-CoA synthetase gene.

Science.gov (United States)

Tamano, Koichi; Bruno, Kenneth S; Koike, Hideaki; Ishii, Tomoko; Miura, Ai; Umemura, Myco; Culley, David E; Baker, Scott E; Machida, Masayuki

2015-04-01

Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).

Improvement of nitrogen accumulation and metabolism in rice (Oryza sativa L.) by the endophyte Phomopsis liquidambari.

Science.gov (United States)

Yang, Bo; Ma, Hai-Yan; Wang, Xiao-Mi; Jia, Yong; Hu, Jing; Li, Xia; Dai, Chuan-Chao

2014-09-01

The fungal endophyte Phomopsis liquidambari can enhance nitrogen (N) uptake and metabolism of rice plants under hydroponic conditions. To investigate the effects of P. liquidambari on N accumulation and metabolism in rice (Oryza sativa L.) under field conditions during the entire growing season (S1, the seedling stage; S2, the tillering stage; S3, the heading stage; S4, the ripening stage), we utilized pot experiments to examine metabolic and physiological levels in both shoot and root tissues of rice, with endophyte (E+) and without endophyte (E-), in response to three different N levels. We found that under low-N treatment, P. liquidambari symbiosis increased the rice yield and N use efficiency by 12% and by 11.59%, respectively; that the total N contents in E+ rice plants at the four growth stages were separately increased by 29.05%, 14.65%, 21.06% and 18.38%, respectively; and that the activities of nitrate reductase and glutamine synthetase in E+ rice roots and shoots were significantly increased by fungal infection during the S1 to S3 stages. Moreover, P. liquidambari significantly increased the free NH4(+), NO3(-), amino acid and soluble protein contents in infected rice tissues under low-N treatment during the S1 to S3 stages. The obtained results offer novel data concerning the systemic changes induced by P. liquidambari in rice during the entire growth period and confirm the hypothesis that the rice-P. liquidambari interaction improved the N accumulation and metabolism of rice plants, consequently increasing rice N utilization in nutrient-limited soil. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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.

Nickel-Catalyzed Synthesis of Primary Aryl and Heteroaryl Amines via C–O Bond Cleavage

KAUST Repository

Yue, Huifeng

2017-03-13

A nickel-catalyzed protocol for the conversion of aryl and heteroaryl alcohol derivatives to primary and secondary aromatic amines via C(sp2)-O bond cleavage is described. The new amination protocol can be applied to a range of substrates bearing diverse functional groups and uses readily available benzophenone imines as an effective nitrogen source.

Nickel-Catalyzed Synthesis of Primary Aryl and Heteroaryl Amines via C–O Bond Cleavage

KAUST Repository

Yue, Huifeng; Guo, Lin; Liu, Xiangqian; Rueping, Magnus

2017-01-01

A nickel-catalyzed protocol for the conversion of aryl and heteroaryl alcohol derivatives to primary and secondary aromatic amines via C(sp2)-O bond cleavage is described. The new amination protocol can be applied to a range of substrates bearing diverse functional groups and uses readily available benzophenone imines as an effective nitrogen source.

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)

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...

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

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.

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.

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

Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

Science.gov (United States)

León-Del-Río, Alfonso; Valadez-Graham, Viviana; Gravel, Roy A

2017-08-21

The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

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.

An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

Science.gov (United States)

Kalb, Daniel; Heinekamp, Thorsten; Schieferdecker, Sebastian; Nett, Markus; Brakhage, Axel A; Hoffmeister, Dirk

2016-10-04

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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...

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.

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.

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

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

Regulation of Azorhizobium caulinodans ORS571 nitrogen fixation (NIF/FIX) genes

NARCIS (Netherlands)

Stigter, J.

1994-01-01

Biological nitrogen fixation is the microbial process by which atmospheric dinitrogen (N ₂ ) is reduced to ammonia. In all microbes studied, dinitrogen reduction is catalyzed by a highly conserved enzyme complex, called nitrogenase.

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

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....

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....

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.

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 ...

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.

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.

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.)

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.

Disruption of the nitrogen regulatory gene AcareA in Acremonium chrysogenum leads to reduction of cephalosporin production and repression of nitrogen metabolism.

Science.gov (United States)

Li, Jinyang; Pan, Yuanyuan; Liu, Gang

2013-12-01

AcareA, encoding a homologue of the fungal nitrogen regulatory GATA zinc-finger proteins, was cloned from Acremonium chrysogenum. Gene disruption and genetic complementation revealed that AcareA was required for nitrogen metabolism and cephalosporin production. Disruption of AcareA resulted in growth defect in the medium using nitrate, uric acid and low concentration of ammonium, glutamine or urea as sole nitrogen source. Transcriptional analysis showed that the transcription of niaD/niiA was increased drastically when induced with nitrate in the wild-type and AcareA complemented strains but not in AcareA disruption mutant. Consistent with the reduction of cephalosporin production, the transcription of pcbAB, cefD2, cefEF and cefG encoding the enzymes for cephalosporin production was reduced in AcareA disruption mutant. Band shift assays showed that AcAREA bound to the promoter regions of niaD, niiA and the bidirectional promoter region of pcbAB-pcbC. Sequence analysis showed that all the AcAREA binding sites contain the consensus GATA elements. These results indicated that AcAREA plays an important role both in the regulation of nitrogen metabolism and cephalosporin production in A. chrysogenum. Copyright © 2013 Elsevier Inc. All rights reserved.

Isotopic and enzymatic analyses of planktonic nitrogen utilisation in the vicinity of Cape Sines (Portugal) during weak upwelling activity

Science.gov (United States)

Slawyk, Gerd; Coste, Bernard; Collos, Yves; Rodier, Martine

1997-01-01

Using measurements of 15N uptake and activities of nitrate reductase and glutamine synthetase, the utilization of nitrogenous nutrients by microplankton in the Portuguese upwelling area was investigated. During this cruise the euphotic zone of coastal waters was in most cases bisected by a nitracline forming two layers. Total inorganic nitrogen uptake rates (NH 4+ + NO 3-) in the upper mixed and nitrate-impoverished layer ranged from 0.1 to 0.8 nM h -1 and were primarily supported by regenerated (ammonium) nitrogen (62-97%), whereas they varied between 0.9 and 10.4 nM h -1 in the deep nitrate-rich layer and were mainly driven by new (nitrate) nitrogen (52-82%). Depth profiles of Chl a-specific uptake rates for ammonium and nitrate paralleled those of absolute uptake rates, i.e. values of VNH 4+Chl were highest (up to 16.1 nmol μg -1 h -1) in nitrate-poor surface waters while values of VNO 3-Chl were maximum (up to 8.4 nmol μg -1 h -1)within the nitracline. This latter vertical ordering of planktonic nitrogen nutrition was consistent with an aged upwelling situation. However, applying several indices of cell metabolism and nutritional status, such as 15N uptake/enzyme activity, surge uptake internally controlled uptake, and V maxChl/K t ratios, we were able to demonstrate that the phytoplankton assemblages inhabiting the nutrient-impoverished upper layer still bore the signature of physically mediated nitrogen (nitrate) supply generated by active upwelling that had occurred during the week before our visit to the area. This signature was the most evident in samples from the station furthest inshore and faded with distance from shore as a result of the deepening of the nitrate isopleths (weakening of upwelling activity), which showed the same offshore trend. The appearance of nitrate-rich waters at the surface, after a strong pulse of upwelling favourable winds just before the end of the cruise, led to a five-fold increase in average (over the euphotic zone

Effects of Organic and Inorganic Nitrogen on the Growth and Production of Domoic Acid by Pseudo-nitzschia multiseries and P. australis (Bacillariophyceae) in Culture.

Science.gov (United States)

Martin-Jézéquel, Véronique; Calu, Guillaume; Candela, Leo; Amzil, Zouher; Jauffrais, Thierry; Séchet, Véronique; Weigel, Pierre

2015-11-26

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment.

[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

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

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

Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction.

Science.gov (United States)

Gaudin, Zachary; Cerveau, Delphine; Marnet, Nathalie; Bouchereau, Alain; Delavault, Philippe; Simier, Philippe; Pouvreau, Jean-Bernard

2014-01-21

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa.

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...

Copper-catalyzed aerobic oxidative C-H functionalization of substituted pyridines: synthesis of imidazopyridine derivatives.

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Yu, Jipan; Jin, Yunhe; Zhang, Hao; Yang, Xiaobo; Fu, Hua

2013-12-02

A novel, efficient, and practical method for the synthesis of imidazopyridine derivatives has been developed through the copper-catalyzed aerobic oxidative C-H functionalization of substituted pyridines with N-(alkylidene)-4H-1,2,4-triazol-4-amines. The procedure occurs by cleavage of the N-N bond in the N-(alkylidene)-4H-1,2,4-triazol-4-amines and activation of an aryl C-H bond in the substituted pyridines. This is the first example of the preparation of imidazopyridine derivatives by using pyridines as the substrates by transition-metal-catalyzed C-H functionalization. This method should provide a novel and efficient strategy for the synthesis of other nitrogen heterocycles. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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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.

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

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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.

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

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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...

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

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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.

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

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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.

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

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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.

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

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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).

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

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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.

Differential gene expression in Rhododendron fortunei roots colonized by an ericoid mycorrhizal fungus and increased nitrogen absorption and plant growth

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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.

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...

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

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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.

Acetate Activation in Methanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase

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Stefanie Berger

2012-01-01

Full Text Available The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM=0.27±0.05 mM that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell.

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

Transition metal-catalyzed carbocyclization of nitrogen and oxygen-tethered 1,n-enynes and diynes: synthesis of five or six-membered heterocyclic compounds.

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Zhang, Di-Han; Zhang, Zhen; Shi, Min

2012-10-25

Cycloisomerization of 1,n-enynes and diynes is a powerful method in organic synthesis to access heterocyclic compounds and has drawn increasing attention from organic chemists. In this paper, we attempted to summarize our recent results on the transition metal-catalyzed cycloisomerization to synthesize five or six-membered heterocyclic compounds using 1,n-enynes and diynes having a propargylic ester moiety. First, we will describe the synthesis of 2,3-disubstituted 3-pyrrolines via gold catalyzed cycloisomerization of 1,6-diynes. In addition, we will also disclose a novel silver catalyzed tandem 1,3-acyloxy migration/Mannich-type addition/elimination of the sulfonyl group of N-sulfonylhydrazone-propargylic esters to 5,6-dihydropyridazin-4-one derivatives. Furthermore, we will introduce three interesting examples of the synthesis of bicyclic compounds via titanium or rhodium catalyzed carbocyclization of enynes. In this context, we have presented that 1,n-enynes and diynes containing propargylic esters are highly reactive and useful starting materials for the cycloisomerization catalyzed by a transition metal catalyst.

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)

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.

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...

Kinetics of aggregation growth with competition between catalyzed birth and catalyzed death

International Nuclear Information System (INIS)

Wang Haifeng; Gao Yan; Lin Zhenquan

2008-01-01

An aggregation growth model of three species A, B and C with the competition between catalyzed birth and catalyzed death is proposed. Irreversible aggregation occurs between any two aggregates of the like species with the constant rate kernels I n (n = 1,2,3). Meanwhile, a monomer birth of an A species aggregate of size k occurs under the catalysis of a B species aggregate of size j with the catalyzed birth rate kernel K(k,j) = Kkj v and a monomer death of an A species aggregate of size k occurs under the catalysis of a C species aggregate of size j with the catalyzed death rate kernel L(k,j)=Lkj v , where v is a parameter reflecting the dependence of the catalysis reaction rates of birth and death on the size of catalyst aggregate. The kinetic evolution behaviours of the three species are investigated by the rate equation approach based on the mean-field theory. The form of the aggregate size distribution of A species a k (t) is found to be dependent crucially on the competition between the catalyzed birth and death of A species, as well as the irreversible aggregation processes of the three species: (1) In the v k (t) satisfies the conventional scaling form; (2) In the v ≥ 0 case, the competition between the catalyzed birth and death dominates the process. When the catalyzed birth controls the process, a k (t) takes the conventional or generalized scaling form. While the catalyzed death controls the process, the scaling description of the aggregate size distribution breaks down completely

GATOR1 regulates nitrogenic cataplerotic reactions of the mitochondrial TCA cycle.

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Chen, Jun; Sutter, Benjamin M; Shi, Lei; Tu, Benjamin P

2017-11-01

The GATOR1 (SEACIT) complex consisting of Iml1-Npr2-Npr3 inhibits target of rapamycin complex 1 (TORC1) in response to amino acid insufficiency. In glucose medium, Saccharomyces cerevisiae mutants lacking the function of this complex grow poorly in the absence of amino acid supplementation, despite showing hallmarks of increased TORC1 signaling. Such mutants sense that they are amino acid replete and thus repress metabolic activities that are important for achieving this state. We found that npr2Δ mutants have defective mitochondrial tricarboxylic acid (TCA)-cycle activity and retrograde response. Supplementation with glutamine, and especially aspartate, which are nitrogen-containing forms of TCA-cycle intermediates, rescues growth of npr2Δ mutants. These amino acids are then consumed in biosynthetic pathways that require nitrogen to support proliferative metabolism. Our findings revealed that negative regulators of TORC1, such as GATOR1 (SEACIT), regulate the cataplerotic synthesis of these amino acids from the TCA cycle, in tune with the amino acid and nitrogen status of cells.

Kinetics of 15NH4+ assimilation in Zea mays

International Nuclear Information System (INIS)

Magalhaes, J.R.; Ju, G.C.; Rich, P.J.; Rhodes, D.

1990-01-01

Comparative studies of 15 NH 4 + assimilation were undertaken with a GDH1-null mutant of Zea mays and a related (but not strictly isogenic) GDH1-positive wild type from which this mutant was derived. The kinetics of 15 NH 4 + assimilation into free amino acids and total reduced nitrogen were monitored in both roots and shoots of 2-week-old seedlings supplied with 5 millimolar 99% ( 15 NH 4 ) 2 SO 4 via the aerated root medium in hydroponic culture over a 24-h period. The GDH1-null mutant, with a 10- to 15-fold lower total root GDH activity in comparison to the wild type, was found to exhibit a 40 to 50% lower rate of 15 NH 4 + assimilation into total reduced nitrogen. The lower rates of 15 NH 4 + assimilation in the mutant was associated with lower rates of labeling of several free amino acids (including glutamate, glutamine-amino N, aspartate, asparagine-amino N, and alanine) in both roots and shoots of the mutant in comparison to the wild type. Qualitatively, these labeling kinetics appear consistent with a reduced flux of 15 N via glutamate in the GDH1-null mutant. However, the responses of the two genotypes to the potent inhibitor of glutamine synthetase, methionine sulfoximine, and differences in morphology of the two genotypes (particularly a lower shoot:root ratio in the GDH1-null mutant) urge caution in concluding that GDH1 is solely responsible for these differences in ammonia assimilation rate

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

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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.

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.

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

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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.

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 ...

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

Heterocycles by Transition Metals Catalyzed Intramolecular Cyclization of Acetylene Compounds

International Nuclear Information System (INIS)

Vizer, S.A.; Yerzhanov, K.B.; Dedeshko, E.C.

2003-01-01

Review shows the new strategies in the synthesis of heterocycles, having nitrogen, oxygen and sulfur atoms, via transition metals catalyzed intramolecular cyclization of acetylenic compounds on the data published at the last 30 years, Unsaturated heterocyclic compounds (pyrroles and pyrroline, furans, dihydro furans and benzofurans, indoles and iso-indoles, isoquinolines and isoquinolinones, aurones, iso coumarins and oxazolinone, lactams and lactones with various substitutes in heterocycles) are formed by transition metals, those salts [PdCl 2 , Pd(OAc) 2 , HgCl 2 , Hg(OAc) 2 , Hg(OCOCF 3 ) 2 , AuCl 3 ·2H 2 O, NaAuCl 4 ·2H 2 O, CuI, CuCl], oxides (HgO) and complexes [Pd(OAc) 2 (PPh 3 )2, Pd(PPh 3 ) 4 , PdCl 2 (MeCN) 2 , Pd(OAc ) 2 /TPPTS] catalyzed intramolecular cyclization of acetylenic amines, amides, ethers, alcohols, acids, ketones and βdiketones. More complex hetero polycyclic systems typical for natural alkaloids can to obtain similar. Proposed mechanisms of pyrroles, isoquinolines, iso indoles and indoles, benzofurans and iso coumarins, thiazolopyrimidinones formation are considered. (author)

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...

Aerobic Pd-Catalyzed sp3 C–H Olefination: A Route to Both N-Heterocyclic Scaffolds and Alkenes

Science.gov (United States)

Stowers, Kara J.; Fortner, Kevin C.

2011-01-01

This communication describes a new method for the Pd/polyoxometalate-catalyzed aerobic olefination of unactivated sp3 C–H bonds. Nitrogen heterocycles serve as directing groups, and air is used as the terminal oxidant. The products undergo reversible intramolecular Michael addition, which protects the mono-alkenylated product from over-functionalization. Hydrogenation of the Michael adducts provides access to bicyclic nitrogen-containing scaffolds that are prevalent in alkaloid natural products. Additionally, the cationic Michael adducts undergo facile elimination to release α,β-unsaturated olefins, which can be elaborated in numerous C–C and C–heteroatom bond-forming reactions. PMID:21476513

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.

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

Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta "Gold Standard" Leaves.

Science.gov (United States)

Yu, Juanjuan; Zhang, Jinzheng; Zhao, Qi; Liu, Yuelu; Chen, Sixue; Guo, Hongliang; Shi, Lei; Dai, Shaojun

2016-03-08

Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta "Gold Standard" is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta "Gold Standard", as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta "Gold Standard". For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves.

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.

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.

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.

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.

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.

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)

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

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

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

Energy Technology Data Exchange (ETDEWEB)

Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M. (UMM); (HWMRI)

2016-09-05

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.

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

ASTROCYTIC CONTROL OF BIOSYNTHESIS AND TURNOVER OF THE NEUROTRANSMITTERS GLUTAMATE AND GABA

Directory of Open Access Journals (Sweden)

Arne eSchousboe

2013-08-01

Full Text Available Glutamate and GABA are the quantitatively major neurotransmitters in the brain mediating excitatory and inhibitory signaling, respectively. These amino acids are metabolically interrelated and at the same time they are tightly coupled to the intermediary metabolism including energy homeostasis. Astrocytes play a pivotal role in the maintenance of the neurotransmitter pools of glutamate and GABA since only these cells express pyruvate carboxylase (PC, the enzyme required for de novo synthesis of the two amino acids. Such de novo synthesis is obligatory to compensate for catabolism of glutamate and GABA related to oxidative metabolism when the amino acids are used as energy substrates. This, in turn, is influenced by the extent to which the cycling of the amino acids between neurons and astrocytes may occur. This cycling is brought about by the glutamate/GABA – glutamine cycle the operation of which involves the enzymes glutamine synthetase (GS and glutaminase (PAG together with the plasma membrane transporters for glutamate, GABA and glutamine. The distribution of these proteins between neurons and astrocytes determines the efficacy of the cycle and it is of particular importance that GS is exclusively expressed in astrocytes. It should be kept in mind that the operation of the cycle is associated with movement of ammonia nitrogen between the two cell types and different mechanisms which can mediate this have been proposed.This review is intended to delineate the above mentioned processes and to discuss quantitatively their relative importance in the homeostatic mechanisms responsible for the maintenance of optimal conditions for the respective neurotransmission processes to operate.

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

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...

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...

Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

Science.gov (United States)

Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

2017-01-27

The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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...

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.

Combined use of δ¹³C, δ18O and δ15N tracks nitrogen metabolism and genotypic adaptation of durum wheat to salinity and water deficit.

Science.gov (United States)

Yousfi, Salima; Serret, Maria Dolores; Márquez, Antonio José; Voltas, Jordi; Araus, José Luis

2012-04-01

• Accurate phenotyping remains a bottleneck in breeding for salinity and drought resistance. Here the combined use of stable isotope compositions of carbon (δ¹³C), oxygen (δ¹⁸O) and nitrogen (δ¹⁵N) in dry matter is aimed at assessing genotypic responses of durum wheat under different combinations of these stresses. • Two tolerant and two susceptible genotypes to salinity were grown under five combinations of salinity and irrigation regimes. Plant biomass, δ¹³C, δ¹⁸O and δ¹⁵N, gas-exchange parameters, ion and N concentrations, and nitrate reductase (NR) and glutamine synthetase (GS) activities were measured. • Stresses significantly affected all traits studied. However, only δ¹³C, δ¹⁸O, δ¹⁵N, GS and NR activities, and N concentration allowed for clear differentiation between tolerant and susceptible genotypes. Further, a conceptual model explaining differences in biomass based on such traits was developed for each growing condition. • Differences in acclimation responses among durum wheat genotypes under different stress treatments were associated with δ¹³C. However, except for the most severe stress, δ¹³C did not have a direct (negative) relationship to biomass, being mediated through factors affecting δ¹⁸O or N metabolism. Based upon these results, the key role of N metabolism in durum wheat adaptation to salinity and water stress is highlighted. No claim to original US government works. New Phytologist © 2012 New Phytologist Trust.

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.

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.

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.

Nitrogen large area proportional counter with gas regeneration

International Nuclear Information System (INIS)

Leidner, L.; Sadri, E.

1984-10-01

A nitrogen large area proportional chamber with gas regeneration is introduced to measure alpha and beta/gamma activites. In contrast to the flow counters used till now the new detector is independent of an external gas supply. The gas amplification factor of nitrogen keeps constant up to an impurity of 2% of O 2 . Oxygen diffusing through unavoidable leakages into the counting gas is removed by an activated catalyzer using low temperature copper oxidation. Humidty is adsorbed by a molecular sieve. The closed counter consists of three components: the actual detector, a gas purification cartridge and a gas circulating pump. Finally, the report describes long run experiments being carried out with prototypes. (orig./HP) [de

Platinum-Catalyzed, Terminal-Selective C(sp(3))-H Oxidation of Aliphatic Amines.

Science.gov (United States)

Lee, Melissa; Sanford, Melanie S

2015-10-14

This Communication describes the terminal-selective, Pt-catalyzed C(sp(3))-H oxidation of aliphatic amines without the requirement for directing groups. CuCl2 is employed as a stoichiometric oxidant, and the reactions proceed in high yield at Pt loadings as low as 1 mol%. These transformations are conducted in the presence of sulfuric acid, which reacts with the amine substrates in situ to form ammonium salts. We propose that protonation of the amine serves at least three important roles: (i) it renders the substrates soluble in the aqueous reaction medium; (ii) it limits binding of the amine nitrogen to Pt or Cu; and (iii) it electronically deactivates the C-H bonds proximal to the nitrogen center. We demonstrate that this strategy is effective for the terminal-selective C(sp(3))-H oxidation of a variety of primary, secondary, and tertiary amines.

Nitrogen acquisition, transport and metabolism in intact ectomycorrhizal associations studied by 15N stable isotope techniques

International Nuclear Information System (INIS)

Ek, H.

1993-05-01

The focus of this thesis is on the external mycelium and its role in nitrogen uptake, assimilation and translocation. Tree seedlings in association with ectomycorrhizal fungi were grown in observation chambers. The fungal mycelium were fed with 15-N ammonium or 15-N nitrate or a combination of both. The effects of Collembola on the ectomycorrhizal symbiosis were also studied. The results demonstrates an important role of the external mycelium of Paxillus involutus not only in the uptake but also in the assimilation of ammonium into a variety of different amino acids, primarily glutamine but also glutamic acid, aspartic acid, and alanine, immediately after uptake. The results indicate that ammonium is assimilated by GS and GOGAT or GDH in the mycelium at the uptake site. When nitrate was added to the mycelium as the sole nitrogen source nitrate was reduced in the mycelium and the product assimilated into amino acids. When ammonium nitrate was supplied to the fungal mycelium nitrate was taken up the fungus and transferred to the plant, however, apparently no assimilation of nitrate occurred in the external mycelium. Ammonium or an assimilation product, such as glutamine, probably represses nitrate reductase (NR) but not nitrate uptake and transfer in P. involutus. P. involutus nitrogen uptake and transfer to the associated mycorrhizal pine was up to 76% higher when low numbers of the Collembola Onychiurus armatus were present compared to when they were completely absent. This was probably an indirect effect as P. involutus hyphal growth rate and extramatrical biomass increased at a low Collembola density. At high Collembola densities P. involutus hyphal growth rate was retarded. (74 refs.)

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.

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....

Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

Science.gov (United States)

Matussek, Karl; Moritz, Patrick; Brunner, Nina; Eckerskorn, Christoph; Hensel, Reinhard

1998-01-01

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction. PMID:9811660

Cloning, sequencing, and expression of the gene encoding cyclic 2, 3-diphosphoglycerate synthetase, the key enzyme of cyclic 2, 3-diphosphoglycerate metabolism in Methanothermus fervidus.

Science.gov (United States)

Matussek, K; Moritz, P; Brunner, N; Eckerskorn, C; Hensel, R

1998-11-01

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction.

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.

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.

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

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...

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)

Trichodesmium’s strategies to alleviate phosphorus limitation in the future acidified oceans.

Science.gov (United States)

Spungin, Dina; Berman-Frank, Ilana; Levitan, Orly

2014-06-01

Global warming may exacerbate inorganic nutrient limitation, including phosphorus (P), in the surface waters of tropical oceans that are home to extensive blooms of the marine diazotrophic cyanobacterium, Trichodesmium. We examined the combined effects of P limitation and pCO(2), forecast under ocean acidification scenarios, on Trichodesmium erythraeum IMS101 cultures. We measured nitrogen acquisition,glutamine synthetase activity, C uptake rates, intracellular Adenosine Triphosphate (ATP) concentration and the pool sizes of related key proteins. Here, we present data supporting the idea that cellular energy re-allocation enables the higher growth and N(2) fixation rates detected in Trichodesmium cultured under high pCO(2). This is reflected in altered protein abundance and metabolic pools. Also modified are particulate organic carbon and nitrogen production rates,enzymatic activities, and cellular ATP concentrations. We suggest that adjusting these cellular pathways to changing environmental conditions enables Trichodesmium to compensate for low P availability and to thrive in acidified oceans. Moreover, elevated pCO(2) could provide Trichodesmium with a competitive dominance that would extend its niche, particularly in P-limited regions of the tropical and subtropical oceans.