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

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

2017-05-01

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

Apoptosis induced by glufosinate ammonium in the neuroepithelium of developing mouse embryos in culture.

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Watanabe, T

1997-01-24

Glufosinate ammonium structurally resembles glutamate and blocks glutamine synthetase. Glufosinate was recently found to be dysmorphogenic in mammals in vitro. The present study examined the cell death induced specifically by glufosinate in the neuroepithelium of mouse embryos. Electron micrograph revealed characteristic chromatin condensation and segregation, extracellular apoptotic bodies, and cell fragments phagocytosed in macrophages in the neuroepithelium of the brain vesicle and neural tube. Moreover neuroepithelial cells undergoing DNA fragmentation were histochemically identified. DNA gel electrophoresis of the neuroepithelial layer revealed a DNA ladder. These observations demonstrate that glufosinate specifically induced apoptosis in the neuroepithelium of embryos.

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

NARCIS (Netherlands)

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

2016-01-01

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

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.

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

DEFF Research Database (Denmark)

Vurrakula, Swathi

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

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

Science.gov (United States)

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

2017-05-01

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

Ammonium assmilation in spruce ectomycorrhizas

International Nuclear Information System (INIS)

Chalot, M.; Brun, A.; Botton, B.; Stewart, G.

1990-01-01

Assimilation of labelled NH 4 + into amino acids has been followed in ectomycorrhizal roots of spruce. Over an 18 h period of NH 4 + feeding, Gln, Glu and Ala became the most abundant amino acids. Gln was also the most highly labelled amino acid during the experiment, followed by Glu and Ala. This result indicates that Gln synthesis is an important ammonium utilization reaction in spruce mycorrhizas. Addition of MSX to NH 4 + fed mycorrhizas caused an inhibition of Gln accumulation with a corresponding increase in Glu, Ala and Asn levels. The supply of MSX induced a sharp diminution of 15 N enrichment in both amino and amido groups of glutamine. In contrast, the 15 N incorporation into Glu and derivatives (Ala and Asp) remained very high. This study demonstrates that the fungal glutamate dehydrogenase is quite operative in spruce ectomycorrhizas since it is able to sustain ammonium assimilation when glutamine synthetase is inhibited

Cytosolic glutamine synthetase in barley

DEFF Research Database (Denmark)

Thomsen, Hanne Cecilie

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

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

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

2008-01-04

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

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

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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.

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.

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

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

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

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

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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.

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.

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

Science.gov (United States)

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

2014-03-01

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

New perspectives on glutamine synthetase in grasses.

Science.gov (United States)

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

2011-02-01

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

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.

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

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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.

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.

Nutrient removal by Chlorella vulgaris F1068 under cetyltrimethyl ammonium bromide induced hormesis.

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Zhou, Qiongzhi; Li, Feng; Ge, Fei; Liu, Na; Kuang, Yangduo

2016-10-01

Toxicants are generally harmful to biotechnology in wastewater treatment. However, trace toxicant can induce microbial hormesis, but to date, it is still unknown how this phenomenon affects nutrient removal during municipal wastewater treatment process. Therefore, this study focused on the effects of hormesis induced by cetyltrimethyl ammonium bromide (CTAB), a representative quaternary ammonium cationic surfactant, on nutrient removal by Chlorella vulgaris F1068. Results showed that when the concentration of CTAB was less than 10 ng/L, the cellular components chlorophyll a, proteins, polysaccharides, and total lipids increased by 10.11, 58.17, 38.78, and 11.87 %, respectively, and some enzymes in nutrient metabolism of algal cells, such as glutamine synthetase (GS), acid phosphatase (ACP), H(+)-ATPase, and esterase, were also enhanced. As a result, the removal efficiencies of ammonia nitrogen (NH4 (+)) and total phosphorus (TP) increased by 14.66 and 8.51 %, respectively, compared to the control during a 7-day test period. The underlying mechanism was mainly due to an enhanced photosynthetic activity of C. vulgaris F1068 indicated by the increase in chlorophyll fluorescence parameters (the value of Fv/Fm, ΦII, Fv/Fo, and rETR increased by 12.99, 7.56, 25.59, and 8.11 %, respectively) and adenylate energy charge (AEC) (from 0.68 to 0.72). These results suggest that hormesis induced by trace toxicants could enhance the nutrient removal, which would be further considered in the design of municipal wastewater treatment processes. Graphical abstract The schematic mechanism of C. vulgaris F1068 under CTAB induced hormesis. Green arrows ( ) represent the increase and the red arrow ( ) represents the decrease.

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.

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.

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

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+.

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

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 +

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.

Characterization of seizures induced by acute exposure to an organophosphate herbicide, glufosinate-ammonium.

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Calas, André-Guilhem; Perche, Olivier; Richard, Olivier; Perche, Astrid; Pâris, Arnaud; Lauga, Fabien; Herzine, Ameziane; Palomo, Jennifer; Ardourel, Marie-Yvonne; Menuet, Arnaud; Mortaud, Stéphane; Pichon, Jacques; Montécot-Dubourg, Céline

2016-05-04

Glufosinate-ammonium (GLA), the active component of a widely used herbicide, induces convulsions in rodents and humans. In mouse, intraperitoneal treatment with 75 mg/kg GLA generates repetitive tonic-clonic seizures associated with 100% mortality within 72 h after treatment. In this context, we characterized GLA-induced seizures, their histological consequences and the effectiveness of diazepam treatment. Epileptic discharges on electroencephalographic recordings appeared simultaneously in the hippocampus and the cerebral cortex. Diazepam treatment at 6 h immediately stopped the seizures and prevented animal death. However, intermittent seizures were recorded on electroencephalogram from 6 h after diazepam treatment until 24 h, but had disappeared after 15 days. In our model, neuronal activation (c-Fos immunohistochemistry) was observed 6 h after GLA exposure in the dentate gyrus, CA1, CA3, amygdala, piriform and entorhinal cortices, indicating the activation of the limbic system. In these structures, Fluoro-Jade C and Cresyl violet staining did not show neuronal suffering. However, astroglial activation was clearly observed at 24 h and 15 days after GLA treatment in the amygdala, piriform and entorhinal cortices by PCR quantitative, western blot and immunohistochemistry. Concomitantly, glutamine synthetase mRNA expression (PCR quantitative), protein expression (western blot) and enzymatic activity were upregulated. In conclusion, our study suggests that GLA-induced seizures: (a) involved limbic structures and (b) induced astrocytosis without neuronal degeneration as an evidence of a reactive astrocyte beneficial effect for neuronal protection.

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.

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.

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

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

Analysis of reproductive toxicity and classification of glufosinate-ammonium.

Science.gov (United States)

Schulte-Hermann, Rolf; Wogan, Gerald N; Berry, Colin; Brown, Nigel A; Czeizel, Andrew; Giavini, Erminio; Holmes, Lewis B; Kroes, Robert; Nau, Heinz; Neubert, Diether; Oesch, Franz; Ott, Tilmann; Pelkonen, Olavi; Robert-Gnansia, Elisabeth; Sullivan, Frank M

2006-04-01

animals is identified-inhibition of glutamine synthetase. Glutamine is essential to the viability of the embryo. The embryo is dependent on a maternal source of the amino acid. For embryo lethality to occur, a significant reduction of maternal glutamine is required. Such reduction in maternal glutamine depends on a significant inhibition of glutamine synthetase by GA. This can only occur when the mother is exposed to very high levels of GA. SPECIFICALLY: a. The reproductive toxicity of GA is confined to very short, early stages of reproduction, during which the conceptus is dependent on maternal glutamine; and b. In order for the effect to occur, significant reduction in maternal blood glutamine level is required, which in turn depends on a significant inhibition of glutamine synthetase, induced by high levels of GA in the maternal system. There is no evidence for accumulation of GA in the mammalian organism beyond a factor of two and no evidence for its metabolic toxification. To raise a concern in humans, women would have to be exposed to GA during the very limited time frame of preimplantation or implantation and the exposure would have to be to the exceedingly high levels necessary to alter the maternal metabolism and, correspondingly, result in glutamine levels in maternal tissue and blood plasma being drastically reduced. There is no basis to suggest that such exposures would occur under conditions of normal handling and use. SPECIFICALLY: a. Under conditions of normal handling and use, operators would never be exposed to GA levels that could potentially inhibit glutamine synthetase to the extent that this inhibition could impair preimplantation or implantation. b. All acceptable exposure measurements and predictive calculations confirm this conclusion, and in fact demonstrate that reasonably foreseeable exposure of workers would be to levels significantly below the AOEL. c. The evidence is also clear that there is no reproductive toxicity hazard to workers upon

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.

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.)

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 +/.

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)

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.

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...

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

Science.gov (United States)

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

2011-01-01

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

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

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

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.

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

Science.gov (United States)

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

2011-09-01

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

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)

Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

Science.gov (United States)

Meza, Beatriz; de-Bashan, Luz E; Bashan, Yoav

2015-01-01

Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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)

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.

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

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

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

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

Science.gov (United States)

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

2014-05-01

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

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

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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.

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...

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.

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.

Glutamine alimentation in catabolic state.

Science.gov (United States)

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

2001-09-01

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

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

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

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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

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.

Decoupling of ammonium regulation and ntcA transcription in the diazotrophic marine cyanobacterium Trichodesmium sp. IMS101.

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Post, Anton F; Rihtman, Branko; Wang, Qingfeng

2012-03-01

Nitrogen (N) physiology in the marine cyanobacterium Trichodesmium IMS101 was studied along with transcript accumulation of the N-regulatory gene ntcA and of two of its target genes: napA (nitrate assimilation) and nifH (N(2) fixation). N(2) fixation was impaired in the presence of nitrite, nitrate and urea. Strain IMS101 was capable of growth on these combined N sources at ammonium. Whereas ecologically relevant N concentrations (2-20 μM) suppressed growth and assimilation, much higher concentrations were required to affect transcript levels. Transcripts of nifH accumulated under nitrogen-fixing conditions; these transcript levels were maintained in the presence of nitrate (100 μM) and ammonium (20 μM). However, nifH transcript levels were below detection at ammonium concentrations >20 μM. napA mRNA was found at low levels in both N(2)-fixing and ammonium-utilizing filaments, and it accumulated in filaments grown with nitrate. The positive effect of nitrate on napA transcription was abolished by ammonium additions of >200 μM. This effect was restored upon addition of the glutamine synthetase inhibitor L-methionin-DL-sulfoximine. Surprisingly, ntcA transcript levels remained high in the presence of ammonium, even at elevated concentrations. These findings indicate that ammonium repression is decoupled from transcriptional activation of ntcA in Trichodesmium IMS101.

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].

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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.

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

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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.

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

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

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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.

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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.

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.

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

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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

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

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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.

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

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

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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.

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.

The relationship between the violet pigment PP-V production and intracellular ammonium level in Penicillium purpurogenum.

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Kojima, Ryo; Arai, Teppei; Matsufuji, Hiroshi; Kasumi, Takafumi; Watanabe, Taisuke; Ogihara, Jun

2016-12-01

Penicillium purpurogenum is the fungus that produces an azaphilone pigment. However, details about the pigment biosynthesis pathway are unknown. The violet pigment PP-V is the one of the main pigments biosynthesized by this fungus. This pigment contains an amino group in a pyran ring as its core structure. We focused on this pigment and examined the relationship between intracellular ammonium concentration and pigment production using glutamine as a nitrogen source. The intracellular ammonium level decreased about 1.5-fold in conditions favoring PP-V production. Moreover, P. purpurogenum was transferred to medium in which it commonly produces the related pigment PP-O after cultivating it in the presence or absence of glutamine to investigate whether this fungus biosynthesizes PP-V using surplus ammonium in cells. Only mycelia cultured in medium containing 10 mM glutamine produced the violet pigment, and simultaneously intracellular ammonium levels decreased under this condition. From comparisons of the amount of PP-V that was secreted with quantity of surplus intracellular ammonium, it is suggested that P. purpurogenum maintains ammonium homeostasis by excreting waste ammonium as PP-V.

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

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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.

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes.

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Johansen, Maja L; Bak, Lasse K; Schousboe, Arne; Iversen, Peter; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Gjedde, Albert; Ott, Peter; Waagepetersen, Helle S

2007-06-01

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and alpha-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the alpha-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the alpha-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-13C]glucose (2.5 mM) and isoleucine (1 mM) or [U-13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-13C]glucose. Labeling in glutamate and aspartate from [U-13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially

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

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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.

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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.

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.

Azolla-Anabaena Relationship 1

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Meeks, John C.; Steinberg, Nisan A.; Enderlin, Carol S.; Joseph, Cecillia M.; Peters, Gerald A.

1987-01-01

The major radioactive products of the fixation of [13N]N2 by Azolla caroliniana Willd.-Anabaena azollae Stras. were ammonium, glutamine, and glutamate, plus a small amount of alanine. Ammonium accounted for 70 and 32% of the total radioactivity recovered after fixation for 1 and 10 minutes, respectively. The presence of a substantial pool of [13N]N2-derived 13NH4+ after longer incubation periods was attributed to the spatial separation between the site of N2-fixation (Anabaena) and a second, major site of assimilation (Azolla). Initially, glutamine was the most highly radioactive organic product formed from [13N]N2, but after 10 minutes of fixation glutamate had 1.5 times more radiolabel than glutamine. These kinetics of radiolabeling, along with the effects of inhibitors of glutamine synthetase and glutamate synthase on assimilation of exogenous and [13N]N2-derived 13NH4+, indicate that ammonium assimilation occurred by the glutamate synthase cycle and that glutamate dehydrogenase played little or no role in the synthesis of glutamate by Azolla-Anabaena. PMID:16665538

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

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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.

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.

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

Ginger-supplemented diet ameliorates ammonium nitrate-induced ...

African Journals Online (AJOL)

The present study was designed to evaluate the capacity of ginger to repair the oxidative stress induced by ammonium nitrate. 50 male rats were divided into 5 groups; they underwent an oral treatment of ammonium nitrate and/or ginger (N mg/kg body weight + G% in diet) during 30 days. Group I served as control (C); ...

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...

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

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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.

Glufosinate aerogenic exposure induces glutamate and IL-1 receptor dependent lung inflammation.

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Maillet, Isabelle; Perche, Olivier; Pâris, Arnaud; Richard, Olivier; Gombault, Aurélie; Herzine, Ameziane; Pichon, Jacques; Huaux, Francois; Mortaud, Stéphane; Ryffel, Bernhard; Quesniaux, Valérie F J; Montécot-Dubourg, Céline

2016-11-01

Glufosinate-ammonium (GLA), the active component of an herbicide, is known to cause neurotoxicity. GLA shares structural analogy with glutamate. It is a powerful inhibitor of glutamine synthetase (GS) and may bind to glutamate receptors. Since these potentials targets of GLA are present in lung and immune cells, we asked whether airway exposure to GLA may cause lung inflammation in mice. A single GLA exposure (1 mg/kg) induced seizures and inflammatory cell recruitment in the broncho-alveolar space, and increased myeloperoxidase (MPO), inducible NO synthase (iNOS), interstitial inflammation and disruption of alveolar septae within 6-24 h. Interleukin 1β (IL-1β) was increased and lung inflammation depended on IL-1 receptor 1 (IL-1R1). We demonstrate that glutamate receptor pathway is central, since the N-methyl-D-aspartate (NMDA) receptor inhibitor MK-801 prevented GLA-induced lung inflammation. Chronic exposure (0.2 mg/kg 3× per week for 4 weeks) caused moderate lung inflammation and enhanced airway hyperreactivity with significant increased airway resistance. In conclusion, GLA aerosol exposure causes glutamate signalling and IL-1R-dependent pulmonary inflammation with airway hyperreactivity in mice. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

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.

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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

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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...

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

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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...

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

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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.

Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans

International Nuclear Information System (INIS)

Rocha, Rita; Barbosa Pereira, Pedro José; Santos, Manuel A. S.; Macedo-Ribeiro, Sandra

2010-01-01

The seryl-tRNA synthetase from C. albicans was crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and diffraction data were collected to 2.0 Å resolution at a synchrotron source. The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2–3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Å resolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS

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.

Dysregulation of the Glutamine Transporter Slc38a3 (SNAT3 and Ammoniagenic Enzymes in Obese, Glucose-Intolerant Mice

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Stephanie M. Busque

2014-08-01

Full Text Available Background/Aims: Uric acid nephrolithiasis is prevalent among patients with type 2 diabetes and metabolic syndrome; it is correlated with an acidic urine and lower urinary ammonium excretion and is likely associated with insulin resistance. Insulin stimulates ammoniagenesis in renal cell lines via increased phosphate-dependent glutaminase (PDG activity and glutamine metabolism. Ammonium excretion into the proximal tubule is mediated at least in part by the Na+/H+-exchanger NHE3 and in the collecting duct involving the Rhesus protein RhCG. Here we tested, whether obesity and insulin resistance in a diet-induced mouse model could contribute to deranged ammonium excretion. Methods: Obesity was induced by diet in mice and the impact on key molecules of proximal tubular ammoniagenesis and urinary acid excretion tested. Results: Diet-induced obesity was confirmed by pathological intraperitoneal glucose tolerance tests (IPGTT. Three groups of mice were compared: control mice; obese, glucose-intolerant with abnormal IPGTT (O-GI; or moderate weight with normal IPGTT (Non-Responders, NR. Basal urinary ammonium excretion did not differ among groups. However, acid loading increased urinary ammonium excretion in all groups, but to a lesser extent in the O-GI group. SNAT3 mRNA expression was enhanced in both obese groups. PDG expression was elevated only in acid-loaded O-GI mice, whereas PEPCK was enhanced in both O-GI and NR groups given NH4CI. NHE activity in the brush border membrane of the proximal tubule was strongly reduced in the O-GI group whereas RhCG expression was similar. Conclusion: In sum, obesity and glucose intolerance impairs renal ammonium excretion in response to NH4CI feeding most likely through reduced NHE activity. The stimulation of SNAT3 and ammoniagenic enzyme expression may be compensatory but futile.

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.

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

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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|

Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

NARCIS (Netherlands)

Lamers, W. H.; Zonneveld, D.; Charles, R.

1984-01-01

Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is

De Novo Glutamine Synthesis

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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

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.

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.

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

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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_000962 [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

Elevated ammonium levels

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Søgaard, Rikke; Novak, Ivana; MacAulay, Nanna

2012-01-01

was not enhanced. The ammonium-induced stimulation of EAAT3 increased with increasing extracellular pH, suggesting that the gaseous form NH(3) mediates the effect. An ammonium-induced intracellular alkalinization was excluded as the cause of the enhanced EAAT3 activity because 1) ammonium acidified the oocyte...

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

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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.

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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.

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

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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

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.

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.

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.

Ammonium-induced calcium mobilization in 1321N1 astrocytoma cells

International Nuclear Information System (INIS)

Hillmann, Petra; Koese, Meryem; Soehl, Kristina; Mueller, Christa E.

2008-01-01

High blood levels of ammonium/ammonia (NH 4 + /NH 3 ) are associated with severe neurotoxicity as observed in hepatic encephalopathy (HE). Astrocytes are the main targets of ammonium toxicity, while neuronal cells are less vulnerable. In the present study, an astrocytoma cell line 1321N1 and a neuroblastoma glioma hybrid cell line NG108-15 were used as model systems for astrocytes and neuronal cells, respectively. Ammonium salts evoked a transient increase in intracellular calcium concentrations ([Ca 2+ ] i ) in astrocytoma (EC 50 = 6.38 mM), but not in NG108-15 cells. The ammonium-induced increase in [Ca 2+ ] i was due to an intracellular effect of NH 4 + /NH 3 and was independent of extracellular calcium. Acetate completely inhibited the ammonium effect. Ammonium potently reduced calcium signaling by G q protein-coupled receptors (H 1 and M3) expressed on the cells. Ammonium (5 mM) also significantly inhibited the proliferation of 1321N1 astrocytoma cells. While mRNA for the mammalian ammonium transporters RhBG and RhCG could not be detected in 1321N1 astrocytoma cells, both transporters were expressed in NG108-15 cells. RhBG and RhBC in brain may promote the excretion of NH 3 /NH 4 + from neuronal cells. Cellular uptake of NH 4 + /NH 3 was mainly by passive diffusion of NH 3 . Human 1321N1 astrocytoma cells appear to be an excellent, easily accessible human model for studying HE, which can substitute animal studies, while NG108-15 cells may be useful for investigating the role of the recently discovered Rhesus family type ammonium transporters in neuronal cells. Our findings may contribute to the understanding of pathologic ammonium effects in different brain cells, and to the treatment of hyperammonemia

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.

Glufosinate ammonium induces convulsion through N-methyl-D-aspartate receptors in mice.

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Matsumura, N; Takeuchi, C; Hishikawa, K; Fujii, T; Nakaki, T

2001-05-18

Glufosinate ammonium, a broad-spectrum herbicide, causes convulsion in rodents and humans. Because of the structural similarities between glufosinate and glutamate, the convulsion induced by glufosinate ammonium may be ascribed to glutamate receptor activation. Three N-methyl-D-asparate (NMDA) receptor antagonists, dizocilpine, LY235959, and Compound 40, and an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist, NBQX, were coadministrated with glufosinate ammonium (80 mg/kg, intraperitoneally) in mice. Statistical analyses showed that the NMDA receptor antagonists markedly inhibited the convulsions, while the AMPA/kainate receptor antagonist had no effect on the convulsion. These results suggest that the convulsion caused by glufosinate ammonium is mediated through NMDA receptors.

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.

The relationship between the violet pigment PP-V production and intracellular ammonium level in Penicillium purpurogenum

OpenAIRE

Kojima, Ryo; Arai, Teppei; Matsufuji, Hiroshi; Kasumi, Takafumi; Watanabe, Taisuke; Ogihara, Jun

2016-01-01

Penicillium purpurogenum is the fungus that produces an azaphilone pigment. However, details about the pigment biosynthesis pathway are unknown. The violet pigment PP-V is the one of the main pigments biosynthesized by this fungus. This pigment contains an amino group in a pyran ring as its core structure. We focused on this pigment and examined the relationship between intracellular ammonium concentration and pigment production using glutamine as a nitrogen source. The intracellular ammonium...

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.

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

Oral supplementations with L-glutamine or L-alanyl-L-glutamine do not change metabolic alterations induced by long-term high-fat diet in the B6.129F2/J mouse model of insulin resistance.

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Bock, Patricia Martins; Krause, Mauricio; Schroeder, Helena Trevisan; Hahn, Gabriela Fernandes; Takahashi, Hilton Kenji; Schöler, Cinthia Maria; Nicoletti, Graziella; Neto, Luiz Domingos Zavarize; Rodrigues, Maria Inês Lavina; Bruxel, Maciel Alencar; Homem de Bittencourt, Paulo Ivo

2016-01-01

In this work, we aimed to investigate the effects of long-term supplementations with L-glutamine or L-alanyl-L-glutamine in the high-fat diet (HFD)-fed B6.129SF2/J mouse model over insulin sensitivity response and signaling, oxidative stress markers, metabolism and HSP70 expression. Mice were fed in a standard low-fat diet (STA) or a HFD for 20 weeks. In the 21th week, mice from the HFD group were allocated in five groups and supplemented for additional 8 weeks with different amino acids: HFD control group (HFD-Con), HFD + dipeptide L-alanyl-L-glutamine group (HFD-Dip), HFD + L-alanine group (HFD-Ala), HFD + L-glutamine group (HFD-Gln), or the HFD + L-alanine + L-glutamine (in their free forms) group (HFD-Ala + Gln). HFD induced higher body weight, fat pad, fasted glucose, and total cholesterol in comparison with STA group. Amino acid supplementations did not induce any modifications in these parameters. Although insulin tolerance tests indicated insulin resistance in all HFD groups, amino acid supplementations did not improve insulin sensitivity in the present model. There were also no significant differences in the immunocontents of insulin receptor, Akt, and Toll-like receptor-4. Notably, total 70 kDa heat shock protein (HSP72 + HSP73) contents in the liver was markedly increased in HFD-Con group as compared to STA group, which might suggest that insulin resistance is only in the beginning. Apparently, B6.129SF2/J mice are more resistant to the harmful effects of HFD through a mechanism that may include gut adaptation, reducing the absorption of nutrients, including amino acids, which may explain the lack of improvements in our intervention.

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.

Ammonium inhibition of nitrogenase activity in Herbaspirillum seropedicae

Energy Technology Data Exchange (ETDEWEB)

Fu, H.; Burris, R.H. (Univ. of Wisconsin, Madison (USA))

1989-06-01

The effect of oxygen, ammonium ion, and amino acids on nitrogenase activity in the root-associated N{sub 2}-fixing bacterium Herbaspirillum seropedicae was investigated in comparison with Azospirillum spp. and Rhodospirillum rubrum. H. seropedicae is microaerophilic, and its optimal dissolved oxygen level is from 0.04 to 0.2 kPa for dinitrogen fixation but higher when it is supplied with fixed nitrogen. No nitrogenase activity was detected when the dissolved O{sub 2} level corresponded to 4.0 kPa. Ammonium, a product of the nitrogenase reaction, reversible inhibited nitrogenase activity when added to derepressed cell cultures. However, the inhibition of nitrogenase activity was only partial even with concentrations of ammonium chloride as high as 20 mM. Amides such as glutamine and asparagine partially inhibited nitrogenase activity, but glutamate did not. Nitrogenase in crude extracts prepared from ammonium-inhibited cells showed activity as high as in extracts from N{sub 2}-fixing cells. The pattern of the dinitrogenase and the dinitrogenase reductase revealed by the immunoblotting technique did not change upon ammonium chloride treatment of cells in vivo. No homologous sequences were detected with the draT-draG probe from Azospirillum lipoferum. There is no clear evidence that ADP-ribosylation of the dinitrogenase reductase is involved in the ammonium inhibition of H. seropedicae. The uncoupler carbonyl cyanide m-chlorophenylhydrazone decreased the intracellular ATP concentration and inhibited the nitrogenase activity of whole cells. The ATP pool was significantly disturbed when cultures were treated with ammonium in vivo.

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

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.

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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.

Arginyl-glutamine dipeptide or docosahexaenoic acid attenuates hyperoxia-induced small intestinal injury in neonatal mice.

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Li, Nan; Ma, Liya; Liu, Xueyan; Shaw, Lynn; Li Calzi, Sergio; Grant, Maria B; Neu, Josef

2012-04-01

Supplementation studies of glutamine, arginine, and docosahexaenoic acid (DHA) have established the safety of each of these nutrients in neonates; however, the potential for a more stable and soluble dipeptide, arginyl-glutamine (Arg-Gln) or DHA with anti-inflammatory properties, to exert benefits on hyperoxia-induced intestinal injury has not been investigated. Arg-Gln dipeptide has been shown to prevent retinal damage in a rodent model of oxygen-induced injury. The objective of the present study was to investigate whether Arg-Gln dipeptide or DHA could also attenuate markers of injury and inflammation to the small intestine in this same model. Seven-day-old mouse pups were placed with their dams in 75% oxygen for 5 days. After 5 days of hyperoxic exposure (P7-P12), pups were removed from hyperoxia and allowed to recover in atmospheric conditions for 5 days (P12-P17). Mouse pups received Arg-Gln (5g·kg·day) or DHA (5g·kg·day) or vehicle orally started on P12 through P17. Distal small intestine (DSI) histologic changes, myeloperoxidase (MPO), lactate dehydrogenase (LDH), inflammatory cytokines, and tissue apoptosis were evaluated. Hyperoxic mice showed a greater distortion of overall villus structure and with higher injury score (PDHA supplementation groups were more similar to the room air control group. Supplementation of Arg-Gln or DHA reduced hyperoxia-induced MPO activity (PDHA returned LDH activity to the levels of control. Hyperoxia induced apoptotic cell death in DSIs, and both Arg-Gln and DHA reversed this effect (PDHA may limit some inflammatory and apoptotic processes involved in hyperoxic-induced intestinal injury in neonatal mice.

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.)

Glufosinate ammonium-induced pathogen inhibition and defense responses culminate in disease protection in bar-transgenic rice.

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Ahn, Il-Pyung

2008-01-01

Glufosinate ammonium diminished developments of rice (Oryza sativa) blast and brown leaf spot in 35S:bar-transgenic rice. Pre- and postinoculation treatments of this herbicide reduced disease development. Glufosinate ammonium specifically impeded appressorium formation of the pathogens Magnaporthe grisea and Cochliobolus miyabeanus on hydrophobic surface and on transgenic rice. In contrast, conidial germination remained unaffected. Glufosinate ammonium diminished mycelial growth of two pathogens; however, this inhibitory effect was attenuated in malnutrition conditions. Glufosinate ammonium caused slight chlorosis and diminished chlorophyll content; however, these alterations were almost completely restored in transgenic rice within 7 d. Glufosinate ammonium triggered transcriptions of PATHOGENESIS-RELATED (PR) genes and hydrogen peroxide accumulation in transgenic rice and PR1 transcription in Arabidopsis (Arabidopsis thaliana) wild-type ecotype Columbia harboring 35S:bar construct. All transgenic Arabidopsis showed robust hydrogen peroxide accumulation by glufosinate ammonium. This herbicide also induced PR1 transcription in etr1 and jar1 expressing bar; however, no expression was observed in NahG and npr1. Fungal infection did not alter transcriptions of PR genes and hydrogen peroxide accumulation induced by glufosinate ammonium. Infiltration of glufosinate ammonium did not affect appressorium formation of M. grisea in vivo but inhibited blast disease development. Hydrogen peroxide scavengers nullified blast protection and transcriptions of PR genes by glufosinate ammonium; however, they did not affect brown leaf spot progression. In sum, both direct inhibition of pathogen infection and activation of defense systems were responsible for disease protection in bar-transgenic rice.

Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine

DEFF Research Database (Denmark)

Leke, Renata; Bak, Lasse Kristoffer; Anker, Malene

2011-01-01

Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme....... Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways...... in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure...

Irradiation temperature effect on glutamine (spectrophotometric readout) dosimeter

International Nuclear Information System (INIS)

Gupta, B.L.; Narayan, G.R.; Nilekani, S.R.; Bhat, R.M.

2001-01-01

For the dose estimation using glutamine dosimeter, 20 mg L-glutamine powder is dissolved in 10 ml of a solution which contains 2x10 -3 mol dm -3 ferrous ammonium sulphate and 10 -4 mol dm -3 xylenol orange in aerated aqueous 0.033 mol dm -3 sulphuric acid (FX). The plot of absorbance at 549 nm against dose is non-linear, however, the plot of 1/absorbance vs. 1/dose is linear. The slope of this linear plot changes above an absorbance of 0.3 corresponding to a dose of about 15 kGy. The response of the dosimeter is independent of irradiation temperature in the temperature range of 23-30 deg. C within the uncertainty of dose measurement. Below 23 deg. C, the absorbance decreases by 1.23% per deg. sign C decrease in temperature while between 30 and 40 deg. C, the absorbance increases by 0.75% per deg. C increase in temperature. Above 40 deg. C, the absorbance increases by 0.2% per deg. C only. The absorbance is corrected for the irradiation temperature and the dose is read from a calibration graph at 25 deg. C. Either a linear or polynomial relation can be used for the dose calculation

Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.

Science.gov (United States)

Kleijkers, Sander H M; van Montfoort, Aafke P A; Bekers, Otto; Coonen, Edith; Derhaag, Josien G; Evers, Johannes L H; Dumoulin, John C M

2016-06-01

Does ammonium accumulate in commercially available culture media and protein supplements used for in vitro development of human pre-implantation embryos during storage and incubation? Ammonium accumulates in ready-to-use in vitro fertilization (IVF) culture media during storage at 2-8°C and in ready-to-use IVF culture media and protein supplements during incubation at 37°C. Both animal and human studies have shown that the presence of ammonium in culture medium has detrimental effects on embryonic development and pregnancy rate. It is, therefore, important to assess the amount of ammonium accumulation in ready-to-use IVF culture media under conditions that are common in daily practice. Ammonium accumulation was investigated in 15 ready-to-use media, 11 protein-free media and 8 protein supplements. Ammonium was measured by the use of an enzymatic method with glutamate dehydrogenase. To simulate the storage and incubation conditions during IVF treatments, ammonium concentrations were measured at different time-points during storage at 2-8°C for 6 weeks and during incubation at 37°C for 4 days. All ready-to-use, i.e. protein supplemented, culture media showed ammonium accumulation during storage for 6 weeks (ranging from 9.2 to 99.8 µM) and during incubation for 4 days (ranging from 8.4 to 138.6 µM), resulting in levels that might affect embryo development. The protein supplements also showed ammonium accumulation, while the culture media without protein supplementation did not. The main sources of ammonium buildup in ready-to-use culture media were unstable glutamine and the protein supplements. No additional ammonium buildup was found during incubation when using an oil overlay or with the presence of an embryo in the culture droplet. In addition to the unstable glutamine and the protein supplements, other free amino acids might contribute to the ammonium buildup. We did not investigate the deterioration of other components in the media. Break-down of

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...

Ammonium-induced inhibition of ammonium-starved Nitrosomonas europaea cells in soil and sand slurries

NARCIS (Netherlands)

Gerards, S.; Duyts, H.; Laanbroek, H.J.

1998-01-01

Ammonia-oxidising bacteria are poor competitors for limiting amounts of ammonium. Hence, starvation for ammonium seems to be the regular condition for these bacteria in natural environments. Long-term survival in the absence of ammonium will be dependent on the ability to maintain large population

Glutamine prevents oxidative stress in a model of portal hypertension.

Science.gov (United States)

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

2017-07-07

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

Global Transcriptional and Physiological Responses of Saccharomyces cerevisiae to Ammonium, L-Alanine, or L-Glutamine Limitation

DEFF Research Database (Denmark)

Usaite, Renata; Patil, Kiran Raosaheb; Grotkjær, Thomas

2006-01-01

-ammonium in limitation and by growing cells in an excess of ammonium. Cells grown in L-alanine-limited cultures had higher biomass yield per nitrogen mole (19%) than those from ammonium-limited cultures. Whole-genome transcript profiles were analyzed with a genome-scalle metabolic model that suggested increased anabolic...... activity in L-alanine-limited cells. The changes in these cells were found to be focused around pyruvate, acetyl coenzyme A, glyoxylate, and alpha-ketoglutarate via increased levels of ALT1, DAL7, PYC1, GDH2, and ADH5 and decreased levels of GDH3, CIT2, and ACS1 transcripts. The transcript profiles were...

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

Energy Technology Data Exchange (ETDEWEB)

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

1982-04-01

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

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

The role of amino acids on the development of radiation-induced damage of central nervous system

International Nuclear Information System (INIS)

Yamatodani, Atsushi; Yamamoto, Kouichi; Yamamoto, Takashi; Moriyasu, Saeko

2006-01-01

We have found that heavy-ion (carbon) irradiation significantly increased the extracellular glutamate, the major excitatory neurotransmitter in the central nervous system, in the hypothalamus of rats. We also found that the increase of glutamate is dependent on the Ca 2+ ion, suggesting that the increased glutamate is derived from the release from neurons or glial cells. However, the underlying mechanisms of the increase of glutamate release are still unclear. In this study, we investigated that the effects of the glial selective metabolic inhibitor (L-aminoadipatic acid (L-AA), glutamine synthetase inhibitor (methionine sulfoximide (MSO)) and inhibitor of glutamate release from glial cell (carboxyphenylglycine (CPG)) on the increased glutamate measured by in vivo brain microdialysis. L-AA and MSO completely inhibited the radiation-induced increase of glutamate, but CPG did not inhibit the increase. Administration of glutamine recovered the increased extracellular glutamate level in the MSO-treated rats. These results suggested that neurons, but not glial cells, play an important role in the radiation-induced increase of extracellular glutamate. (author)

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

A radiochemical method for carbamoyl-phosphate synthetase I: application to rats fed a hyperproteic diet

OpenAIRE

Arriarán, Sofía; Agnelli, Silvia; Fernández López, José Antonio; Remesar Betlloch, Xavier; Alemany, Marià, 1946-

2012-01-01

A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium w...

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...

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...

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.

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.

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...

Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

Science.gov (United States)

Leke, Renata; Bak, Lasse K; Anker, Malene; Melø, Torun M; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Portela, Luis V; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

2011-04-01

Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

Antioxidant Potential of Momordica Charantia in Ammonium Chloride-Induced Hyperammonemic Rats

Directory of Open Access Journals (Sweden)

A. Justin Thenmozhi

2011-01-01

Full Text Available The present study was aimed to investigate the antioxidant potential of Momordica charantia fruit extract (MCE in ammonium chloride-induced (AC hyperammonemic rats. Experimental hyperammonemia was induced in adult male Wistar rats (180–200 g by intraperitoneal injections of ammonium chloride (100 mg kg−1 body weight thrice a week. The effect of oral administration (thrice a week for 8 consecutive weeks of MCE (300 mg kg−1 body weight on blood ammonia, plasma urea, serum liver marker enzymes and oxidative stress biomarkers in normal and experimental animals was analyzed. Hyperammonemic rats showed a significant increase in the activities of thiobarbituric acid reactive substances, hydroperoxides and liver markers (alanine transaminase, aspartate transaminase and alkaline phosphatase, and the levels of glutathione peroxidase, superoxide dismutase, catalase and reduced glutathione were decreased in the liver and brain tissues. Treatment with MCE normalized the above-mentioned changes in hyperammonemic rats by reversing the oxidant-antioxidant imbalance during AC-induced hyperammonemia, and offered protection against hyperammonemia. Our results indicate that MCE exerting the antioxidant potentials and maintaining the cellular integrity of the liver tissue could offer protection against AC-induced hyperammonemia. However, the exact underlying mechanism is yet to be investigated, and examination of the efficacy of the active constituents of the M. charantia on hyperammonemia is desirable.

Glufosinate Ammonium-Induced Pathogen Inhibition and Defense Responses Culminate in Disease Protection in bar-Transgenic Rice1[C

Science.gov (United States)

Ahn, Il-Pyung

2008-01-01

Glufosinate ammonium diminished developments of rice (Oryza sativa) blast and brown leaf spot in 35S:bar-transgenic rice. Pre- and postinoculation treatments of this herbicide reduced disease development. Glufosinate ammonium specifically impeded appressorium formation of the pathogens Magnaporthe grisea and Cochliobolus miyabeanus on hydrophobic surface and on transgenic rice. In contrast, conidial germination remained unaffected. Glufosinate ammonium diminished mycelial growth of two pathogens; however, this inhibitory effect was attenuated in malnutrition conditions. Glufosinate ammonium caused slight chlorosis and diminished chlorophyll content; however, these alterations were almost completely restored in transgenic rice within 7 d. Glufosinate ammonium triggered transcriptions of PATHOGENESIS-RELATED (PR) genes and hydrogen peroxide accumulation in transgenic rice and PR1 transcription in Arabidopsis (Arabidopsis thaliana) wild-type ecotype Columbia harboring 35S:bar construct. All transgenic Arabidopsis showed robust hydrogen peroxide accumulation by glufosinate ammonium. This herbicide also induced PR1 transcription in etr1 and jar1 expressing bar; however, no expression was observed in NahG and npr1. Fungal infection did not alter transcriptions of PR genes and hydrogen peroxide accumulation induced by glufosinate ammonium. Infiltration of glufosinate ammonium did not affect appressorium formation of M. grisea in vivo but inhibited blast disease development. Hydrogen peroxide scavengers nullified blast protection and transcriptions of PR genes by glufosinate ammonium; however, they did not affect brown leaf spot progression. In sum, both direct inhibition of pathogen infection and activation of defense systems were responsible for disease protection in bar-transgenic rice. PMID:17981989

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.)

Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1

International Nuclear Information System (INIS)

Iwaki, Jun; Suzuki, Ryuichiro; Fujimoto, Zui; Momma, Mitsuru; Kuno, Atsushi; Hasegawa, Tsunemi

2005-01-01

Tyrosyl-tRNA synthetase from the hyperthermophilic archaeon A. pernix K1 was cloned, purified and crystallized. The crystals belonged to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K. Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K

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 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.

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...

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

Directory of Open Access Journals (Sweden)

Yan-Ren Lin

2016-01-01

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

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.

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

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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.

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

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

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

Detection of an ylide intermediate in the electrochemically-induced Stevens rearrangement of an ammonium salt by in situ UV–vis spectroelectrochemistry

International Nuclear Information System (INIS)

Capobianco, Amedeo; Caruso, Tonino; Palombi, Laura; Peluso, Andrea

2013-01-01

Highlights: ► Mechanistic insights of the electro-induced Stevens rearrangement are provided. ► The reduction of PhCOCH 2 N + (CH 3 ) 2 CH 2 Ph is ascribed to a one-electron transfer process. ► An electrogenerated ammonium ylide has been detected by UV-spectroelectrochemistry. -- Abstract: The electrochemically-induced Stevens rearrangement of 2-(benzyldimethyl)ammonium acetophenone has been investigated by in situ UV–vis spectroelectrochemistry. Voltammetric analysis and absorption spectra recorded during the potentiostatic reduction indicate that the reaction proceeds via a one-electron transfer with a Platinum cathode and generation of an ammonium ylide intermediate

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.

Chronotherapeutic effect of fisetin on expression of urea cycle enzymes and inflammatory markers in hyperammonaemic rats.

Science.gov (United States)

Subramanian, Perumal; Jayakumar, Murugesan; Jayapalan, Jaime Jacqueline; Hashim, Onn Haji

2014-12-01

Elevated blood ammonia leads to hyperammonaemia that affects vital central nervous system (CNS) functions. Fisetin, a naturally occurring flavonoid, exhibits therapeutic benefits, such as anti-cancer, anti-diabetic, anti-oxidant, anti-angiogenic, neuroprotective and neurotrophic effects. In this study, the chronotherapeutic effect of fisetin on ammonium chloride (AC)-induced hyperammonaemic rats was investigated, to ascertain the time point at which the maximum drug effect is achieved. The anti-hyperammonaemic potential of fisetin (50mg/kg b.w. oral) was analysed when administered to AC treated (100mg/kg b.w. i.p.) rats at 06:00, 12:00, 18:00 and 00:00h. Amelioration of pathophysiological conditions by fisetin at different time points was measured by analysing the levels of expression of liver urea cycle enzymes (carbamoyl phosphate synthetase-I (CPS-I), ornithine transcarbamoylase (OTC) and argininosuccinate synthetase (ASS)), nuclear transcription factor kappaB (NF-κB p65), brain glutamine synthetase (GS) and inducible nitric oxide synthase (iNOS) by Western blot analysis. Fisetin increased the expression of CPS-I, OTC, ASS and GS and decreased iNOS and NF-κB p65 in hyperammonaemic rats. Fisetin administration at 00:00h showed more significant effects on the expression of liver and brain markers, compared with other time points. Fisetin could exhibit anti-hyperammonaemic effect owing to its anti-oxidant and cytoprotective influences. The temporal variation in the effect of fisetin could be due to the (i) chronopharmacological, chronopharmacokinetic properties of fisetin and (ii) modulations in the endogenous circadian rhythms of urea cycle enzymes, brain markers, redox enzymes and renal clearance during hyperammonaemia by fisetin. However, future studies in these lines are necessitated. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

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

Energy Technology Data Exchange (ETDEWEB)

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

2012-09-01

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

[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

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.

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

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...

CO2 laser photo-induced decomposition of ammoniated ammonium ions

International Nuclear Information System (INIS)

Ikezoe, Yasumasa; Soga, Takesi; Suzuki, Kazuya; Moriyama, Noboru; Ohno, Shin-ichi

1995-01-01

Photo-induced decomposition of ammoniated (clustered) ammonium ions was studied using a CO 2 laser to excite vibrational levels of the cluster ion. A tandem mass spectrometer (TMS) was installed with two quadrupole mass filters, a corona discharge ionization chamber, and a series of einzel lenses. Cluster ions of NH 4 + ·nNH 3 with n=1-7 were formed in TMS, and found to decompose at the frequency of 1077 cm -1 to an extent in proportional to laser intensity. CO 2 laser between 925 and 1055 do not decompose the cluster ions with laser intensities examined. (author)

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.

Science.gov (United States)

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

2017-11-10

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

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

International Nuclear Information System (INIS)

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

1998-01-01

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

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.

Introduction to the Glutamate-Glutamine Cycle.

Science.gov (United States)

Sonnewald, Ursula; Schousboe, Arne

2016-01-01

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

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.).

Science.gov (United States)

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.

Deamidation Reactions of Asparagine- and Glutamine-Containing Dipeptides Investigated by Ion Spectroscopy

NARCIS (Netherlands)

Kempkes, L.J.M.; Martens, J.; Grzetic, J.; Berden, G.; Oomens, J.

2016-01-01

Deamidation is a major fragmentation channel upon activation by collision induced dissociation (CID) for protonated peptides containing glutamine (Gln) and asparagine (Asn) residues. Here, we investigate these NH3-loss reactions for four Asn- and Gln-containing protonated peptides in terms of the

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.

Adaptation to rhizosphere acidification is a necessary prerequisite for wheat (Triticum aestivum L.) seedling resistance to ammonium stress.

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Wang, Feng; Gao, Jingwen; Tian, Zhongwei; Liu, Yang; Abid, Muhammad; Jiang, Dong; Cao, Weixing; Dai, Tingbo

2016-11-01

Because soil acidification accompanies ammonium (NH 4 + ) stress, the tolerance of higher plants to ammonium is associated with their adaptation to root medium acidification. However, the underlying mechanisms of this adaptation have not been fully elucidated. The objective of this study was thus to elucidate the effect of rhizosphere pH on NH 4 + tolerance in different winter wheat cultivars (Triticum aestivum L.). Hydroponic experiments were carried out on two wheat cultivars: AK58 (an NH 4 + -sensitive cultivar) and XM25 (an NH 4 + -tolerant cultivar). Four pH levels resembling acidified (4.0, 5.0, 6.0 and 7.0) were tested and 5 mM NH 4 + nitrogen (AN) was used as a stress treatment, with 5 mM nitrate nitrogen used as a control. The addition of AN led to a severe reduction in biomass and an increase in free NH 4 + , amino acids, and the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in the shoots and roots of the two wheat cultivars. Further decreases in growth medium pH led to further increases in free NH 4 + , but decreases in total amino acids and the activities of GS and NADH-dependent glutamate synthase (NADH-GDH). However, there was less of an increase in free NH 4 + and less of a reduction in the activities of GS and NADH-GDH in the cultivar XM25 compared with AK58. In addition, total soluble sugar content and the root-to-shoot soluble sugar ratio were also decreased by AN treatment, except in the shoots of XM25. Decreasing pH resulted in lower root-to-shoot soluble sugar ratios with greater reductions in the AK58 cultivar. These results indicate that wheat growth was inhibited significantly by the addition of NH 4 + combined with low pH. Low medium pH reduced the capacity for nitrogen assimilation and interrupted carbohydrate transport between the shoot and root. The NH 4 + -tolerant cultivar XM25 was better adapted to low rhizosphere pH due to its increased capacity for assimilating NH 4 + efficiently and thereby avoiding

Influence of endogenous pyrogen on the cerebral prostaglandin-synthetase system.

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Ziel, R; Krupp, P

1976-11-15

The biotransformation of arachidonic acid to prostaglandins in vitro is specifically augmented by endogenous pyrogen to a degree depending on the concentration applied, providing that the microsomal fraction of the cerebral cortex is used as prostaglandin-synthetase system. This effect is inhibited by non-steroidal anti-inflammatory agents. These findings are compatible with the hypothesis that prostaglandins might act as mediators of the febrile reaction induced by endogenous pyrogen.

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 and glutamate as vital metabolites

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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.

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

MRI characterization of structural mouse brain changes in response to chronic exposure to the glufosinate ammonium herbicide.

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Meme, Sandra; Calas, André-Guilhem; Montécot, Céline; Richard, Oliver; Gautier, Hélène; Gefflaut, Thierry; Doan, Bich Thuy; Même, William; Pichon, Jacques; Beloeil, Jean-Claude

2009-10-01

Glufosinate ammonium (GLA) is the active component of herbicides widely used in agriculture, truck farming, or public domains. GLA acts by inhibiting the plant glutamine synthetase (GlnS). It also inhibits mammalian GlnS in vitro and ex vivo. In the central nervous system this enzyme is exclusively localized in glial cells. Whereas acute neurotoxic effects of GLA are well documented, long-term effects during chronic exposure at low doses remain largely undisclosed. In the present work, C57BL/6J mice were treated intraperitoneally with 2.5, 5, and 10 mg/kg of GLA three times a week during 10 weeks. Cerebral magnetic resonance imaging (MRI) experiments were performed at high field (9.4 T) and the images were analyzed with four texture analysis (TA) methods. TA highlighted structural changes in seven brain structures after chronic GLA treatments. Changes are dose dependent and can be seen at a dose as low as 2.5 mg/kg for two areas, namely hippocampus and somatosensorial cortex. Glial fibrillary acidic protein (GFAP) expression in the same seven brain structures and GlnS activity in the hippocampus and cortex areas were also studied. The number of GFAP-positive cells is modified in six out of the seven areas examined. GlnS activity was significantly increased in the hippocampus but not in the cortex. These results indicate some kind of suffering at the cerebral level after chronic GLA treatment. Changes in TA were compared with the modification of the number of GFAP-positive astrocytes in the studied brain areas after GLA treatment. We show that the noninvasive MRI-TA is a sensitive method and we suggest that it would be a very helpful tool that can efficiently contribute to the detection of cerebral alterations in vivo during chronic exposure to xenobiotics.

Glutamine prevents gastric oxidative stress in an animal model of portal hypertension gastropathy.

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Marques, Camila; Mauriz, José L; Simonetto, Douglas; Marroni, Claudio A; Tuñon, María J; González-Gallego, Javier; Marrón, Norma P

2011-01-01

Portal hypertension (PHI) is a clinical syndrome characterized by increases of the blood flow and/or of the vascular resistance in the portal system. A direct consequence of PHI can appearance different lesions on the gastric mucosa and submucosa, cumulatively termed portal hypertensive gastropathy (PHG). To investigate the effects of glutamine on oxidative stress in an experimental model of PHG induced by partial portal vein ligation (PPVL). Portal pressure, transaminase and alkaline phosphatase activity were quantified. Gastric tissue damage was assessed by histological analysis. Oxidative stress was measured by quantification of cytosolic concentration of thiobarbituric acid reactive substances (TBARS), hydroperoxide-initiated chemiluminescence (QL), and nitric oxide (NO) production. Moreover, activities of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were analyzed. Transaminase and alkaline phosphatase activities were not significantly modified by PPVL, indicating absence of liver injury. Histological analysis of gastric sections showed a lost of normal architecture, with edema and vasodilatation. TBARS, QL, and NO production were significantly increased in PPVL animals. A reduction of SOD activity was found. Glutamine administration markedly alleviated histological abnormalities and oxidative stress, normalized SOD activity, and blocked NO overproduction. Our results confirm that the use of molecules with antioxidant capacity can provide protection of the gastric tissue in portal hypertension. Glutamine treatment can be useful to reduce the oxidative damage induced by PHI on gastric tissue.

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.

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

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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*

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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

A diet containing whey protein, glutamine, and TGFbeta modulates gut protein metabolism during chemotherapy-induced mucositis in rats.

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Boukhettala, Nabile; Ibrahim, Ayman; Claeyssens, Sophie; Faure, Magali; Le Pessot, Florence; Vuichoud, Jacques; Lavoinne, Alain; Breuillé, Denis; Déchelotte, Pierre; Coëffier, Moïse

2010-08-01

Mucositis, a common side effect of chemotherapy, is characterized by compromised digestive function, barrier integrity and immune competence. Our aim was to evaluate the impact of a specifically designed diet Clinutren Protect (CP), which contains whey proteins, TGFbeta-rich casein, and free glutamine, on mucositis in rats. Mucositis was induced by three consecutive injections (day 0, day 1, day 2) of methotrexate (2.5 mg/kg). Rats had free access to CP or placebo diets from days -7 to 9. In the placebo diet, whey proteins and TGFbeta-rich casein were replaced by TGFbeta-free casein and glutamine by alanine. Intestinal parameters were assessed at day 3 and 9. Values, expressed as mean +/- SEM, were compared using two-way ANOVA. At day 3, villus height was markedly decreased in the placebo (296 +/- 11 microm) and CP groups (360 +/- 10 microm) compared with controls (464 +/- 27 microm), but more markedly in the placebo as compared to CP group. The intestinal damage score was also reduced in the CP compared with the placebo group. Glutathione content increased in the CP compared with the placebo group (2.2 +/- 0.2 vs. 1.7 +/- 0.2 micromol/g tissue). Gut protein metabolism was more affected in the placebo than in the CP group. The fractional synthesis rate was decreased in the placebo group (93.8 +/- 4.9%/day) compared with controls (121.5 +/- 12.1, P < 0.05), but not in the CP group (106.0 +/- 13.1). In addition, at day 9, rats exhibited improved body weight and food intake recovery in the CP compared to the placebo group. Clinutren Protect feeding reduces intestinal injury in the acute phase of methotrexate-induced mucositis in rats and improves recovery.

A fluorescence-coupled assay for gamma aminobutyric acid (GABA reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

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Joseph E Ippolito

Full Text Available Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA have been implicated in the pathogenesis of high grade neuroendocrine (NE neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1, was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

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

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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

Protective effect of morin on lipid peroxidation and lipid profile in ammonium chloride-induced hyperammonemic rats

OpenAIRE

S Subash; P Subramanian

2012-01-01

Objective: To evaluated the protective effects of morin (3, 5, 7, 2', 4'-pentahydroxyflavone) on lipid peroxidation and lipid levels during ammonium chloride (AC) induced hyperammonemia in experimental rats. Methods: Thirty two male albino Wistar rats, which are weighing between 180-200 g were used for the study. The hyperammonemia was induced by administration of 100 mg/kg body weight (i.p. ) thrice in a week of AC for 8 weeks. Rats were treated with morin at dose (30 mg/kg bo...

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

Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-α in LPS-stimulated monocytes in human whole-blood.

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Raspé, C; Czeslick, E; Weimann, A; Schinke, C; Leimert, A; Kellner, P; Simm, A; Bucher, M; Sablotzki, A

2013-04-01

To investigate the effects of the commonly-used immunomodulators l-glutamine, l-alanine, and the combination of both l-alanyl-l-glutamine (Dipeptamin(®)) on intracellular expression of IL-6, IL-8, and TNF-α during endotoxemia, lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system were investigated by flow cytometry. Whole blood of twenty-seven healthy volunteers was stimulated with LPS and incubated with three different amino acid solutions (1. l-glutamine, 2. l-alanine, 3. l-alanyl-l-glutamine, each concentration 2 mM, 5 mM, incubation time 3 h). CD14(+) monocytes were phenotyped in whole-blood and intracellular expression of cytokines was assessed by flow cytometry. Our investigations showed for the first time in whole blood probes, imitating best physiologically present cellular interactions, that l-glutamine caused a dose-independent inhibitory effect on IL-6 and TNF-α production in human monocytes stimulated with LPS. However, l-alanine had contrary effects on IL-6 expression, significantly upregulating expression of IL-6 in LPS-treated monocytes. The impact of l-alanine on the expression of TNF-α was comparable with glutamine. Neither amino acid was able to affect IL-8 production in LPS-stimulated monocytes. The combination of both did not influence significantly IL-6 and IL-8 expression in monocytes during endotoxemia, however strongly reduced TNF-α production. For the regulation of TNF-α, l-glutamine, l-alanine and the combination of both show a congruent and exponentiated downregulating effect during endotoxemia, for the modulation of IL-6, l-glutamine and l-alanine featured opposite regulation leading to a canceling impact of each other when recombining both amino acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Science.gov (United States)

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

2014-01-01

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

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

2014-11-25

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.

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

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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.

Effects of erythromycin on γ-glutamyl cysteine synthetase and interleukin-1β in hyperoxia-exposed lung tissue of premature newborn rats.

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Cai, Cheng; Qiu, Gang; Gong, Xiaohui; Chen, Yihuan; Zhao, Huanhu

2014-01-01

To explore the effect of erythromycin on hyperoxia-induced lung injury. One-day-old preterm offspring Sprague-Dawley (SD) rats were randomly divided into four groups: group 1, air + sodium chloride; group 2, air + erythromycin;group 3, hyperoxia + sodium chloride; and group 4, hyperoxia + erythromycin. At one, seven, and 14 days of exposure, glutathione (GSH) and interleukin-1 beta (IL-1 beta) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) was used to detect GSH protein. γ-glutamine-cysteine synthetase (γ-GCS) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Compared with group 1, expressions of GSH and γ-GCS mRNA in group 3 were significantly increased at one and seven days of exposure (p < 0.05), but expression of γ-GCS mRNA was significantly reduced at 14 days; expression of IL-1 beta in group 3 was significantly increased at seven days of exposure (p < 0.05), and was significantly reduced at 14 days. Compared with group 3, expressions of GSH and γ-GCS mRNA in group 4 were significantly increased at one, seven, and 14 days of exposure (p < 0.05), but expressions of GSH showed a downward trend at 14 days; expression of IL-1 beta in group 4 was significantly reduced at one and seven days of exposure (p < 0.05). Changes in oxidant-mediated IL-1 beta and GSH are involved in the development of hyperoxia-induced lung injury. Erythromycin may up-regulate the activity of γ-GCS, increasing the expression of GSH, inhibiting the levels of oxidant-mediated IL-1 beta and alleviating hyperoxia-induced lung injury via an antioxidant effect. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

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

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.

Lymphocyte Glucose and Glutamine Metabolism as Targets of the Anti-Inflammatory and Immunomodulatory Effects of Exercise

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Frederick Wasinski

2014-01-01

Full Text Available Glucose and glutamine are important energetic and biosynthetic nutrients for T and B lymphocytes. These cells consume both nutrients at high rates in a function-dependent manner. In other words, the pathways that control lymphocyte function and survival directly control the glucose and glutamine metabolic pathways. Therefore, lymphocytes in different functional states reprogram their glucose and glutamine metabolism to balance their requirement for ATP and macromolecule production. The tight association between metabolism and function in these cells was suggested to introduce the possibility of several pathologies resulting from the inability of lymphocytes to meet their nutrient demands under a given condition. In fact, disruptions in lymphocyte metabolism and function have been observed in different inflammatory, metabolic, and autoimmune pathologies. Regular physical exercise and physical activity offer protection against several chronic pathologies, and this benefit has been associated with the anti-inflammatory and immunomodulatory effects of exercise/physical activity. Chronic exercise induces changes in lymphocyte functionality and substrate metabolism. In the present review, we discuss whether the beneficial effects of exercise on lymphocyte function in health and disease are associated with modulation of the glucose and glutamine metabolic pathways.

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.

Antioxidant properties of glutamine and its role in VEGF-Akt pathways in portal hypertension gastropathy.

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Marques, Camila; Licks, Francielli; Zattoni, Ingrid; Borges, Beatriz; de Souza, Luiz Eduardo Rizzo; Marroni, Claudio Augusto; Marroni, Norma Possa

2013-07-28

To investigate the effects of glutamine on oxidative/nitrosative stress and the vascular endothelial growth factor (VEGF)-Akt-endothelial nitric oxide synthase (eNOS) signaling pathway in an experimental model of portal hypertension induced by partial portal vein ligation (PPVL). Portal hypertension was induced by PPVL. The PPVL model consists of a partial obstruction of the portal vein, performed using a 20 G blunt needle as a guide, which is gently removed after the procedure. PPVL model was performed for 14 d beginning treatment with glutamine on the seventh day. On the fifteenth day, the mesenteric vein pressure was checked and the stomach was removed to test immunoreactivity and oxidative stress markers. We evaluated the expression and the immunoreactivity of proteins involved in the VEGF-Akt-eNOS pathway by Western blotting and immunohistochemical analysis. Oxidative stress was measured by quantification of the cytosolic concentration of thiobarbituric acid reactive substances (TBARS) as well as the levels of total glutathione (GSH), superoxide dismutase (SOD) activity, nitric oxide (NO) production and nitrotyrosine immunoreactivity. All data are presented as the mean ± SE. The production of TBARS and NO was significantly increased in PPVL animals. A reduction of SOD activity was detected in PPVL + G group. In the immunohistochemical analyses of nitrotyrosine, Akt and eNOS, the PPVL group exhibited significant increases, whereas decreases were observed in the PPVL + G group, but no difference in VEGF was detected between these groups. Western blotting analysis detected increased expression of phosphatidylinositol-3-kinase (PI3K), P-Akt and eNOS in the PPVL group compared with the PPVL + G group, which was not observed for the expression of VEGF when comparing these groups. Glutamine administration markedly alleviated oxidative/nitrosative stress, normalized SOD activity, increased levels of total GSH and blocked NO overproduction as well as the formation of

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.

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.

INDUCED METABOLIC ACIDOSIS BY AMMONIUM CHLORIDE: ACTION MECHANISMS, DOSE AND EFFECTS ON ATHLETIC PERFORMANCE

OpenAIRE

Correia-Oliveira, Carlos Rafaell; Kiss, Maria Augusta Peduti Dal’Molin

2018-01-01

ABSTRACT The relationship between metabolic acidosis and athletic performance has been investigated over the years through manipulation of the blood and muscle pH. Among the pH manipulation manners, the ammonium chloride (NH4Cl) is the most widely used chemical component when is intentioned to induce a blood acidosis status prior to exercise. However, there is a lack of studies investigating the action of this substance on athletic performance as only two studies were performed in the last 15...

Proton- and ammonium- sensing by histaminergic neurons controlling wakefulness.

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Yvgenij eYanovsky

2012-04-01

Full Text Available Orexinergic and histaminergic neurons in the posterior hypothalamus are involved in the control of arousal. Extracellular levels of acid /CO2 are fundamental physicochemical signals controlling wakefulness and breathing. Acidification excites orexinergic neurons like the chemosensory neurons in the brain stem. Hypercapnia induces c-Fos expression, a marker for increased neuronal activity, in the rat histaminergic tuberomamillary nucleus (TMN, but the mechanisms of this excitation are unknown. Acid-sensing ion channels (ASICs are gated by protons and also by ammonium. Recordings in rat brain slices revealed now that acidification within the physiological range (pH from 7.3 to 7.0 as well as ammonium chloride (5mM excite histaminergic neurons. We detected variable combinations of 4 known types of ASICs in single TMN neurons, along with the pharmacological properties of pH-induced current. At pH 7.0 however, activation of ASICs in TMN neurons was negligible. Block of type I metabotropic glutamate receptors abolished proton- but not ammonium- induced excitation. Mouse TMN neurons were identified within a novel HDC-Cre transgenic reporter mouse line. In contrast to the rat these lacked pH 7.0-induced excitation and showed only a minimal response to the mGluR I agonist DHPG (0.5µM. Ammonium-induced excitation was similar in mouse and rat. Thus glutamate, which is released by glial cells and orexinergic axons amplifies CO2/acid-induced arousal through the recruitment of the histaminergic system in rat but not in mouse. These results are relevant for the understanding of neuronal mechanisms controlling H+/CO2-induced arousal in hepatic encephalopathy and obstructive sleep apnoea. The new HDC-Cre mouse model will be a useful tool for studying the physiological and pathophysiological roles of the histaminergic system.

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

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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.

Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation.

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Pallavi Sontakke

Full Text Available The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells.

N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction

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Costa Nicola

2007-12-01

Full Text Available Abstract Background HIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC. Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood. Here we investigated on the effect of N-acetylcysteine (NAC, on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS in the imbalanced activity of glutamine synthase (GS, the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders. Results Incubation of Lipari human cultured astroglial cells with gp 120 (0.1–10 nM produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS. This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA. In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5–5 mM, dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells. Conclusion In conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered

Glufosinate (phosphinothricin), a natural amino acid with unexpected herbicidal properties.

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Hoerlein, G

1994-01-01

Glufosinate ammonium (phosphinothricin ammonium) (GLA) is the active ingredient of Basta and several other herbicides used worldwide. It is produced as part of the tripeptide L-phosphinothricyl-L-alanyl-L-alanin, which was first isolated from Streptomyces viridichromogenes or Streptomyces hygroscopicus. Its structure is confirmed by degradation and synthesis. Several processes for the preparation of D,L- and L-phosphinothricin are described. Glufosinate is a structural analog of glutamate and inhibits the glutamine synthetase. The result is a rapid build-up of a high ammonia level and a concomitant depletion of glutamine and several other amino acids in the plant. These effects are accompanied by a rapid decline of photosynthetic CO2-fixation and are followed by chlorosis and desiccation. The results of numerous toxicological studies show that glufosinate ammonium and its commercial formulations are safe for users and consumers under the conditions of recommended use. The fast and complete degradation in soil and surface water prevents movement of residues into groundwater. The toxicological threshold levels for all the nontarget organisms tested are well above the potential exposure levels and therefore do not reflect any hazard for nontarget organisms in the ecosystem. Basta is a nonselective foliar applied herbicide for the control of undesirable mono- and dicotyledonous plants in orchards, vineyards, and plantations for minimum tillage, and as a harvest aid. A synthetic phosphinothricin acetyltransferase (PAT) gene has been introduced via Agrobacterium tumefaciens into dicot crops, such as like tobacco, tomato, spring and winter rapeseed, alfalfa, and several horticultural crops. The PAT gene was also successfully introduced into maize protoplasts that could be regenerated into fertile plants. All transgenic crop plants tolerated a two- to threefold field dosage of Basta.

Determination of ammonium on an integrated microchip with LED-induced fluorescence detection.

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Xue, Shuhua; Uchiyama, Katsumi; Li, Hai-Fang

2012-01-01

A simply fabricated microfluidic device integrated with a fluorescence detection system has been developed for on-line determination of ammonium in aqueous samples. A 365-nm light-emitting diode (LED) as an excitation source and a minor band pass filter were mounted into a polydimethylsiloxane (PDMS)-based microchip for the purpose of miniaturization of the entire analytical system. The ammonium sample reacted with o-phthaldialdehyde (OPA) on-chip with sodium sulfite as reducing reagent to produce a fluorescent isoindole derivative, which can emit fluorescence signal at about 425 nm when excited at 365 nm. Effects of pH, flow rate of solutions, concentrations of OPA-reagent, phosphate and sulfite salt were investigated. The calibration curve of ammonium in the range of 0.018-1.8 microg/mL showed a good linear relationship with R2 = 0.9985, and the detection limit was (S/N = 3) 3.6 x 10(-4) microg/mL. The relative standard deviation was 2.8% (n = 11) by calculating at 0.18 microg/mL ammonium for repeated detection. The system was applied to determine the ammonium concentration in rain and river waters, even extent to other analytes fluorescence detection by the presented device.

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

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.

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

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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.

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.

Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction, but pathologic hypertrophy is reversed by rapamycin

DEFF Research Database (Denmark)

Paul, David S; Grevengoed, Trisha J; Pascual, Florencia

2014-01-01

In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1(H-/-)), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy...... and B-type natriuretic peptide. mTOR activation of the related Acsl3 gene, usually associated with pathologic hypertrophy, was also attenuated in the Acsl1(H-/-) hearts, indicating that alternative pathways of fatty acid activation did not compensate for the loss of Acsl1. Compared to controls, Acsl1(H......-/-) hearts exhibited an 8-fold higher uptake of 2-deoxy[1-(14)C]glucose and a 35% lower uptake of the fatty acid analog 2-bromo[1-(14)C]palmitate. These data indicate that Acsl1-deficiency causes diastolic dysfunction and that mTOR activation is linked to the development of cardiac hypertrophy in Acsl1(H...

The ctenidium of the giant clam, Tridacna squamosa, expresses an ammonium transporter 1 that displays light-suppressed gene and protein expression and may be involved in ammonia excretion.

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Boo, Mel V; Hiong, Kum C; Goh, Enan J K; Choo, Celine Y L; Wong, Wai P; Chew, Shit F; Ip, Yuen K

2018-04-24

Ammonium transporters (AMTs) can participate in ammonia uptake or excretion across the plasma membrane of prokaryotic, plant and invertebrate cells. The giant clam, Tridacna squamosa, harbors nitrogen-deficient symbiotic zooxanthellae, and normally conducts light-enhanced ammonia absorption to benefit the symbionts. Nonetheless, it can excrete ammonia when there is a supply of exogenous nitrogen or exposed to continuous darkness. This study aimed to elucidate the role of AMT1 in the ctenidium of T. squamosa by cloning and characterizing the AMT1/AMT1, determining its subcellular localization, and examining changes in its transcript and protein expression levels in response to light exposure. The cDNA coding sequence of AMT1 from T. squamosa consisted of 1527 bp and encoded 508 amino acids of 54.6 kDa. AMT1-immunofluorescence was detected mainly at the apical epithelium of ctenidial filaments, and it decreased significantly after 12 h of exposure to light. By contrast, the epithelial cells surrounding the tertiary water channels in the ctentidium, which are known to exhibit light-enhanced glutamine synthetase expression and take part in the assimilation of exogenous ammonia in light, did not display any AMT1-immunolabelling. Furthermore, the transcript level and protein abundance of ctenidial AMT1/AMT1 decreased significantly at the 6th and 12th h of light exposure. Taken together, these results indicate that AMT1 might participate in ammonia excretion instead of ammonia absorption and assimilation in T. squamosa. It is probable that the expression levels of AMT1/AMT1 need to be down-regulated during light exposure to achieve light-enhanced ammonia uptake.

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.

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

Nutrition Modulation of Cardiotoxicity and Anticancer Efficacy Related to Doxorubicin Chemotherapy by Glutamine and ω-3 Polyunsaturated Fatty Acids

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Xue, Hongyu; Ren, Wenhua; Denkinger, Melanie; Schlotzer, Ewald; Wischmeyer, Paul E.

2015-01-01

Background Doxorubicin (DOX) has been one of the most effective antitumor agents against a broad spectrum of malignancies. However, DOX-induced cardiotoxicity forms the major cumulative dose-limiting factor. Glutamine and ω-3 polyunsaturated fatty acids (PUFAs) are putatively cardioprotective during various stresses and/or have potential chemosensitizing effects during cancer chemotherapy. Methods Antitumor activity and cardiotoxicity of DOX treatment were evaluated simultaneously in a MatBIII mammary adenocarcinoma tumor-bearing rat model treated with DOX (cumulative dose 12 mg/kg). Single or combined treatment of parenteral glutamine (0.35 g/kg) and ω-3 PUFAs (0.19 g/kg eicosapentaenoic acid and 0.18 g/kg docosahexaenoic acid) was administered every other day, starting 6 days before chemotherapy initiation until the end of study (day 50). Results Glutamine alone significantly prevented DOX-related deterioration of cardiac function, reduced serum cardiac troponin I levels, and diminished cardiac lipid peroxidation while not affecting tumor inhibition kinetics. Single ω-3 PUFA treatment significantly enhanced antitumor activity of DOX associated with intensified tumoral oxidative stress and enhanced tumoral DOX concentration while not potentiating cardiac dysfunction or increasing cardiac oxidative stress. Intriguingly, providing glutamine and ω-3 PUFAs together did not consistently confer a greater benefit; conversely, individual benefits on cardiotoxicity and chemosensitization were mostly attenuated or completely lost when combined. Conclusions Our data demonstrate an interesting differentiality or even dichotomy in the response of tumor and host to single parenteral glutamine and ω-3 PUFA treatments. The intriguing glutamine × ω-3 PUFA interaction observed draws into question the common assumption that there are additive benefits of combinations of nutrients that are beneficial on an individual basis. PMID:25888676

Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound

Directory of Open Access Journals (Sweden)

Das Pralay

2011-05-01

Full Text Available Abstract Background Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. Results When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide, respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. Conclusions We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed.

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.

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

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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.

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

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

Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase

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Cura, Vincent; Olieric, Natacha; Guichard, Alexandre [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Wang, En-Duo [State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031 (China); Moras, Dino [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Eriani, Gilbert [Architecture et Réactivité de l’ARN, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 Rue René Descartes, 67084 Strasbourg (France); Cavarelli, Jean, E-mail: cava@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

2005-10-01

The wild-type editing CP1 domain of A. aeolicus leucyl-tRNA synthetase and two mutant CP1 domains have been overexpressed, purified and crystallized. X-ray diffraction data have been collected to 1.8 Å, which has enabled determination of the structures by molecular replacement. The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 Å. Crystals diffract to beyond 1.8 Å resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 Å resolution. All structures were solved by molecular-replacement techniques.

Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase

International Nuclear Information System (INIS)

Cura, Vincent; Olieric, Natacha; Guichard, Alexandre; Wang, En-Duo; Moras, Dino; Eriani, Gilbert; Cavarelli, Jean

2005-01-01

The wild-type editing CP1 domain of A. aeolicus leucyl-tRNA synthetase and two mutant CP1 domains have been overexpressed, purified and crystallized. X-ray diffraction data have been collected to 1.8 Å, which has enabled determination of the structures by molecular replacement. The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 Å. Crystals diffract to beyond 1.8 Å resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 Å resolution. All structures were solved by molecular-replacement techniques

PDHA1 gene knockout in prostate cancer cells results in metabolic reprogramming towards greater glutamine dependence

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Li, Yaqing; Li, Xiaoran; Li, Xiaoli; Zhong, Yali; Ji, Yasai; Yu, Dandan; Zhang, Mingzhi; Wen, Jian-Guo; Zhang, Hongquan; Goscinski, Mariusz Adam; Nesland, Jahn M.; Suo, Zhenhe

2016-01-01

Alternative pathways of metabolism endowed cancer cells with metabolic stress. Inhibiting the related compensatory pathways might achieve synergistic anticancer results. This study demonstrated that pyruvate dehydrogenase E1α gene knockout (PDHA1 KO) resulted in alterations in tumor cell metabolism by rendering the cells with increased expression of glutaminase1 (GLS1) and glutamate dehydrogenase1 (GLUD1), leading to an increase in glutamine-dependent cell survival. Deprivation of glutamine induced cell growth inhibition, increased reactive oxygen species and decreased ATP production. Pharmacological blockade of the glutaminolysis pathway resulted in massive tumor cells apoptosis and dysfunction of ROS scavenge in the LNCaP PDHA1 KO cells. Further examination of the key glutaminolysis enzymes in human prostate cancer samples also revealed that higher levels of GLS1 and GLUD1 expression were significantly associated with aggressive clinicopathological features and poor clinical outcome. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon alternative energy metabolism and targeting the glutamine anaplerosis of energy metabolism via GLS1 and GLUD1 in cancer cells may offer a potential novel therapeutic strategy. PMID:27462778

MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

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Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

2017-03-01

Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

Science.gov (United States)

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

2014-07-01

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

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...

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

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

Cell Death-Autophagy Loop and Glutamate-Glutamine Cycle in Amyotrophic Lateral Sclerosis

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Shu Yuan

2017-07-01

Full Text Available Although we know that amyotrophic lateral sclerosis (ALS is correlated with the glutamate-mediated corticomotor neuronal hyperexcitability, detailed ALS pathology remains largely unexplained. While a number of drugs have been developed, no cure exists so far. Here, we propose a hypothesis of neuronal cell death—incomplete autophagy positive-feedback loop—and summarize the role of the neuron-astrocyte glutamate-glutamine cycle in ALS. The disruption of these two cycles might ideally retard ALS progression. Cerebrovascular injuries (such as multiple embolization sessions and strokes induce neuronal cell death and the subsequent autophagy. ALS impairs autophagosome-lysosome fusion and leads to magnified cell death. Trehalose rescues this impaired fusion step, significantly delaying the onset of the disease, although it does not affect the duration of the disease. Therefore, trehalose might be a prophylactic drug for ALS. Given that a major part of neuronal glutamate is converted from glutamine through neuronal glutaminase (GA, GA inhibitors may decrease the neuronal glutamate accumulation, and, therefore, might be therapeutic ALS drugs. Of these, Ebselen is the most promising one with strong antioxidant properties.

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.

Shock wave synthesis of amino acids from solutions of ammonium formate and ammonium bicarbonate

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Suzuki, Chizuka; Furukawa, Yoshihiro; Kobayashi, Takamichi; Sekine, Toshimori; Nakazawa, Hiromoto; Kakegawa, Takeshi

2015-07-01

The emergence of life's building blocks, such as amino acids and nucleobases, on the prebiotic Earth was a critical step for the beginning of life. Reduced species with low mass, such as ammonia, amines, or carboxylic acids, are potential precursors for these building blocks of life. These precursors may have been provided to the prebiotic ocean by carbonaceous chondrites and chemical reactions related to meteorite impacts on the early Earth. The impact of extraterrestrial objects on Earth occurred more frequently during this period than at present. Such impacts generated shock waves in the ocean, which have the potential to progress chemical reactions to form the building blocks of life from reduced species. To simulate shock-induced reactions in the prebiotic ocean, we conducted shock-recovery experiments on ammonium bicarbonate solution and ammonium formate solution at impact velocities ranging from 0.51 to 0.92 km/s. In the products from the ammonium formate solution, several amino acids (glycine, alanine, ß-alanine, and sarcosine) and aliphatic amines (methylamine, ethylamine, propylamine, and butylamine) were detected, although yields were less than 0.1 mol % of the formic acid reactant. From the ammonium bicarbonate solution, smaller amounts of glycine, methylamine, ethylamine, and propylamine were formed. The impact velocities used in this study represent minimum cases because natural meteorite impacts typically have higher velocities and longer durations. Our results therefore suggest that shock waves could have been involved in forming life's building blocks in the ocean of prebiotic Earth, and potentially in aquifers of other planets, satellites, and asteroids.

Biochemical characterization of an in-house Coccidioides antigen: perspectives for the immunodiagnosis of coccidioidomycosis.

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Filho, Renato Evando Moreira; Bandeira, Silviane Praciano; Brillhante, Raimunda Sâmia Nogueira; Rocha, Marcos Fábio Gadelha; Vasconcelos, Ilka Maria; Pereira, Mirella Leite; Castelo-Branco, Débora de Souza Collares Maia; Rocha, Francisco Airton Castro; Camargo, Zoilo Pires de; Ramos, Marcio Viana; Cordeiro, Rossana de Aguiar; Sidrim, José Júlio Costa

2012-06-28

The objective of this study was to evaluate the reactivity of an in-house antigen, extracted from a strain of C. posadasii isolated in northeastern Brazil, by radial immunodiffusion and Western blotting, as well as to establish its biochemical characterization. The protein antigen was initially extracted with the use of solid ammonium sulfate and characterized by 1-D electrophoresis. Subsequently, it was tested by means of double radial immunodiffusion and Western blotting. A positive reaction was observed against the antigen by both immunodiagnostic techniques tested on sera from patients suffering from coccidioidomycosis. Besides this, two immunoreactive protein bands were observed and were revealed to be a β-glucosidase and a glutamine synthetase after sequencing of the respective N-terminal regions. Our in-house Coccidioides antigen can be promising as a quick and low-cost diagnostic tool without the risk of direct manipulation of the microorganism.

Treating ammonium-rich wastewater with sludge from water treatment plant to produce ammonium alum

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Wen-Po Cheng

2016-03-01

Full Text Available This study applies a process to treat ammonium-rich wastewater using alum-generated sludge form water purification plant, and gain economic benefit by producing ammonium alum (Al(NH4(SO42·12H2O. The factors affecting production of ammonium alum include molar ratio of ammonium to aluminum concentration, sulfuric acid concentration, mixing speed, mixing time, standing time, and temperature. According to the equation for the ammonium removal reaction, the theoretical quantity of ammonium alum was calculated based on initial and final concentrations of ammonium. Then, the weight of ammonium alum crystal was divided by the theoretical weight to derive the recovery ratio. The optimum sludge and sulfuric acid dosage to treat about 17 g L−1 ammonium wastewater are 300 g L−1 and 100 mL L−1, respectively. The optimal dosage for wastewater is molar ratio of ammonium to aluminum of about 1 due to the aluminum dissolving in acidified wastewater. The ammonium removal efficiency is roughly 70% and the maximum recovery ratio for ammonium alum is 93% when the wastewater is mixed for 10 min at the mixing velocity gradient of 100 s−1. Ammonium alum production or ammonium removal can be enhanced by controlling the reaction at low temperatures.

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

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

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.

Influence of ammonium nitrate on kinetics and mechanism of thermal decomposition of ammonium polyuranates

International Nuclear Information System (INIS)

Karelin, A.I.; Lobas, O.P.; Zhiganov, A.N.; Vasil'ev, K.F.; Zhiganova, A.A.

1988-01-01

The influence of ammonium nitrate on the mechanism and kinetics of dehydration and thermal decomposition of ammonium polyuranates was studied. An appreciable influence of the nitrate ion content in the samples of ammonium polyuranates on the development of thermal stability has been noted. The kinetic parameters of the thermal decomposition of ammonium polyuranates have been evaluated. A mechanism of the dehydration and thermal decomposition of ammonium polyuranates in the presence of ammonium nitrate has been proposed. It was shown that increase in the content of ammonium nitrate in the precipitate of ammonium polyuranates leads to a decrease in the specific surface of uranoso-uranic oxide

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

Solubility of ammonium metavanadate in ammonium carbonate and sodium bicarbonate solutions at 25 deg C

International Nuclear Information System (INIS)

Fedorov, P.I.; Andreev, V.K.; Slotvinskij-Sidak, N.P.

1978-01-01

Solubility at 25 deg C has been studied in the system ammonium metavanadate - sodium bicarbonate - water which is a stable section of the corresponding quaternary mutual system. In the eutonic point the content of ammonium metavanadate is 4.95% and of sodium bicarbonate 12.1%. The crystallization branch of ammonium metavanadate has been studied in the system ammonium metavanadate - ammonium carbonate - water at 25 deg C. Metavanadate solubility attains minimum (0.14%) at ammonium carbonate concentration 2.6%. Three sections have been studied of the quaternary system ammonium - metavanadate - ammonium carbonate - sodium bicarbonate-water at 25 deg C in the crystallization region of ammonium metavanadate at a ratio of sodium bicarbonate to ammonium carbonate 3:1, 1:1, and 1:3. A region of minimum solubility of ammonium metavanadate has been detected (0.1%)

Properties of the ammonium tartrate/EPR dosimeter

International Nuclear Information System (INIS)

Yordanov, N.D.; Gancheva, V.

2004-01-01

The EPR response of γ-irradiated ammonium tartrate on the absorbed dose of γ-rays up to 22 kGy as well as the changes in the shape of the EPR spectrum upon applied modulation amplitude and microwave power are reported. Also the possibility to use ammonium tartrate together with Mn 2+ magnetically diluted in MgO as an internal reference material is evaluated. The influence of the microwave power and the modulation amplitude on their dose response is investigated. The results show that the radiation-induced EPR spectrum of ammonium tartrate, obtained at a low microwave power is complex consisting several patterns and is more easily saturated than the Mn 2+ EPR spectrum. In this case the following settings of the EPR parameters are recommended: H mod ≤0.05 mT and 10≤P MW ≤13 mW. Using these parameters the dosimeters can be considered for use in intercomparisons

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.

Investigation of ammonium nitrate effect on kinetics and mechanism of thermal decomposition of ammonium polyuranates

International Nuclear Information System (INIS)

Karelin, A.I.; Lobas, O.P.; Zhiganov, A.N.; Vasil'ev, K.F.; Zhiganova, A.A.

1987-01-01

A study was made on ammonium nitrate effect on the mechanism and kinetics of dehydration and thermal decomposition of ammonium polyuranates. Sufficient effect of nitrate ion content in ammonium polyuranate samples on their thermal stability was noted. Kinetic parameters of thermal decomposition of ammonium polyuranates were evaluated. Mechanism of dehydration and thermal decomposition of ammonium polyuranates in the presence of ammonium nitrate was suggested. It was shown that increase of ammonium nitrate content in ammonium polyuranate precipitate resulted to reduction of the specific surface of prepared uranium mixed oxide

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.

Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

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Mounira Tlili

2015-01-01

Full Text Available The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP, we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC and cytokines (IL-1α and TNF-α in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders.

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.

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.

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

Science.gov (United States)

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.

Construction of hybrid peptide synthetases by module and domain fusions.

Science.gov (United States)

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

2000-05-23

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

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.

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.

Ammonium nitrate explosion hazards

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Negovanović Milanka

2015-01-01

Full Text Available Ammonium nitrate (AN primarily is used as a fertilizer but it is also very important compound in the production of industrial explosives. The application of ammonium nitrate in the production of industrial explosives was related with the early era of Nobel dynamite and widely increased with the appearance of blasting agents such as ANFO and Slurry, in the middle of the last Century. Throughout the world millions of tons of ammonium nitrate are produced annually and handled without incident. Although ammonium nitrate generally is used safely, accidental explosions involving AN have high impact resulting in loss of lives and destruction of property. The paper presents the basic properties of ammonium nitrate as well as hazards in handling of ammonium nitrate in order to prevent accidents. Several accidents with explosions of ammonium nitrate resulted in catastrophic consequences are listed in the paper as examples of non-compliance with prescribed procedures.

Mitochondrial Sirt3 supports cell proliferation by regulating glutamine-dependent oxidation in renal cell carcinoma

International Nuclear Information System (INIS)

Choi, Jieun; Koh, Eunjin; Lee, Yu Shin; Lee, Hyun-Woo; Kang, Hyeok Gu; Yoon, Young Eun; Han, Woong Kyu; Choi, Kyung Hwa; Kim, Kyung-Sup

2016-01-01

Clear cell renal carcinoma (RCC), the most common malignancy arising in the adult kidney, exhibits increased aerobic glycolysis and low mitochondrial respiration due to von Hippel-Lindau gene defects and constitutive hypoxia-inducible factor-α expression. Sirt3 is a major mitochondrial deacetylase that mediates various types of energy metabolism. However, the role of Sirt3 as a tumor suppressor or oncogene in cancer depends on cell types. We show increased Sirt3 expression in the mitochondrial fraction of human RCC tissues. Sirt3 depletion by lentiviral short-hairpin RNA, as well as the stable expression of the inactive mutant of Sirt3, inhibited cell proliferation and tumor growth in xenograft nude mice, respectively. Furthermore, mitochondrial pyruvate, which was used for oxidation in RCC, might be derived from glutamine, but not from glucose and cytosolic pyruvate, due to depletion of mitochondrial pyruvate carrier and the relatively high expression of malic enzyme 2. Depletion of Sirt3 suppressed glutamate dehydrogenase activity, leading to impaired mitochondrial oxygen consumption. Our findings suggest that Sirt3 plays a tumor-progressive role in human RCC by regulating glutamine-derived mitochondrial respiration, particularly in cells where mitochondrial usage of cytosolic pyruvate is severely compromised. -- Highlights: •Sirt3 is required for the maintenance of RCC cell proliferation. •Mitochondrial usage of cytosolic pyruvate is severely compromised in RCC. •Sirt3 supports glutamine-dependent oxidation in RCC.

Mitochondrial Sirt3 supports cell proliferation by regulating glutamine-dependent oxidation in renal cell carcinoma

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Choi, Jieun; Koh, Eunjin; Lee, Yu Shin; Lee, Hyun-Woo; Kang, Hyeok Gu [Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Yoon, Young Eun; Han, Woong Kyu [Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Choi, Kyung Hwa [Department of Urology, CHA Bundang Medical Center, CHA University, Seongnam 463-712 (Korea, Republic of); Kim, Kyung-Sup, E-mail: KYUNGSUP59@yuhs.ac [Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

2016-06-03

Clear cell renal carcinoma (RCC), the most common malignancy arising in the adult kidney, exhibits increased aerobic glycolysis and low mitochondrial respiration due to von Hippel-Lindau gene defects and constitutive hypoxia-inducible factor-α expression. Sirt3 is a major mitochondrial deacetylase that mediates various types of energy metabolism. However, the role of Sirt3 as a tumor suppressor or oncogene in cancer depends on cell types. We show increased Sirt3 expression in the mitochondrial fraction of human RCC tissues. Sirt3 depletion by lentiviral short-hairpin RNA, as well as the stable expression of the inactive mutant of Sirt3, inhibited cell proliferation and tumor growth in xenograft nude mice, respectively. Furthermore, mitochondrial pyruvate, which was used for oxidation in RCC, might be derived from glutamine, but not from glucose and cytosolic pyruvate, due to depletion of mitochondrial pyruvate carrier and the relatively high expression of malic enzyme 2. Depletion of Sirt3 suppressed glutamate dehydrogenase activity, leading to impaired mitochondrial oxygen consumption. Our findings suggest that Sirt3 plays a tumor-progressive role in human RCC by regulating glutamine-derived mitochondrial respiration, particularly in cells where mitochondrial usage of cytosolic pyruvate is severely compromised. -- Highlights: •Sirt3 is required for the maintenance of RCC cell proliferation. •Mitochondrial usage of cytosolic pyruvate is severely compromised in RCC. •Sirt3 supports glutamine-dependent oxidation in RCC.

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

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.

Crystallization characteristics of ammonium uranyl carbonate (AUC) in ammonium carbonate solutions

International Nuclear Information System (INIS)

Kim, T.J.; Jeong, K.C.; Park, J.H.; Chang, I.S.; Choi, C.S.

1994-01-01

Ammonium carbonate solutions with an excessive amount of NH 3 were produced in a commercial AUC (ammonium uranyl carbonate) conversion plant. In this study the AUC crystals, precipitated with uranyl nitrate and ammonium carbonate solutions prepared in the laboratory, were characterized to determine the feasibility of recycling ammonium carbonate solution. The AUC crystals were easily agglomerated with the increasing concentration of CO 3 2- and mole ratio of NH 4 + /CO 3 2- in ammonium carbonate solution. Effects of a mixing system for the solution in the AUC crystallizer and the feed location of the solution onthe agglomeration of AUC crystals were also studied along with the effects of agglomerated AUC powders on UO 2 powders. Finally, the feasibility of manufacturing UO 2 fuel with a sintered pellet density of 10.52 g/cm 3 , using the AUC powders generated in this experiment, was demonstrated. (orig.)

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.

Hyperammonemia following glufosinate-containing herbicide poisoning: a potential marker of severe neurotoxicity.

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Mao, Yan-Chiao; Wang, Jiaan-Der; Hung, Dong-Zong; Deng, Jou-Fang; Yang, Chen-Chang

2011-01-01

Glufosinate-ammonium (GLA) is the active ingredient of certain widely used non-selective contact herbicides ("e.g.," Basta). Although it is thought to be much less toxic to humans than to plants, deliberate ingestion of GLA could still lead to serious effects ("e.g.," neurotoxicity) or even death. Three cases presented with delayed-onset neurotoxicity including stupor, delirium, seizures, coma, and amnesia after ingesting large amount of Basta. Considering that GLA could irreversibly inhibit glutamine synthetase (GS) in plants, we performed serial measurements of serum ammonia in those patients and revealed marked hyperammonemia in all of them. All patients recovered with the sequelae of persistent amnesia after receiving intensive care and hemodialysis. We speculated that the occurrence of hyperammonemia might at least be partially related to GS inhibition in humans. Moreover, hyperammonemia could serve as a potential marker of severe neurotoxicity, especially prolonged amnesia, following massive ingestion of GLA-containing herbicides. The possible dose-response relation between GLA exposure and serum ammonia level, however, needs more investigations.

Biochemical Characterization of an In-House Coccidioides Antigen: Perspectives for the Immunodiagnosis of Coccidioidomycosis

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Renato Evando Moreira Filho

2012-06-01

Full Text Available The objective of this study was to evaluate the reactivity of an in-house antigen, extracted from a strain of C. posadasii isolated in northeastern Brazil, by radial immunodiffusion and Western blotting, as well as to establish its biochemical characterization. The protein antigen was initially extracted with the use of solid ammonium sulfate and characterized by 1-D electrophoresis. Subsequently, it was tested by means of double radial immunodiffusion and Western blotting. A positive reaction was observed against the antigen by both immunodiagnostic techniques tested on sera from patients suffering from coccidioidomycosis. Besides this, two immunoreactive protein bands were observed and were revealed to be a β-glucosidase and a glutamine synthetase after sequencing of the respective N-terminal regions. Our in-house Coccidioides antigen can be promising as a quick and low-cost diagnostic tool without the risk of direct manipulation of the microorganism.

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

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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.

Modulation of the nuclear factor-kappa B (NF-κB) signalling pathway by glutamine in peritoneal macrophages of a murine model of protein malnutrition.

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da Silva Lima, Fabiana; Rogero, Marcelo Macedo; Ramos, Mayara Caldas; Borelli, Primavera; Fock, Ricardo Ambrósio

2013-06-01

Protein malnutrition affects resistance to infection by impairing the inflammatory response, modifying the function of effector cells, such as macrophages. Recent studies have revealed that glutamine-a non-essential amino acid, which could become conditionally essential in some situations like trauma, infection, post-surgery and sepsis-is able to modulate the synthesis of cytokines. The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappa B (NF-κB) signalling pathway of peritoneal macrophages from malnourished mice. Two-month-old male Balb/c mice were submitted to protein-energy malnutrition (n = 10) with a low-protein diet containing 2 % protein, whereas control mice (n = 10) were fed a 12 % protein-containing diet. The haemogram and analysis of plasma glutamine and corticosterone were evaluated. Peritoneal macrophages were pre-treated in vitro with glutamine (0, 0.6, 2 and 10 mmol/L) for 24 h and then stimulated with 1.25 μg LPS for 30 min, and the synthesis of TNF-α and IL-1α and the expression of proteins related to the NF-κB pathway were evaluated. Malnourished animals had anaemia, leucopoenia, lower plasma glutamine and increased corticosterone levels. TNF-α production of macrophages stimulated with LPS was significantly lower in cells from malnourished animals when cultivated in supraphysiological (2 and 10 mmol/L) concentrations of glutamine. Further, glutamine has a dose-dependent effect on the activation of macrophages, in both groups, when stimulated with LPS, inducing a decrease in TNF-α and IL-1α production and negatively modulating the NF-κB signalling pathway. These data lead us to infer that the protein malnutrition state interferes with the activation of macrophages and that higher glutamine concentrations, in vitro, have the capacity to act negatively in the NF-κB signalling pathway.

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

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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.

Regulations of enzymes in animals: effects of developmental processes, cancer and radiation. Progress report IX, 1 May 1974--31 April 1975

International Nuclear Information System (INIS)

Knox, W.E.

1975-01-01

Investigations of the properties of variant forms of emnzymes in rat tissues were continued. Two glutamyltransferases, one which remains associated with glutamine synthetase and the other which can be separated from it, were purified. A new assay method forglutaminase activity was established which facilitated further characterization of the 3 isozymes and their concentration in normal and neoplastic tissues. Studies of arginase led to the demonstration of the role that the new variant of arginase plays in proline synthesis in mammary gland. An inhibitor of asparagine synthetase, which is absent from fetal liver and tumors, was discovered in adult rat liver. Peptidyl proline hydroxylase (an essential enzyme in collagen synthesis) was identified as one of the most sensitive indicators of neoplastic growth. The spectrum of experimental, transplantable rat tumors was extended to a series of salivary gland tumors and a radiation-induced lymphoma. (U.S.)

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.

Disruption of the Glutamate–Glutamine Cycle Involving Astrocytes in an Animal Model of Depression for Males and Females

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Rappeneau, Virginie; Blaker, Amanda; Petro, Jeff R.; Yamamoto, Bryan K.; Shimamoto, Akiko

2016-01-01

Background: Women are twice as likely as men to develop major depression. The brain mechanisms underlying this sex disparity are not clear. Disruption of the glutamate–glutamine cycle has been implicated in psychiatric disturbances. This study identifies sex-based impairments in the glutamate–glutamine cycle involving astrocytes using an animal model of depression. Methods: Male and female adult Long-Evans rats were exposed to chronic social defeat stress (CSDS) for 21 days, using a modified resident-intruder paradigm. Territorial aggression was used for males and maternal aggression was used for females to induce depressive-like deficits for intruders. The depressive-like phenotype was assessed with intake for saccharin solution, weight gain, estrous cycle, and corticosterone (CORT). Behaviors displayed by the intruders during daily encounters with residents were characterized. Rats with daily handling were used as controls for each sex. Ten days after the last encounter, both the intruders and controls were subjected to a no-net-flux in vivo microdialysis to assess glutamate accumulation and extracellular glutamine in the nucleus accumbens (NAc). The contralateral hemispheres were used for determining changes in astrocytic markers, including glial fibrillary acidic protein (GFAP) and glutamate transporter-1 (GLT-1). Results: Both male and female intruders reduced saccharin intake over the course of CSDS, compared to their pre-stress period and to their respective controls. Male intruders exhibited submissive/defensive behaviors to territorial aggression by receiving sideways threats and bites. These males showed reductions in striatal GLT-1 and spontaneous glutamine in the NAc, compared to controls. Female intruders exhibited isolated behaviors to maternal aggression, including immobility, rearing, and selfgrooming. Their non-reproductive days were extended. Also, they showed reductions in prefrontal and accumbal GFAP+ cells and prefrontal GLT-1, compared to

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

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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.

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

Reduction of quaternary ammonium-induced ocular surface toxicity by emulsions: an in vivo study in rabbits.

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Liang, H; Brignole-Baudouin, F; Rabinovich-Guilatt, L; Mao, Z; Riancho, L; Faure, M O; Warnet, J M; Lambert, G; Baudouin, C

2008-01-31

To evaluate and compare the toxicological profiles of two quaternary ammonium compounds (QAC), benzalkonium chloride (BAK), and cetalkonium chloride (CKC), in standard solution or cationic emulsion formulations in rabbit eyes using newly developed in vivo and ex vivo experimental approaches. Seventy eyes of 35 adult male New Zealand albino rabbits were used in this study. They were randomly divided into five groups: 50 microl of phosphate-buffered saline (PBS), PBS containing 0.02% BAK or 0.002% CKC (BAK Sol and CKC Sol, respectively), and emulsion containing 0.02% BAK or 0.002% CKC (BAK Em and CKC Em, respectively) were applied to rabbit eyes 15 times at 5-min intervals. The ocular surface changes induced by these eye drops were investigated using slit-lamp examination, flow cytometry (FCM), impression cytology (IC) on conjunctiva, and corneal in vivo confocal microscopy (IVCM). Standard immunohistology in cryosections was also examined for cluster of differentiation (CD) 45+ infiltrating and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL)+ apoptotic cells. Clinical observations and IVCM showed that the highest toxicity was induced by BAK Sol, characterized by damaged corneal epithelium and a high level of inflammatory infiltration. BAK Em and CKC Sol presented moderate effects, and CKC Em showed the lowest toxicity with results similar to those of PBS. Conjunctival imprints analyzed by FCM showed a higher expression of RLA-DR and TNFR1 markers in BAK Sol-instilled eyes than in all other groups, especially at 4 h. Immunohistology was correlated with in vivo and ex vivo findings and confirmed this toxicity profile. A high level of infiltration of CD45+ inflammatory cells and TUNEL+ apoptotic cells was observed in limbus and conjunctiva, especially in QAC solution-receiving eyes compared to QAC emulsion-instilled eyes. The acute administration of 15 instillations at 5 min intervals was a rapid and efficient model to assess quaternary

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...

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

Science.gov (United States)

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

2013-01-01

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

Crystallisation of mixtures of ammonium nitrate, ammonium sulphate and soot

NARCIS (Netherlands)

Dougle, P.G.; Veefkind, J.P.; Brink, H.M. ten

1998-01-01

Crystallisation of laboratory aerosols of ammonium nitrate and of internal mixtures of this salt with ammonium sulphate were investigated using humidity controlled nephelometry. The aerosol was produced via nebulizing of solutions and then dried to 25% RH, which is a realistic minimum value for

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.

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

Activity of the lactate-alanine shuttle is independent of glutamate-glutamine cycle activity in cerebellar neuronal-astrocytic cultures

DEFF Research Database (Denmark)

Bak, Lasse K; Sickmann, Helle M; Schousboe, Arne

2004-01-01

The glutamate-glutamine cycle describes the neuronal release of glutamate into the synaptic cleft, astrocytic uptake, and conversion into glutamine, followed by release for use as a neuronal glutamate precursor. This only explains the fate of the carbon atoms, however, and not that of the ammonia....... Recently, a role for alanine has been proposed in transfer of ammonia between glutamatergic neurons and astrocytes, denoted the lactate-alanine shuttle (Waagepetersen et al. [ 2000] J. Neurochem. 75:471-479). The role of alanine in this context has been studied further using cerebellar neuronal cultures...... and corresponding neuronal-astrocytic cocultures. A superfusion paradigm was used to induce repetitively vesicular glutamate release by N-methyl-D-aspartate (NMDA) in the neurons, allowing the relative activity dependency of the lactate-alanine shuttle to be assessed. [(15)N]Alanine (0.2 mM), [2-(15)N]/[5-(15)N...

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

Parallel Changes in Intracellular Water Volume and pH Induced by NH3/NH4+ Exposure in Single Neuroblastoma Cells

Directory of Open Access Journals (Sweden)

Víctor M. Blanco

2013-12-01

not sufficient to trigger these responses. Inhibition of glutamine synthetase partially blocked the NH4Cl-induced swelling. Conclusions: A quantitative description of the osmotic changes produced by exposure to NH3/NH4+ in single neurons and glial cells shows that ∼35 to 45% of the initial cell swelling can be explained by intracellular accumulation of NH4+ due to rapid permeation and protonation of NH3. Another∼23% of the swelling can be accounted for by rapid glutamine accumulation. The results are discussed in terms of basic cell physiology and their potential relevance to the pathophysiology of hyperammonemic cellular brain edema.

Rat liver responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia

Directory of Open Access Journals (Sweden)

H.M. de Souza

2001-06-01

Full Text Available Hepatic responsiveness to gluconeogenic substrates during insulin-induced hypoglycemia was investigated. For this purpose, livers were perfused with a saturating concentration of 2 mM glycerol, 5 mM L-alanine or 5 mM L-glutamine as gluconeogenic substrates. All experiments were performed 1 h after an ip injection of saline (CN group or 1 IU/kg of insulin (IN group. The IN group showed higher (P<0.05 hepatic glucose production from glycerol, L-alanine and L-glutamine and higher (P<0.05 production of L-lactate, pyruvate and urea from L-alanine and L-glutamine. In addition, ip injection of 100 mg/kg glycerol, L-alanine and L-glutamine promoted glucose recovery. The results indicate that the hepatic capacity to produce glucose from gluconeogenic precursors was increased during insulin-induced hypoglycemia.

49 CFR 176.415 - Permit requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers.

Science.gov (United States)

2010-10-01

... nitrates, and certain ammonium nitrate fertilizers. 176.415 Section 176.415 Transportation Other... requirements for Division 1.5, ammonium nitrates, and certain ammonium nitrate fertilizers. (a) Except as... Captain of the Port (COTP). (1) Ammonium nitrate UN1942, ammonium nitrate fertilizers containing more than...

Association of IDDM and attenuated response of 2',5'-oligoadenylate synthetase to yellow fever vaccine

DEFF Research Database (Denmark)

Bonnevie-Nielsen, V; Larsen, M L; Frifelt, J J

1989-01-01

Basal and yellow fever vaccination-induced 2',5'-oligoadenylate synthetase (2',5'A) activity was determined in blood mononuclear cells (peripheral blood lymphocytes [PBLs]) from insulin-dependent diabetes mellitus (IDDM) and matched control subjects. The live attenuated yellow fever vaccine...... represented a primary stimulus in all subjects. First, basal 2',5'A activity increased severalfold in response to yellow fever vaccination. In IDDM subjects, this increase was significantly lower (P = .025). Second, the 2',5'A activity increased proportionately to the higher basal 2',5'A activity in IDDM...

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

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....

Waterproofing Materials for Ammonium Nitrate

OpenAIRE

R.S. Damse

2004-01-01

This study explores the possibility of overcoming the problem of hygroscopicity of ammonium nitrate by coating the particles with selected waterproofing materials. Gravimetric analysis ofthe samples of ammonium nitrate coated with eight different waterproofing materials, vis-a-vis, uncoated ammonium nitrate, were conducted at different relative humidity and exposuretime. The results indicate that mineral jelly is the promising waterproofing material for ammonium nitrate among the materials te...

N114S mutation causes loss of ATP-induced aggregation of human phosphoribosylpyrophosphate synthetase 1

International Nuclear Information System (INIS)

Liu Honglin; Peng, Xiaohui; Zhao Fang; Zhang Guobin; Tao Ye; Luo Zhaofeng; Li Yang; Teng Maikun; Li Xu; Wei Shiqiang

2009-01-01

This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of α-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO 4 3- allosteric site where PO 4 3- functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

49 CFR 176.410 - Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures.

Science.gov (United States)

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures. 176.410 Section 176.410 Transportation Other Regulations Relating to... nitrate and ammonium nitrate mixtures. (a) This section prescribes requirements to be observed with...

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.

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.

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)

Glutamine Attenuates Acute Lung Injury Caused by Acid Aspiration

Directory of Open Access Journals (Sweden)

Chih-Cheng Lai

2014-08-01

Full Text Available Inadequate ventilator settings may cause overwhelming inflammatory responses associated with ventilator-induced lung injury (VILI in patients with acute respiratory distress syndrome (ARDS. Here, we examined potential benefits of glutamine (GLN on a two-hit model for VILI after acid aspiration-induced lung injury in rats. Rats were intratracheally challenged with hydrochloric acid as a first hit to induce lung inflammation, then randomly received intravenous GLN or lactated Ringer’s solution (vehicle control thirty min before different ventilator strategies. Rats were then randomized to receive mechanical ventilation as a second hit with a high tidal volume (TV of 15 mL/kg and zero positive end-expiratory pressure (PEEP or a low TV of 6 mL/kg with PEEP of 5 cm H2O. We evaluated lung oxygenation, inflammation, mechanics, and histology. After ventilator use for 4 h, high TV resulted in greater lung injury physiologic and biologic indices. Compared with vehicle treated rats, GLN administration attenuated lung injury, with improved oxygenation and static compliance, and decreased respiratory elastance, lung edema, extended lung destruction (lung injury scores and lung histology, neutrophil recruitment in the lung, and cytokine production. Thus, GLN administration improved the physiologic and biologic profiles of this experimental model of VILI based on the two-hit theory.

Electrospray ionization tandem mass spectrometry of ammonium cationized polyethers.

Science.gov (United States)

Nasioudis, Andreas; Heeren, Ron M A; van Doormalen, Irene; de Wijs-Rot, Nicolette; van den Brink, Oscar F

2011-05-01

Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine's degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers. © American Society for Mass Spectrometry, 2011

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...

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

Disruption of the glutamate-glutamine cycle involving astrocytes in an animal model of depression for males and females

Directory of Open Access Journals (Sweden)

Virginie Rappeneau

2016-12-01

Full Text Available Background: Women are twice as likely as men to develop major depression (MD. The brain mechanisms underlying this sex disparity are not clear. Disruption of the glutamate-glutamine cycle has been implicated in psychiatric disturbances. This study identifies sex-based impairments in the glutamate-glutamine cycle involving astrocytes using an animal model of depression. Methods: Male and female adult Long-Evans rats were exposed to chronic social defeat stress (CSDS for 21 days, using a modified resident-intruder paradigm. Territorial aggression was used for males and maternal aggression was used for females to induce depressive-like deficits for intruders. The depressive-like phenotype was assessed with intake for saccharin solution, weight gain, estrous cycle, and corticosterone (CORT. Behaviors displayed by the intruders during daily encounters with residents were characterized. Rats with daily handling were used as controls for each sex. Ten days after the last encounter, both the intruders and controls were subjected to a no-net-flux in vivo microdialysis to assess glutamate accumulation and extracellular glutamine in the nucleus accumbens (NAc. The contralateral hemispheres were used for determining changes in astrocytic markers, including glial fibrillary acidic protein (GFAP and glutamate transporter-1 (GLT-1. Results: Both male and female intruders reduced saccharin intake over the course of CSDS, compared to their pre-stress period and to their respective controls. Male intruders exhibited submissive/defensive behaviors to territorial aggression by receiving sideways threats and bites. These males showed reductions in striatal GLT-1 and spontaneous glutamine in the NAc, compared to controls. Female intruders exhibited isolated behaviors to maternal aggression, including immobility, rearing, and self-grooming. Their non-reproductive days were extended. Also, they showed reductions in prefrontal and accumbal GFAP+ cells and prefrontal GLT

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.

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.

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...

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

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.

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 salinity induced by ammonium sulfate fertilizer on root and shoot growth of highbush blueberry

Science.gov (United States)

Ammonium sulfate fertilizer is commonly used in highbush blueberry (Vaccinium corymbosum L.), but due to a high salt index, it often causes salt damage, particularly in young plants, when too much of the fertilizer is applied. A study was done to determine the sensitivity of blueberry to ammonium su...

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.

Effects of Aromatic Ammoniums on Methyl Ammonium Lead Iodide Hybrid Perovskite Materials

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Jianli Yang

2017-01-01

Full Text Available The introduction of bulky ammoniums into methyl ammonium lead iodide hybrid perovskites (MAPbI3 has emerged as a promising strategy to improve the properties of these materials. In the present work, we studied the effects of several aromatic ammoniums onto the structural, electronic, and optical properties of MAPbI3. Although powder XRD data suggest that the bulky cations are not involved in the bulk phase of the MAPbI3, a surprisingly large effect of the bulky cations onto the photoluminescence properties was observed.

Novel Ammonium Metal Borohydrides

DEFF Research Database (Denmark)

Grinderslev, Jakob; Jepsen, Lars Haahr; Cerny, Radovan

, it cannot store hydrogen reversibly. Recently, the first ammonium metal borohydride, NH4Ca(BH4)3 was published, which may be considered as substitution of K+ by NH4+ in KCa(BH4)3, due to the similar sizes of NH4+ and K+[1]. This compound successfully stabilizes NH4BH4. In the present work, a series of novel...... halide-free ammonium metal borohydrides is presented, which have the chemical compositions (NH4)xM(BH4)n+x. The ammonium metal borohydrides are synthesized by cryomilling of NH4BH4 – M(BH4)n (M = Li, Na, K, Mg, Sr, Y, Mn, La, Gd) in different ratios. A new range of ammonium metal borohydrides is formed......, and the crystal structures and thermal decompositions are investigated. Mixtures of NH4BH4 - NaBH4 do not react, while solid solutions, K1-x(NH4)xBH4, are formed for NH4BH4 - KBH4. For the other composites, novel ammonium metal borohydrides are formed. Several of these structures have been solved from high...

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

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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

A comparative study of low pH stress in E. coli and S. typhimurium, and a comparative study of the inducibility of lysyl-tRNA synthetase in the enterobacteriaceae

International Nuclear Information System (INIS)

Hickey, E.W.

1988-01-01

Lysyl-tRNA synthetase (LRS) in Escherichia coli is coded by two genes, one constitutive, and the other inducible. The commonness of inducibility of this enzyme in prokaryotes was first tested in eight members of the Enterobacteriaceae using culture conditions known to induce it in E. coli. LRS was found to be inducible in Salmonella Typhimurium, Citrobacter freundii, Klebsiella pneumoniae and Enterobacter aerogenes, but not in Serratia marcescens, Proteus mirabilis, Proteus vulgaris or Morganella morganii. The results also indicated that LRS was not induced in E. coli grown in defined medium (SMM) at an external pH (pH 0 ) of 5.0, whereas, it was induced in S. typhimurium under this condition. Further investigation of low pH 0 induced behavior in E. coli and S. typhimurium by quantitation of H 2 35 SO 4 labeled proteins from two dimensional polyacrylamide gels of whole cell sonic extracts showed that at least twenty proteins were induced from 2- to 16-fold in S. typhimurium grown at pH 0 5.0 or shifted from growth at pH 0 7.0 to 5.0. Internal pH (pH i ) changes occurring during steady state growth at low pH 0 , and on shifting from pH 0 7.0 to 5.0, were measured using 14 C-benzoic acid uptake

Interactions of MK-801 with glutamate-, glutamine- and methamphetamine-evoked release of [3H]dopamine from striatal slices

International Nuclear Information System (INIS)

Bowyer, J.F.; Scallet, A.C.; Holson, R.R.; Lipe, G.W.; Slikker, W. Jr.; Ali, S.F.

1991-01-01

The interactions of MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine], glutamate and glutamine with methamphetamine (METH)-evoked release of [ 3 H]dopamine were assessed in vitro to determine whether MK-801 inhibition of METH neurotoxicity might be mediated presynaptically, and to evaluate the effects of glutamatergic stimulation on METH-evoked dopamine release. MK-801 inhibition of glutamate- or METH-evoked dopamine release might reduce synaptic dopamine levels during METH exposure and decrease the formation of 6-hydroxydopamine or other related neurotoxins. Without Mg 2+ present, 40 microM and 1 mM glutamate evoked a N-methyl-D-aspartate receptor-mediated [ 3 H]dopamine and [ 3 H]metabolite (tritium) release of 3 to 6 and 12 to 16% of total tritium stores, respectively, from striatal slices. With 1.50 mM Mg 2+ present, 10 mM glutamate alone or in combination with the dopamine uptake blocker nomifensine released only 2.1 or 4.2%, respectively, of total tritium stores, and release was only partially dependent on N-methyl-D-aspartate-type glutamate receptors. With or without 1.50 mM Mg 2+ present, 0.5 or 5 microM METH evoked a substantial release of tritium (5-8 or 12-21% of total stores, respectively). METH-evoked dopamine release was not affected by 5 microM MK-801 but METH-evoked release was additive with glutamate-evoked release. Without Mg 2+ present, 1 mM glutamine increased glutamate release and induced the release of [ 3 H]dopamine and metabolites. Both 0.5 and 5 microM METH also increased tritium release with 1 mM glutamine present. When striatal slices were exposed to 5 microM METH this glutamine-evoked release of glutamate was increased more than 50%

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

Glutamate/glutamine metabolism coupling between astrocytes and glioma cells: neuroprotection and inhibition of glioma growth.

Science.gov (United States)

Yao, Pei-Sen; Kang, De-Zhi; Lin, Ru-Ying; Ye, Bing; Wang, Wei; Ye, Zu-Cheng

2014-07-18

Glioma glutamate release has been shown to promote the growth of glioma cells and induce neuronal injuries from epilepsy to neuronal death. However, potential counteractions from normal astrocytes against glioma glutamate release have not been fully evaluated. In this study, we investigated the glutamate/glutamine cycling between glioma cells and astrocytes and their impact on neuronal function. Co-cultures of glioma cells with astrocytes (CGA) in direct contact were established under different mix ratio of astrocyte/glioma. Culture medium conditioned in these CGAs were sampled for HPLC measurement, for neuronal ratiometric calcium imaging, and for neuronal survival assay. We found: (1) High levels of glutaminase expression in glioma cells, but not in astrocytes, glutaminase enables glioma cells to release large amount of glutamate in the presence of glutamine. (2) Glutamate levels in CGAs were directly determined by the astrocyte/glioma ratios, indicating a balance between glioma glutamate release and astrocyte glutamate uptake. (3) Culture media from CGAs of higher glioma/astrocyte ratios induced stronger neuronal Ca(2+) response and more severe neuronal death. (4) Co-culturing with astrocytes significantly reduced the growth rate of glioma cells. These results indicate that normal astrocytes in the brain play pivotal roles in glioma growth inhibition and in reducing neuronal injuries from glioma glutamate release. However, as tumor growth, the protective role of astrocytes gradually succumb to glioma cells. Copyright © 2014 Elsevier Inc. All rights reserved.

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...

21 CFR 184.1138 - Ammonium chloride.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ammonium chloride. 184.1138 Section 184.1138 Food... Specific Substances Affirmed as GRAS § 184.1138 Ammonium chloride. (a) Ammonium chloride (NH4Cl, CAS Reg. No. 12125-02-9) is produced by the reaction of sodium chloride and an ammonium salt in solution. The...

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 ...

Peptides derivatized with bicyclic quaternary ammonium ionization tags. Sequencing via tandem mass spectrometry.

Science.gov (United States)

Setner, Bartosz; Rudowska, Magdalena; Klem, Ewelina; Cebrat, Marek; Szewczuk, Zbigniew

2014-10-01

Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1-azabicyclo[2.2.2]octane (ABCO) or 1,4-diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI-MS) and longer retention times on the reverse-phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision-induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a- and b-type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision-induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI-MS/MS sequencing of peptides. Copyright © 2014 John Wiley & Sons, Ltd.

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

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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.

Proteomic and metabolomic responses in hepatopancreas of Mytilus galloprovincialis challenged by Micrococcus luteus and Vibrio anguillarum.

Science.gov (United States)

Wu, Huifeng; Ji, Chenglong; Wei, Lei; Zhao, Jianmin; Lu, Hongjian

2013-12-06

The outbreak of pathogens can induce diseases and lead to massive mortalities of aquaculture animals including fish, mollusk and shrimp. In this work, the responses induced by Micrococcus luteus and Vibrio anguillarum were investigated in hepatopancreas of mussel Mytilus galloprovincialis using proteomics and metabolomics. Metabolic biomarkers demonstrated that M. luteus and V. anguillarum injections could induce osmotic stress and disturbance in energy metabolism. And the uniquely and more markedly altered metabolic biomarkers (glutamine, succinate, aspartate, glucose, ATP, homarine and tyrosine) indicated that V. anguillarum could cause more severe disturbances in osmotic regulation and energy metabolism. The differentially altered proteins meant that M. luteus and V. anguillarum induced different effects in mussels. However, the common proteomic biomarkers, arginine kinase and small heat shock protein, demonstrated that these two bacteria induced similar effects including oxidative stress and disturbance in energy metabolism in M. galloprovincialis. In addition, some metabolic biomarkers, ATP and glutamine, were confirmed by related proteins including arginine kinase, ATP synthase, nucleoside diphosphate kinase and glutamine synthetase in bacteria-challenged mussels. This study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of environmental pathogens to the marine mussel M. galloprovincialis. The outbreak of pathogens can lead to diseases and massive mortalities of aquaculture animals including fish, mollusk and shrimp. The mussel M. galloprovincialis distributes widely along the Bohai coast and is popularly consumed as delicious seafood by local residents. This bivalve has become one of the important species in marine aquaculture industry in China. Therefore a study on pathogen-induced effects is necessary. In the present study, an integrated metabolomic and proteomic approach was used to elucidate the

IMMUNOHISTOCHEMICAL APPROACH REVEALS LOCALIZATION OF CYSTATHIONINE-?-LYASE AND CYSTATHIONINE-ß-SYNTHETASE IN ETHANOL-INDUCED GASTRIC MUCOSA DAMAGE IN MICE

Directory of Open Access Journals (Sweden)

Jand-Venes Rolim MEDEIROS

2013-04-01

Full Text Available Context Hydrogen sulphide (H2S has been proved to be a neuromodulator and contributes to the maintenance of gastric mucosal integrity in damage caused by anti-inflammatory nonsteroidal drugs. Previously, we demonstrated that H2S synthesis is essential to gastric protection against ethanol. Objective To better understanding the role of H2S and the detailed localization of its production in both normal and injured stomach due to ethanol injection, we studied the expression of cystathionine-γ-lyase (CSE and cystathionine-β-synthetase (CBS isoforms in gastric mucosa of mice treated with saline or 50% ethanol. Methods Mice were treated by gavage with saline or 50% ethanol (0.5 mL/25 g. After 1 hour, mice were sacrificed, and gastric tissue was evaluated by histological and immunohistochemical analysis specific for CSE and CBS. Results We have demonstrated a non-specific expression of CBS in the normal gastric mucosa and expression of CSE occurring mainly in the parietal cells of the animals treated with ethanol. Conclusion Thus, we demonstrated that the expression of CBS appears to be constitutive and diffuse across the gastric epithelium, while the expression of CSE appears to be induced in parietal cells by damage agents such as ethanol.

Novel Ammonium Metal Borohydrides

DEFF Research Database (Denmark)

Grinderslev, Jakob; Jepsen, Lars Haahr; Cerny, Radovan

Ammonium borohydride, NH4BH4, has a very high gravimetric (ρm = 24.5 wt% H2) and volumetric (157.3 g·H2/L) hydrogen content and releases 18.4 wt% H2 below 170 °C. However, NH4BH4 is metastable at RT and ambient pressure, with a half-life of ~6 h. The decomposition is strongly exothermic; therefore......, it cannot store hydrogen reversibly. Recently, the first ammonium metal borohydride, NH4Ca(BH4)3 was published, which may be considered as substitution of K+ by NH4+ in KCa(BH4)3, due to the similar sizes of NH4+ and K+[1]. This compound successfully stabilizes NH4BH4. In the present work, a series of novel...... halide-free ammonium metal borohydrides is presented, which have the chemical compositions (NH4)xM(BH4)n+x. The ammonium metal borohydrides are synthesized by cryomilling of NH4BH4 – M(BH4)n (M = Li, Na, K, Mg, Sr, Y, Mn, La, Gd) in different ratios. A new range of ammonium metal borohydrides is formed...

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: A fluorine-19 NMR study

International Nuclear Information System (INIS)

Chu, Wenchy; Horowitz, J.

1991-01-01

Interactions of 5-fluorouracil-substituted Escherichia coli tRNA Val with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene. Apparent K M and V max values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNA Val . Binding of VRS to (FUra)tRNA Val induces structural perturbations that are reflected in selective changes in the 19 F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNA Val along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNA Val , suggesting conformational changes in this part of the molecule. No 19 F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNA Val that has been proposed as a common intermediate in the aminoacylation reaction

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.

Ps laser pulse induced stimulated Raman scattering of ammonium nitrate dissolved in water

Science.gov (United States)

Kumar, V. Rakesh; Kiran, P. Prem

2018-04-01

An intense picosecond laser pulse focused into a liquid medium generates a shock wave in the focal region. This shock wave while propagating into the medium varies the pressure and temperature of the liquid locally leading to the appearance of novel phases which are manifested by the appearance of Raman peaks. We present the phase changes of ammonium nitrate (AN) dissolved in water by studying the forward and backward stimulated Raman Scattering (FSRS and BSRS) signals due to propagation of 30 ps laser pulse induced shockwaves. The dominant peak corresponding to the NO3- symmetric stretching mode is observed with a Raman shift of 1045 cm-1 which represents phase IV of AN with an orthogonal crystalline structure. Apart from this peak, the dominant mode of liquid phase of water with a Raman shift of 3400 cm-1 and an ice VII peak at a Raman shift of 3050 cm-1 confirming the pressure of 10 GPa is observed. The effect of the concentration and input energy on the appearance of the phases will be presented.

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

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...

Simultaneous desulfurization and denitrification by microwave reactor with ammonium bicarbonate and zeolite

International Nuclear Information System (INIS)

Wei Zaishan; Lin Zhehang; Niu Hejingying; He Haiming; Ji Yongfeng

2009-01-01

Microwave reactor with ammonium bicarbonate (NH 4 HCO 3 ) and zeolite was set up to study the simultaneous removal of sulfur dioxide (SO 2 ) and nitrogen oxides (NO x ) from flue gas. The results showed that the microwave reactor filled with NH 4 HCO 3 and zeolite could reduce SO 2 to sulfur with the best desulfurization efficiency of 99.1% and reduce NO x to nitrogen with the best NO x purifying efficiency of 86.5%. Microwave desulfurization and denitrification effect of the experiment using ammonium bicarbonate and zeolite together is much higher than that using ammonium bicarbonate or zeolite only. NO x concentration has little effect on denitrification but has no influence on desulfurization, SO 2 concentration has no effect on denitrification. The optimal microwave power and empty bed residence time (EBRT) on simultaneous desulfurization and dentrification are 211-280 W and 0.315 s, respectively. The mechanism for microwave reduced desulfurization and denitrification can be described as the microwave-induced catalytic reduction reaction between SO 2 , NO x and ammonium bicarbonate with zeolite being the catalyst and microwave absorbent

Reduced blood flow increases the in vivo ammonium ion concentration in the RIF-1 tumor

International Nuclear Information System (INIS)

Constantinidis, Ioannis; Gamcsik, Michael P.

1995-01-01

Purpose: Previous studies from our laboratory have suggested that pooling of ammonium in tumor tissues may be caused by its inefficient removal due to the poor vasculature commonly found in tumors. The purpose of these experiments was to validate the relationship between tumor ammonium ion concentration and tumor blood flow, and to determine whether large concentrations of ammonium ion detected by Nuclear Magnetic Resonance (NMR) spectroscopy are either produced within the tumor or simply imported into the tumor through the blood stream. Methods and Materials: To test this hypothesis, we reduced blood flow in subcutaneously grown Radiation Induced Fibrosarcoma-1 (RIF-1) tumors, either by creating partial ischemia with a bolus injection of hydralazine or by occlusion with surgical sutures. 14 N and 31 P NMR spectroscopy were used to detect the presence of ammonium, and to assess the bioenergetic status of the tumors, respectively. Results: A correlation between ammonium ion concentration and (PCr(P i )) ratio was established for untreated tumors. An increase in the in vivo tumor ammonium ion concentration was observed for every tumor that experienced a reduction in blood flow caused by either hydralazine injection or suture ligation. Changes in ammonium ion concentration paralleled changes in the bioenergetics of hydralazine-treated tumors. Conclusion: Our results support the hypothesis that a reduction in tumor blood flow is responsible for the accumulation of ammonium in tumors, and that detected ammonium originated from within the tumor

Quaternary ammonium compounds – New occupational hazards

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Agnieszka Lipińska-Ojrzanowska

2014-10-01

Full Text Available Quaternary ammonium compounds (QACs, quats belong to organic ionic chemical agents which display unique properties of both surfactants and disinfectants. Their wide distribution in the work environment and also in private households brings about new occupational hazards. This paper reviews reports about the health effects of QACs. QACs could play a role of sensitizers and irritants to the skin and mucous membranes. It is suspected that particular QACs can display an immunologic crossreactivity between each other and with other chemical compounds containing ammonium ion, such as muscle relaxants widely used in anesthesia. They may promote the development of airway allergy, however, the background mechanisms are still unclear and need to be further investigated. Until now, a few cases of occupational asthma induced by QACs have been described and their involvement in contact dermatitis has been documented. The possibility of anaphylaxis due to QACs cannot be excluded as well. Med Pr 2014;65(5:675–682

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

Science.gov (United States)

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

2012-01-01

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

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

Science.gov (United States)

Mulchandani, A; Bassi, A S

1996-01-01

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

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

DEFF Research Database (Denmark)

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

2012-01-01

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

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.

Science.gov (United States)

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

2012-12-01

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

Efeito da suplementação com L-alanil-L-glutamina sobre a resposta de hipersensibilidade do tipo tardio em ratos submetidos ao treinamento intenso Effect of L-glutamine and L-alanyl-L-glutamine supplementation on the response to delayed-type hypersensitivity test (DTH in rats submitted to intense training

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Marcelo Macedo Rogero

2002-12-01

Full Text Available O treinamento intenso e o exercício exaustivo podem ocasionar imunossupressão em atletas por meio da diminuição da concentração plasmática de glutamina. O presente estudo verificou inicialmente o efeito da suplementação com L-glutamina e L-alanilL-glutamina sobre a resposta ao teste de hipersensibilidade do tipo tardio (HTT em ratos submetidos ao treinamento intenso em natação durante seis semanas. Posteriormente, foi avaliado o efeito dessas intervenções nutricionais sobre a contagem total e porcentual de leucócitos e concentração sérica de anticorpos IgG anti-albumina de soro bovino, em animais submetidos ao teste de exaustão e recuperados durante o período de 3 horas. Não houve efeito do treinamento e da suplementação sobre a resposta ao teste de HTT. Animais suplementados apresentaram maior concentração de glutamina no plasma (PIntense training and exhaustive exercise may cause immunesupression in athletes by reducing plasma glutamine concentration. Initially, this study verified the effect of L-glutamine and L-alanyl-L-glutamine supplementation on the response to delayed-type hypersensitivity test (DTH in rats submitted to intense swimming training for six weeks. Later on, we assessed the effect of these nutritional interventions on total and differential white blood cell counts and on concentration of anti-bovine serum albumin IgG antibodies, in animals submitted to exhaustion test and a three-hour recovery period. There was no effect of training and supplementation on the response to DTH. Supplemented animals presented greatest plasma glutamine concentration (p<0.05, though this increase in glutaminemia did not interfere on the serum IgG antibody concentration. The recovery period after intense exercise resulted in decreased glutaminemia as compared with the values obtained immediately after exhaustion test (p<0.05. Increase in corticosterone levels induced by strenuous exercises led to leukocytosis, neutrophilia

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-01-10

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

Ammonium generation during SRAT cycle

International Nuclear Information System (INIS)

Hsu, C.W.

1992-01-01

During the IDMS noble-metal demonstration runs ammonium nitrate deposition was found in the vessel vent system of the feed preparation area. In the bench-scale experiments of studying the hydrogen generation during the sludge treatment cycle, ammonium ion production was also monitored. It was found that: During a simulation of the DWPF Cold Chemical Runs SRAT cycle no detectable amount of ammonium ions was generated when treating a non-noble-metal containing sludge simulant according to the nitric acid flowsheet. Ammonium ions were generated during the SRAT-SME cycle when treating the noble-metal containing sludge with either formic acid or nitric acid/late-washing PHA. This is due to the reaction between formic acid and nitrate catalyzed by the noble metals in the sludge simulant. Ammonium ion production closely followed the hydrogen evolution from the catalytic decomposition of formic acid. This report summarizes the results of the production of ammonia during the SRAT cycle

A comparative study of low pH stress in E. coli and S. typhimurium, and a comparative study of the inducibility of lysyl-tRNA synthetase in the enterobacteriaceae

Energy Technology Data Exchange (ETDEWEB)

Hickey, E.W.

1988-01-01

Lysyl-tRNA synthetase (LRS) in Escherichia coli is coded by two genes, one constitutive, and the other inducible. The commonness of inducibility of this enzyme in prokaryotes was first tested in eight members of the Enterobacteriaceae using culture conditions known to induce it in E. coli. LRS was found to be inducible in Salmonella Typhimurium, Citrobacter freundii, Klebsiella pneumoniae and Enterobacter aerogenes, but not in Serratia marcescens, Proteus mirabilis, Proteus vulgaris or Morganella morganii. The results also indicated that LRS was not induced in E. coli grown in defined medium (SMM) at an external pH (pH{sub 0}) of 5.0, whereas, it was induced in S. typhimurium under this condition. Further investigation of low pH{sub 0} induced behavior in E. coli and S. typhimurium by quantitation of H{sub 2} {sup 35}SO{sub 4} labeled proteins from two dimensional polyacrylamide gels of whole cell sonic extracts showed that at least twenty proteins were induced from 2- to 16-fold in S. typhimurium grown at pH{sub 0} 5.0 or shifted from growth at pH{sub 0} 7.0 to 5.0. Internal pH (pH{sub i}) changes occurring during steady state growth at low pH{sub 0}, and on shifting from pH{sub 0} 7.0 to 5.0, were measured using {sup 14}C-benzoic acid uptake.

Alanyl-glutamine attenuates 5-fluorouracil-induced intestinal mucositis in apolipoprotein E-deficient mice

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C.V. Araújo

2015-06-01

Full Text Available Apolipoprotein E (APOE=gene, apoE=protein is a known factor regulating the inflammatory response that may have regenerative effects during tissue recovery from injury. We investigated whether apoE deficiency reduces the healing effect of alanyl-glutamine (Ala-Gln treatment, a recognized gut-trophic nutrient, during tissue recovery after 5-FU-induced intestinal mucositis. APOE-knockout (APOE-/- and wild-type (APOE+/+ C57BL6J male and female mice (N=86 were given either Ala-Gln (100 mM or phosphate buffered saline (PBS by gavage 3 days before and 5 days after a 5-fluorouracil (5-FU challenge (450 mg/kg, via intraperitoneal injection. Mouse body weight was monitored daily. The 5-FU cytotoxic effect was evaluated by leukometry. Intestinal villus height, villus/crypt ratio, and villin expression were monitored to assess recovery of the intestinal absorptive surface area. Crypt length, mitotic, apoptotic, and necrotic crypt indexes, and quantitative real-time PCR for insulin-like growth factor-1 (IGF-1 and B-cell lymphoma 2 (Bcl-2 intestinal mRNA transcripts were used to evaluate intestinal epithelial cell turnover. 5-FU challenge caused significant weight loss and leukopenia (P<0.001 in both mouse strains, which was not improved by Ala-Gln. Villus blunting, crypt hyperplasia, and reduced villus/crypt ratio (P<0.05 were found in all 5-FU-challenged mice but not in PBS controls. Ala-Gln improved villus/crypt ratio, crypt length and mitotic index in all challenged mice, compared with PBS controls. Ala-Gln improved villus height only in APOE-/- mice. Crypt cell apoptosis and necrotic scores were increased in all mice challenged by 5-FU, compared with untreated controls. Those scores were significantly lower in Ala-Gln-treated APOE+/+ mice than in controls. Bcl-2 and IGF-1 mRNA transcripts were reduced only in the APOE-/--challenged mice. Altogether our findings suggest APOE-independent Ala-Gln regenerative effects after 5-FU challenge.

Alanyl-glutamine attenuates 5-fluorouracil-induced intestinal mucositis in apolipoprotein E-deficient mice

Energy Technology Data Exchange (ETDEWEB)

Araújo, C.V. [Laboratório da Biologia da Cicatrização, Ontogenia e Nutrição de Tecidos, INCT - Instituto de Biomedicina do Semiárido Brasileiro, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Lazzarotto, C.R. [Laboratório de Biologia Molecular e do Desenvolvimento, Universidade de Fortaleza, Fortaleza, CE (Brazil); Aquino, C.C.; Figueiredo, I.L.; Costa, T.B.; Oliveira Alves, L.A. de [Laboratório da Biologia da Cicatrização, Ontogenia e Nutrição de Tecidos, INCT - Instituto de Biomedicina do Semiárido Brasileiro, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Ribeiro, R.A. [Laboratório da Inflamação e Câncer, INCT - Instituto de Biomedicina do Semiárido Brasileiro, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Bertolini, L.R. [Laboratório de Biologia Molecular e do Desenvolvimento, Universidade de Fortaleza, Fortaleza, CE (Brazil); Lima, A.A.M. [Laboratório de Doenças Infecciosas, INCT - Instituto de Biomedicina do Semiárido Brasileiro, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Brito, G.A.C. [Laboratório da Inflamação e Câncer, INCT - Instituto de Biomedicina do Semiárido Brasileiro, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Oriá, R.B. [Laboratório da Biologia da Cicatrização, Ontogenia e Nutrição de Tecidos, INCT - Instituto de Biomedicina do Semiárido Brasileiro, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil)

2015-04-28

Apolipoprotein E (APOE=gene, apoE=protein) is a known factor regulating the inflammatory response that may have regenerative effects during tissue recovery from injury. We investigated whether apoE deficiency reduces the healing effect of alanyl-glutamine (Ala-Gln) treatment, a recognized gut-trophic nutrient, during tissue recovery after 5-FU-induced intestinal mucositis. APOE-knockout (APOE{sup -/-}) and wild-type (APOE{sup +/+}) C57BL6J male and female mice (N=86) were given either Ala-Gln (100 mM) or phosphate buffered saline (PBS) by gavage 3 days before and 5 days after a 5-fluorouracil (5-FU) challenge (450 mg/kg, via intraperitoneal injection). Mouse body weight was monitored daily. The 5-FU cytotoxic effect was evaluated by leukometry. Intestinal villus height, villus/crypt ratio, and villin expression were monitored to assess recovery of the intestinal absorptive surface area. Crypt length, mitotic, apoptotic, and necrotic crypt indexes, and quantitative real-time PCR for insulin-like growth factor-1 (IGF-1) and B-cell lymphoma 2 (Bcl-2) intestinal mRNA transcripts were used to evaluate intestinal epithelial cell turnover. 5-FU challenge caused significant weight loss and leukopenia (P<0.001) in both mouse strains, which was not improved by Ala-Gln. Villus blunting, crypt hyperplasia, and reduced villus/crypt ratio (P<0.05) were found in all 5-FU-challenged mice but not in PBS controls. Ala-Gln improved villus/crypt ratio, crypt length and mitotic index in all challenged mice, compared with PBS controls. Ala-Gln improved villus height only in APOE{sup -/-} mice. Crypt cell apoptosis and necrotic scores were increased in all mice challenged by 5-FU, compared with untreated controls. Those scores were significantly lower in Ala-Gln-treated APOE{sup +/+} mice than in controls. Bcl-2 and IGF-1 mRNA transcripts were reduced only in the APOE{sup -/-}-challenged mice. Altogether our findings suggest APOE-independent Ala-Gln regenerative effects after 5-FU

Alanyl-glutamine attenuates 5-fluorouracil-induced intestinal mucositis in apolipoprotein E-deficient mice

International Nuclear Information System (INIS)

Araújo, C.V.; Lazzarotto, C.R.; Aquino, C.C.; Figueiredo, I.L.; Costa, T.B.; Oliveira Alves, L.A. de; Ribeiro, R.A.; Bertolini, L.R.; Lima, A.A.M.; Brito, G.A.C.; Oriá, R.B.

2015-01-01

Apolipoprotein E (APOE=gene, apoE=protein) is a known factor regulating the inflammatory response that may have regenerative effects during tissue recovery from injury. We investigated whether apoE deficiency reduces the healing effect of alanyl-glutamine (Ala-Gln) treatment, a recognized gut-trophic nutrient, during tissue recovery after 5-FU-induced intestinal mucositis. APOE-knockout (APOE -/- ) and wild-type (APOE +/+ ) C57BL6J male and female mice (N=86) were given either Ala-Gln (100 mM) or phosphate buffered saline (PBS) by gavage 3 days before and 5 days after a 5-fluorouracil (5-FU) challenge (450 mg/kg, via intraperitoneal injection). Mouse body weight was monitored daily. The 5-FU cytotoxic effect was evaluated by leukometry. Intestinal villus height, villus/crypt ratio, and villin expression were monitored to assess recovery of the intestinal absorptive surface area. Crypt length, mitotic, apoptotic, and necrotic crypt indexes, and quantitative real-time PCR for insulin-like growth factor-1 (IGF-1) and B-cell lymphoma 2 (Bcl-2) intestinal mRNA transcripts were used to evaluate intestinal epithelial cell turnover. 5-FU challenge caused significant weight loss and leukopenia (P<0.001) in both mouse strains, which was not improved by Ala-Gln. Villus blunting, crypt hyperplasia, and reduced villus/crypt ratio (P<0.05) were found in all 5-FU-challenged mice but not in PBS controls. Ala-Gln improved villus/crypt ratio, crypt length and mitotic index in all challenged mice, compared with PBS controls. Ala-Gln improved villus height only in APOE -/- mice. Crypt cell apoptosis and necrotic scores were increased in all mice challenged by 5-FU, compared with untreated controls. Those scores were significantly lower in Ala-Gln-treated APOE +/+ mice than in controls. Bcl-2 and IGF-1 mRNA transcripts were reduced only in the APOE -/- -challenged mice. Altogether our findings suggest APOE-independent Ala-Gln regenerative effects after 5-FU challenge

Evidence of ammonium ion-exchange properties of natural bentonite and application to ammonium detection.

Science.gov (United States)

Zazoua, A; Kazane, I; Khedimallah, N; Dernane, C; Errachid, A; Jaffrezic-Renault, N

2013-12-01

Ammonium exchange with hybrid PVC-bentonite (mineral montmorillonite clay) thin film was revealed using FTIR spectroscopy, EDX, cyclic voltammetry and electrochemical impedance spectroscopy. The effect of ammonium exchange on the charge transfer resistance of PVC-bentonite hybrid thin film was attributed to a modification of the intersheet distance and hydration of bentonite crystals. The obtained impedimetric ammonium sensor shows a linear range of detection from 10(-4)M to 1M and a detection limit around 10(-6)M. © 2013.

Enhancement of lysyl-tRNA synthetase activity in the Enterobacteriaceae

International Nuclear Information System (INIS)

Hickey, E.W.; Hirshfield, I.

1987-01-01

Lysyl-tRNA synthetase (LRS) in E. coli is coded by two genes, one constitutive, and the other inducible; the latter is a cell stress protein. To determine if this system is wide spread in prokaryotes, the inducibility of LRS was first tested in eight members of the Enterobacteriaceae using cultural conditions known to induce the enzyme in E. coli K-12. Uninduced control cultures were grown to an O.D. of 0.2 at 580 nm in a supplemented minimal medium (SMM), pH 7.0 at 37 0 C. Induction stimuli include: growth in SMM with 3mM Gly-L-Leu; growth in SMM as above, but with the initial pH adjusted to 5.0; or growth in Difco AC Broth to early stationary phase with a concomitant drop in the pH of the medium below 5.5. LRS activity was assayed in whole-cell sonic extracts by the aminoacylation of crude E. coli tRNA by 14 C-lysine at pH 7.8 for three minutes. When E. aerogenes, K. pneumoniae, C. freundii, and S. typhimurium were grown in AC Broth, LRS activity was enhanced 2 to 4 fold. The enzyme is induced 2 to 4 fold in C. freundii and S. typhimurium upon growth at pH 5.0, whereas E. coli, K.; pneumoniae, and E. aerogenes show only a 1.5 fold induction. The peptide Gly-L-Leu enhanced LRS activity only in E. coli. LRS was not found to be inducible in S. marcescens, M. morganii, P. mirabilis, or P. vulgaris by any of the stimuli

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

Science.gov (United States)

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

2012-09-01

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

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.

Science.gov (United States)

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

2018-05-25

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

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

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.

Optimalisation of magnesium ammonium phosphate precipitation and its applicability to the removal of ammonium.

Science.gov (United States)

Demeestere, K; Smet, E; Van Langenhove, H; Galbacs, Z

2001-12-01

Among the physico-chemical abatement technologies, mainly acid scrubbers have been used to control NH3-emission. The disadvantage of this technique is that it yields waste water, highly concentrated in ammonia. In this report, the applicability of the magnesium ammonium phosphate (MAP) process to regenerate the liquid phase, produced by scrubbing NH3-loaded waste gases, was investigated. In the MAP process, ammonium is precipitated as magnesium ammonium phosphate, which can be used as a slow release fertilizer. The influence of a number of parameters, e.g. pH, kinetics, molar ratio NH(+)4/Mg2+/PO(3-)4 on the efficiency of the formation of MAP and on the ammonium removal efficiency was investigated. In this way, optimal conditions were determined for the precipitation reaction. Next to this, interference caused by other precipitation reactions was studied. At aqueous NH(+)4-concentrations of about 600 mg l(-1), ammonium removal efficiencies of 97% could be obtained at a molar ratio NH(+)4/Mg2+/PO(3-)4 of 1/1.5/1.5. To obtain this result, the pH was continuously adjusted to a value of 9 during the reaction. According to this study, it is obvious that the MAP-precipitation technology offers opportunities for ammonium removal from scrubbing liquids. The practical applicability of the MAP-process in waste gas treatment systems, however, should be the subject for further investigations.

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)

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

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.

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.

Removal of NO {sub x} by microwave reactor with ammonium bicarbonate and Ga-A zeolites at low temperature

Energy Technology Data Exchange (ETDEWEB)

Wei, Z.S. [School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275 (China)]. E-mail: weizaishan98@163.com; Du, Z.Y. [School of Light Industry and Chemical Engineering, Guangdong University of Technology, Guangzhou 510006 (China); Lin, Z.H. [School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275 (China); He, H.M. [School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275 (China); Qiu, R.L. [School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275 (China)

2007-08-15

Microwave reactor with the mixture of ammonium bicarbonate (NH{sub 4}HCO{sub 3}) and Ga-A zeolites was set up to study the removal of nitrogen oxides (NO {sub x} ) from waste gas with excess oxygen concentration (14-19%) at low temperature (80-120 deg. C). The results showed that the microwave reactor filled with NH{sub 4}HCO{sub 3} and Ga-A zeolites could reduce NO {sub x} to nitrogen with the best purifying efficiency of 95.45% and the best denitrification amount of 89.28 mg h{sup -1}. The optimal microwave power and residence time (RT) on denitrification was 259-280 W and 0.259 s, respectively. Microwave denitrification effect of the experiment using ammonium bicarbonate and Ga-A zeolites was much higher than that using ammonium bicarbonate or Ga-A zeolites only. The mechanism for microwave-induced NO {sub x} reduction can be explained as the microwave-induced catalytic reaction between NO {sub x} and ammonium bicarbonate with Ga-A zeolites being the catalyst and microwave absorbent.

Glufosinate ammonium selection of transformed Arabidopsis.

Science.gov (United States)

Weigel, Detlef; Glazebrook, Jane

2006-12-01

INTRODUCTIONOne of the most commonly used markers for the selection of transgenic Arabidopsis is resistance to glufosinate ammonium, an herbicide that is sold under a variety of trade names including Basta and Finale. Resistance to glufosinate ammonium is conferred by the bacterial bialophos resistance gene (BAR) encoding the enzyme phosphinotricin acetyl transferase (PAT). This protocol describes the use of glufosinate ammonium to select transformed Arabidopsis plants. The major advantage of glufosinate ammonium selection is that it can be performed on plants growing in soil and does not require the use of sterile techniques.

Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition

International Nuclear Information System (INIS)

Santana, Steven Michael; Kirby, Brian J; Antonyak, Marc A; Cerione, Richard A

2014-01-01

Extracellular shed vesicles (ESVs) facilitate a unique mode of cell–cell communication wherein vesicle uptake can induce a change in the recipient cell's state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells. (paper)

Different rates of synthesis and degradation of two chloroplastic ammonium-inducible NADP-specific glutamate dehydrogenase isoenzymes during induction and deinduction in Chlorella sorokiniana cells

International Nuclear Information System (INIS)

Bascomb, N.F.; Prunkard, D.E.; Schmidt, R.R.

1987-01-01

The kinetics of accumulation (per milliliter of culture) of the α- and β-subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The β-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the α-subunit holoenzyme(s). After 120 minutes, the α-subunit ceased accumulating and thereafter remained at a constant level. From pulse-chase experiments, using 35 SO 4 and immunochemical procedures, the rate of synthesis of the α-subunit was shown to be greater than the β-subunit during the first 80 minutes of induction. The α- and β-subunits had different rates of degradation during the induction period (t/sub 1/2/ = 50 versus 150 minutes, respectively) and during the deinduction period (t/sub 1/2/ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed

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

Directory of Open Access Journals (Sweden)

Yang T. Du

2018-06-01

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

Nitrogen and carbon source balance determines longevity, independently of fermentative or respiratory metabolism in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Santos, Júlia; Leitão-Correia, Fernanda; Sousa, Maria João; Leão, Cecília

2016-04-26

Dietary regimens have proven to delay aging and age-associated diseases in several eukaryotic model organisms but the input of nutritional balance to longevity regulation is still poorly understood. Here, we present data on the role of single carbon and nitrogen sources and their interplay in yeast longevity. Data demonstrate that ammonium, a rich nitrogen source, decreases chronological life span (CLS) of the prototrophic Saccharomyces cerevisiae strain PYCC 4072 in a concentration-dependent manner and, accordingly, that CLS can be extended through ammonium restriction, even in conditions of initial glucose abundance. We further show that CLS extension depends on initial ammonium and glucose concentrations in the growth medium, as long as other nutrients are not limiting. Glutamine, another rich nitrogen source, induced CLS shortening similarly to ammonium, but this effect was not observed with the poor nitrogen source urea. Ammonium decreased yeast CLS independently of the metabolic process activated during aging, either respiration or fermentation, and induced replication stress inhibiting a proper cell cycle arrest in G0/G1 phase. The present results shade new light on the nutritional equilibrium as a key factor on cell longevity and may contribute for the definition of interventions to promote life span and healthy aging.

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

CHRONIC Cd TOXICITY OF BEAN PLANTS CAN BE PARTIALLY REDUCED BY SUPPLY OF AMMONIUM SULPHATE

Directory of Open Access Journals (Sweden)

Andon VASSILEV

2006-05-01

Full Text Available The effect of ammonium sulphate supply on plant Cd uptake, growth and photosynthesis of bean plants (cv. Limburgse vroege grown in Cd-contaminated artifi cial soil was studied. The experiments were performed at controlled conditions in absence or presence of Cd (0 or 50 mg Cd kg-1 soil and with or without supply of ammonium sulphate [0 or 0.687 g (NH42SO4 kg-1]. Cadmium inhibited both growth and photosynthetic activity of bean plants. The supply of ammonium sulphate had no signifi cant effect on plant Cd uptake and growth inhibition, but to some extend, reduced Cd-induced stress and its negative impact on the photosynthetic performance.

Simultaneous desulfurization and denitrification by microwave reactor with ammonium bicarbonate and zeolite

Energy Technology Data Exchange (ETDEWEB)

Wei Zaishan [School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275 (China)], E-mail: weizaishan98@163.com; Lin Zhehang; Niu Hejingying; He Haiming; Ji Yongfeng [School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275 (China)

2009-03-15

Microwave reactor with ammonium bicarbonate (NH{sub 4}HCO{sub 3}) and zeolite was set up to study the simultaneous removal of sulfur dioxide (SO{sub 2}) and nitrogen oxides (NO{sub x}) from flue gas. The results showed that the microwave reactor filled with NH{sub 4}HCO{sub 3} and zeolite could reduce SO{sub 2} to sulfur with the best desulfurization efficiency of 99.1% and reduce NO{sub x} to nitrogen with the best NO{sub x} purifying efficiency of 86.5%. Microwave desulfurization and denitrification effect of the experiment using ammonium bicarbonate and zeolite together is much higher than that using ammonium bicarbonate or zeolite only. NO{sub x} concentration has little effect on denitrification but has no influence on desulfurization, SO{sub 2} concentration has no effect on denitrification. The optimal microwave power and empty bed residence time (EBRT) on simultaneous desulfurization and dentrification are 211-280 W and 0.315 s, respectively. The mechanism for microwave reduced desulfurization and denitrification can be described as the microwave-induced catalytic reduction reaction between SO{sub 2}, NO{sub x} and ammonium bicarbonate with zeolite being the catalyst and microwave absorbent.

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

Ammonium-induced impairment of axonal growth is prevented through glial creatine.

OpenAIRE

Braissant, O.; Henry, H.; Villard, A.M.; Zurich, M.G.; Loup, M.; Eilers, B.; Parlascino, G.; Matter, E.; Boulat, O.; Honegger, P.; Bachmann, C.

2002-01-01

Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cot...

Urea cycle pathway targeted therapeutic action of naringin against ammonium chloride induced hyperammonemic rats.

Science.gov (United States)

Ramakrishnan, Arumugam; Vijayakumar, Natesan

2017-10-01

Ammonia is a well-known neurotoxin that causes liver disease and urea cycle disorder. Excessive ammonia content in the blood leads to hyperammonemic condition and affects both excitatory and inhibitory neurotransmission including brain edema and coma. Naringin, a plant bioflavonoid present in various citrus fruits and mainly extracted from the grape fruit. This study was designed to assess the protective effect of naringin on ammonium chloride (NH 4 Cl) induced hyperammonemic rats. Experimental hyperammonemia was induced by intraperitoneal injections (i.p) of NH 4 Cl (100mg/kg body weight (b.w.)) thrice a week for 8 consecutive weeks. Hyperammonemic rats were treated with naringin (80mg/kg b.w.) via oral gavage. Naringin administration significantly augmented the level of blood ammonia and plasma urea. Naringin also upregulate the expression of urea cycle enzymes such as carbamoyl phosphate synthase I (CPS I) and ornithine transcarbamylase (OTC), arininosuccinate synthase (ASS), argininosuccinate lyase (ASL) and arginase I (ARG) and metabotropic glutamate receptors (mGluRs) such as mGluRs I and mGluRs V and down regulate the expression of inflammatory markers like tumor necrosis factor (TNF-α), nuclear factor kappa B (NF-kB), Interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS). In addition, to this, the protective effect of naringin was also revealed through the immunohistochemical changes in tissues. Thus our present study result suggest that naringin modulates the expression of proteins involved in urea cycle pathway and suppresses the expression of inflammatory markers and acts as a potential agent to treat condition in rats. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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...

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

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.

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.

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

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

Ammonium photo-production by heterocytous cyanobacteria: potentials and constraints.

Science.gov (United States)

Grizeau, Dominique; Bui, Lan Anh; Dupré, Catherine; Legrand, Jack

2016-08-01

Over the last decades, production of microalgae and cyanobacteria has been developed for several applications, including novel foods, cosmetic ingredients and more recently biofuel. The sustainability of these promising developments can be hindered by some constraints, such as water and nutrient footprints. This review surveys data on N2-fixing cyanobacteria for biomass production and ways to induce and improve the excretion of ammonium within cultures under aerobic conditions. The nitrogenase complex is oxygen sensitive. Nevertheless, nitrogen fixation occurs under oxic conditions due to cyanobacteria-specific characteristics. For instance, in some cyanobacteria, the vegetative cell differentiation in heterocyts provides a well-adapted anaerobic microenvironment for nitrogenase protection. Therefore, cell cultures of oxygenic cyanobacteria have been grown in laboratory and pilot photobioreactors (Dasgupta et al., 2010; Fontes et al., 1987; Moreno et al., 2003; Nayak & Das, 2013). Biomass production under diazotrophic conditions has been shown to be controlled by environmental factors such as light intensity, temperature, aeration rate, and inorganic carbon concentration, also, more specifically, by the concentration of dissolved oxygen in the culture medium. Currently, there is little information regarding the production of extracellular ammonium by heterocytous cyanobacteria. This review compares the available data on maximum ammonium concentrations and analyses the specific rate production in cultures grown as free or immobilized filamentous cyanobacteria. Extracellular production of ammonium could be coupled, as suggested by recent research on non-diazotrophic cyanobacteria, to that of other high value metabolites. There is little information available regarding the possibility for using diazotrophic cyanobacteria as cellular factories may be in regard of the constraints due to nitrogen fixation.

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

Science.gov (United States)

Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

2015-10-01

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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...

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

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...

Producing ammonium chloride from coal or shale

Energy Technology Data Exchange (ETDEWEB)

Christenson, O L

1921-02-25

Process of producing ammonium chloride consists of mixing the substance to be treated with a chloride of an alkali or alkaline earth metal, free silica, water and free hydrochloric acid, heating the mixture until ammonium chloride distills off and collecting the ammonium chloride.

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.

Ammonium nitrate-potassium nitrate system

Energy Technology Data Exchange (ETDEWEB)

Cady, H.H.

1981-01-01

A portion of the binary phase diagram for the system ammonium nitrate-potassium nitrate has been determined from -55/sup 0/C to 185/sup 0/C. Results are presented for the ammonium-nitrate-rich end of the system up to 30 wt% potassium nitrate.

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

Directory of Open Access Journals (Sweden)

Zhang Yang

2015-01-01

Full Text Available This report describes biomass production and compositional changes of developing castor seeds in response to change in the nitrogen resource (glutamine of the medium. During the early developmental period (24-36 days after pollination, oil was found to initially accumulate in the developing seeds. Carbohydrates and oil were inversely related after glutamine provision (35 mM, in the culture medium. [U-14C] sucrose labeling was used to investigate the effect of metabolic fluxes among different storage materials. Addition of glutamine led to a 7% increase of labeling in lipids and an inverse decrease of labeling in carbohydrates. It was postulated that changes in the glutamine concentration in the medium are likely to influence the partitioning of resources between the various storage products, especially carbohydrates and oil. These observations will contribute to a better understanding of assimilate partitioning in developing castor seeds and the development of molecular strategies to improve castor bean seed quality and plant breeding studies.

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....

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

Science.gov (United States)

Marroquin-Guzman, Margarita; Wilson, Richard A

2015-04-01

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

Directory of Open Access Journals (Sweden)

Margarita Marroquin-Guzman

2015-04-01

Full Text Available Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

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.

Effect of Electroacupuncture on the Expression of Glycyl-tRNA Synthetase and Ultrastructure Changes in Atrophied Rat Peroneus Longus Muscle Induced by Sciatic Nerve Injection Injury

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Meng Wang

2016-01-01

Full Text Available Glycyl-tRNA synthetase (GlyRS is one of the key enzymes involved in protein synthesis. Its mutations have been reported to cause Charcot-Marie-Tooth disease which demonstrates muscular atrophy in distal extremities, particularly manifested in peroneus muscles. In this situation, the dysfunctions of mitochondria and sarcoplasmic reticulum (SR affect energy supply and excitation-contraction coupling of muscle fibers, therefore resulting in muscular atrophy. Although the treatment of muscular atrophy is a global urgent problem, it can be improved by electroacupuncture (EA treatment. To investigate the mechanism underlying EA treatment improving muscular atrophy, we focused on the perspective of protein synthesis by establishing a penicillin injection-induced sciatic nerve injury model. In our model, injured rats without treatment showed decreased sciatic functional index (SFI, decreased peroneus longus muscle weight and muscle fiber cross-sectional area, aggregated mitochondria with vacuoles appearing, swollen SR, and downregulated mRNA and protein expression levels of GlyRS and myosin heavy chain IIb (MHC-IIb. The injured rats with EA treatment showed significant recovery. These results indicated that EA stimulation can alleviate peroneus longus muscular atrophy induced by iatrogenic sciatic nerve injury through promoting the recovery of GlyRS and muscle ultrastructure and increasing muscle protein synthesis.

Protective effect of morin on lipid peroxidation and lipid profile in ammonium chloride-induced hyperammonemic rats

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S Subash

2012-04-01

Full Text Available Objective: To evaluated the protective effects of morin (3, 5, 7, 2', 4'-pentahydroxyflavone on lipid peroxidation and lipid levels during ammonium chloride (AC induced hyperammonemia in experimental rats. Methods: Thirty two male albino Wistar rats, which are weighing between 180-200 g were used for the study. The hyperammonemia was induced by administration of 100 mg/kg body weight (i.p. thrice in a week of AC for 8 weeks. Rats were treated with morin at dose (30 mg/kg body weight via intragastric intubations together with AC. At the end of experimental duration, blood ammonia, plasma urea, lipid peroxidation indices [thiobarbituric acid reactive substances, hydroperoxides and lipid levels (cholesterol, triglycerides, free fatty acids and phospholipids] in serum and tissues were analysed to evaluate the antiperoxidative and antilipidemic effects of morin. Results: Ammonia, urea, lipid peroxidative indices and lipid levels were significantly increased in AC administered group. Morin treatment resulted in positive modulation of ammonia, urea, lipid peroxidative indices and lipid levels. Morin administration to normal rats did not exhibit any significant changes in any of the parameters studied. Conclusions: It can be concluded that the beneficial effect of morin on ammonia, urea, lipid peroxidative indices and lipid levels could be due to its antioxidant property.

Combined Effects of Tauroursodeoxycholic Acid and Glutamine on Bacterial Translocation in Obstructive Jaundiced Rats

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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.

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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)

Quantitative analysis of 15N labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives

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The enteral metabolism of glutamine and citrulline are intertwined because, while glutamine is one of the main fuel sources for the enterocyte, citrulline is one of its products. It has been shown that the administration of 15N labeled glutamine results in the incorporation of the 15N label into cit...

Quantitative analysis of 15N-labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives.

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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.

Development of an Improved Mammalian Overexpression Method for Human CD62L

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Brown, Haley A.; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D.

2014-01-01

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402

Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

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Sharma, Arvind; Sharma, Amit

2015-02-01

The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

Seryl-tRNA Synthetases in Translation and Beyond

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Marko Močibob

2016-06-01

Full Text Available For a long time seryl-tRNA synthetases (SerRSs stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS, exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases. These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place

Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases

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