Alteration of Striatal Glutamate Release After Glutamine Synthetase Inhibition

The effect of the glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), on glutamate levels in, and glutamate release from, rat striatal tissue was examined. Tissue levels of glutamate were unchanged 24 h after an intraventricular injection of MSO, but tissue glutamine levels were decreased 50%. Calcium-dependent, potassium-stimulated glutamate release was diminished in tissue prisms from animals pretreated with MSO compared to controls. The decreased release of glutamate correlated over time with the inhibition of GS following an intraventricular injection of MSO. The maximum diminution of calcium-dependent, potassium-stimulated glutamate release (50%) and the maximum inhibition of GS activity (51%) were observed 24 h after MSO. The addition of 0.5 mM glutamine to the perfusion medium completely reversed the effects of MSO pretreatment on calcium-dependent, potassium-stimulated glutamate release. Since GS is localized in glial cells and the measured glutamate release is presumed to occur from neurons, the data support the contention that astroglial glutamine synthesis is an important contributor to normal neuronal neurotransmitter release.     

Improvement of Vero cell growth in glutamate-based culture by supplementing ammoniagenic compounds

Ammonia has been identified as one of the most inhibitory substances for mammalian cells. We have attempted to develop a less-ammoniagenic medium for the growth of Vero cells by substitution of glutamine with glutamate. In spite of reduced ammonia formation, Vero cells cultured in glutamate-based medium (DMEM-glu) could not grow normally as in glutamine-based medium (DMEM-gln). After Vero cells adapted to DMEM-glu, alanine was consumed instead of accumulated and both asparagine and glutamine were almost undetectable, indicating the lacking for aminonitrogen. By supplementing NH₄Cl, the growth was significantly improved and the cellular uptake of glutamate from medium was greatly increased. However the growth was still not restored to the level in DMEM-gln, likely due to ammonia toxicity. Asparagine was chosen to support the growth of Vero cells in DMEM-glu, formulating DMEM-glu-asn. This replacement reduced ammonia formation by 79% and increased cell yields by 34% compared with DMEM-gln. After Vero cells adapted to DMEM-glu-asn, glutamine synthetase (GS) activity was elevated by 3.8 folds compared with control in DMEM-gln. In DMEM-glu-asn Vero cell growth was arrested by the specific GS inhibitor, methionine sulphoximine. This arrest affirmed the essential role of GS in glutamine synthesis and disconfirmed the potential role of asparagine synthase (AS) in glutamine formulation (also asparagine utilization).     

Effects of the Convulsant Methionine Sulfoximine on Striatal Dopamine Metabolism

Experiments were conducted to investigate the effects of the convulsant L-methionine-DL-sulfoximine (MSO) on striatal dopamine (DA) metabolism. Intraventricular injections of MSO produced a transient increase in striatal DA release followed by inhibition of DA release for up to 3 days, which paralleled the inhibition by MSO of the enzyme glutamine synthetase (GS). DA synthesis was decreased for up to 24 h after injection of MSO, but returned to normal within 3 days after MSO administration. Intrastriatal injections of MSO produced a pronounced decrease in striatal DA release and inhibition of striatal GS activity 24 h postinjection but, unlike intraventricular MSO, did not produce behavioral convulsions. Glutamate-DA interactions may be responsible for the observed effects.     

Regulation of Neurotransmitter Aspartate Metabolism by Glial Glutamine Synthetase

Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+-dependent, K+-stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium-dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.     

不同浓度的NO^—3和NH^＋4对小麦根谷氨酰胺合成酶及其相关酶的影响

利用酶活性测定和 Northern分子杂交等技术 ,研究了小麦幼苗根在不同浓度的 Na NO3 和(NH4) 2 SO4的供应下 ,其谷氨酰胺合成酶 (GS)、天冬酰胺合成酶 (AS)、谷氨酸脱氢酶 (GDH)、硝酸还原酶 (NR)以及 GS- m RNA的变化。结果表明 :NH 4 处理的小麦 ,其根部 GS活性比 NO-3 处理的高 ;高浓度处理的比低浓度处理的高 ;Northern杂交结果说明 GS- m RNA转录量与 GS活性一致 ;3mmol/ L NO-3促进了 AS的活性。AS酶活性变化与 GS酶活性变化无明显依赖关系。在实验的条件下 ,没能测出 GDH的活性 ,不同浓度的 NO-3 和 NH 4 处理对 NR活性没有明显的规律。     

Influence of Different Concentrations of NO-3 and NH+4 on the Activity of Glutamine Synthetase and Other Relevant Enzymes of Nitrogen Metabolism in Wheat Roots

Influence of different concentrations of NO3 and NH+ on the activity of glutamine synthetase (GS), asparagine synthetase (AS), glutamate dehydrogenase (GDH), nitrate reductase (NR) and the changes of GS-mRNA in wheat roots have been studied with enzymes activity assay and Northern blot. The results showed that the higher GS activity was found in roots of wheat when NH+4-N was the sole nitrogen source than when NO3-N was the sole nitrogen source. GS-mRNA of Northern blot was simillar to GS activity. 3 mmol/L NO3- promoted the activity of AS. The change of AS was independent of the change of GS. GDH activity was not been detected, and change in regulation of NR activity was not found.     

Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation

Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO₃ assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO₃ appears to be limited by the activity of GS. Moreover although in normal cells NH₃ can completely inhibit NO₃ uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO₃ assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS glutamine synthetase - GS_s glutamine synthetase, synthetase activity - GS_t glutamine synthetase, transferase activity - NR nitrate reductase - NIR nitrite reductase - GDH glutamate dehydrogenase - CHX cycloheximide - MSO L-methionine-DL-sulfoximine - FAD flavine adenine dinucleotide     

Glutamine synthetase induction by glucocorticoids in the glucocorticoid-sensitive human leukemic cell line CEM-C7

Treatment of CEM-C7 cells with glucocorticoids produces a 2.5-fold increase in the activity of the enzyme glutamine synthetase (GS). This increase is specific for steroids with glucocorticoid activity adn occurs over a range of steroid concentrations consistent with a receptor-mediated mechanism. Half-maximal and maximal inductions by dexamethasone (dex) occur at 2 X 10(-8) M and 2 X 10(-7) M dex, respectively, concentrations approximately equal to those necessary to produce half and full occupancy of glucocorticoid receptors. GS activity began to increase 1 hour after dex treatment and was complete by 12 hours. This is well before any of the growth inhibitory or cytolytic effects of dex on this cell line occur. This increase was dependent on the presence of glucocorticoid receptors and required both RNA and protein synthesis. Removal of dex following stimulation to maximal levels resulted in a decrease of GS activity to preinduced levels with a half-time of 5 hours. Glutamine deprivation of cells resulted in increased GS activity. However, even in the total absence of glutamine, dex treatment elicited a 2.0-2.5-fold increase in GS activity, ruling out inhibition of glutamine uptake as a mechanism for the dex-induced increase. Experiments with 5'-bromodeoxyuridine (BrdU) demonstrated that GS elevation was sensitive to BrdU substitution of DNA, while dex-induced growth inhibition was not. Therefore GS elevation and growth inhibition in this cell line appear to be independently expressed steroid responses.     

Effects of glutamine and asparagine on recombinant antibody production using CHO‐GS cell lines

A unique and nontraditional approach using glutamine and asparagine supplements for CHO‐glutamine synthetase (GS) cell lines was studied. In our experiments, we found that a decrease in pH and an increase in cell death occurred in production phase of a GS cell line, leading to reduced antibody expression and lower antibody yields. The experimental results and the statistical analysis (ANOVA) indicated that additions of glutamine and asparagine in the basal and feed media were effective to buffer the cell culture pH, reduce lactate generation, maintain a higher cell viability profile, and improve antibody productivity. In bench‐top bioreactors, glutamine and asparagine supplementation helped to prevent cell death, improve antibody yield, and reduce base usage. Glutamine is normally excluded from culture media for GS cell lines to prevent the bypass of selection pressure. In this study, however, the addition of glutamine did not affect cell population homogeneity, protein quality, or decrease antibody yield of two GS cell lines. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1457–1468, 2014     

NITROGEN METABOLISM AND AMINO ACID NUTRITION IN THE SOIL ALGA STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

Nitrate-grown cells of Stichococcus bacillaris Naeg. (UTEX 314) contained much higher activities of glutamine synthetase (GS) and NADPH-glutamate dehydrogenase (GDH) than ammonium-grown cells. Methylamine, a non-metabolizable ammonium analog, caused a decrease in GS activity in nitrate-grown cells suggesting that GS is regulated by the size of the endogenous ammonium pool. The decrease in GS observed in methylammonium-loaded nitrate-grown cells was accompanied by an increase in NADPH-GDH activity. Stichococcus bacillaris can be grown in the presence of methionine sulfoximine (MSX), a potent inhibitor of GS. However, only a fraction of a control cell population showed a requirement for glutamine or arginine for growth following MSX addition. Fully adapted MSX-grown cells were indistinguishable from control cells in their ability to photosynthesize and utilize amino acids as nitrogen sources. Alanine, arginine, asparagine, glutamine, glycine and proline were good nitrogen sources, and maximum capacity for amino acid transport was developed in cells grown on these amino acids. Compared to nitrate-grown cells the activity of GS in ammo acid-grown cells was low, whereas NADPH-GDH was very active. The activity of NADH-GDH in amino acid-grown cells was highest under heterotrophic conditions.     

Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl

The effects of NaCl on changes in ammonium level and enzyme activities of ammonium assimilation in roots growth of rice (Oryza sativa L.) seedlings were investigated. NaCl was effective in inhibiting root growth and stimulated the accumulation of ammonium in roots. Accumulation of ammonium in roots preceded inhibition of root growth caused by NaCl. Both effects caused by NaCl are reversible. Exogenous ammonium chloride and methionine sulfoximine (MSO), which caused ammonium accumulation in roots, inhibited root growth of rice seedlings. NaCl decreased glutamine synthetase and glutamate synthase activities in roots, but increased glutamate dehydrogenase activity. The growth inhibition of roots by NaCl or MSO could be reversed by the addition of L-glutamic acid or L-glutamine. The current results suggest that disturbance of ammonium assimilation in roots may be involved in regulating root growth reduction caused by NaCl.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSO methionine sulfoximine     

Up-regulation and localization of asparagine synthetase in tomato leaves infected by the bacterial pathogen Pseudomonas syringae

Nitrogen metabolism is one aspect of basic metabolism, which is still quite unknown in the field of plant-pathogen interactions. Evidence derived from previous studies conducted in our laboratory strongly suggests that during microbial pathogenesis an important nitrogen mobilization process takes place in diseased tissues. Here we describe the expression pattern of asparagine synthetase (AS; EC 6.3.5.4) in tomato leaves infected by the bacterial pathogen Pseudomonas syringae pv. tomato. Using an homologous AS cDNA probe isolated by RT-PCR from infected leaves, we have observed a high level induction of AS expression during the course of infection. Concomitantly, a single AS polypeptide also accumulated in response to bacterial infection. Furthermore, immunohistochemical analysis of AS in infected leaves revealed a strong immunostaining in phloem cells of the main vascular bundles and in secondary veins of the leaf blade. These data correlate with those previously reported for expression of a cytosolic isoform of glutamine synthetase (GS1) also induced during development of the infectious process. Taken together, our results suggest the existence of a GS1/AS pathway representing a metabolic route for transferring ammonium released from protein catabolism into asparagine, an amino acid that may have a major role in nitrogen mobilization from diseased tissues.     

Glutamine-stimulated modification and degradation of glutamine synthetase in hepatoma tissue culture cells

Effects of glutamine on glutamine synthetase (GS) activity of hepatoma tissue culture (HTC) cells were studied with the aid of a specific goat anti-rat GS serum. Immunodiffusion and immunoelectrophoretic tests show that rat liver GS and HTC cell GS are immunologically similar but not identical. Immunotitrations of HTC cell extracts demonstrate that in cells incubated in high concentrations (5 mM) of glutamine, a cross-reacting form of GS with a decreased enzyme-specific activity accumulates. On prolonged incubation of cells in high glutamine, there is net degradation of GS to form immunologically inactive products. Radioimmunoprecipitation experiments show that glutamine acts by accelerating the degradation of preformed GS.     

Expression and Functional Characterization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Recombinant <Emphasis Type="SmallCaps">l</Emphasis>.Asparaginase

Recombinant _l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS–PAGE. The activity of the purified L.ASNase was enhanced by Mg²⁺ and inhibited by Zn²⁺ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as _L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.     

光呼吸和谷氨酰胺合成酶抑制剂对水稻冠层NH3挥发的影响

在营养液培养条件下,对两个不同氮效率基因型水稻品种扬稻6号和武育粳3号采用光呼吸抑制剂异烟肼（INH）和谷氨酰胺合成酶（GS）抑制剂蛋氨酸亚砜亚胺（MSO）处理,研究其对水稻光合速率、光呼吸速率、GS酶活性及冠层的NH。挥发速率的影响。结果发现：（1）MSO导致剑叶光合速率下降,光呼吸速率升高;INH导致光呼吸速率显著下降,同时一定程度上引起光合速率降低。（2）MSO处理显著降低了GS酶活性,相应地引起NH。挥发速率增加;INH在一定程度上导致NH。挥发速率降低。（3）扬稻6号NH。挥发速率比武育粳3号低的生理原因是光呼吸速率较低和GS酶活性较高。     

Effects of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica.

The addition of exogenous L-methionine-DL-sulphoximine (MSO) to N₂-fixing cultures of the blue-green alga Anabaena cylindrica results in over half of the newly fixed NH₃ being released into the medium. MSO also inhibits glutamine synthetase (GS) activity, has negligible effect on alanine dehydrogenase activity, and glutamate dehydrogenase activity under N₂-fixing conditions is negligible. In the presence of MSO, intracellular pools of glutamate and glutamine decrease, those of aspartate and alanine + glycine show little change, and the NH₃ pool increases. MSO alleviates the inhibitory effect of exogenous NH₄⁺ on nitrogenase synthesis and heterocyst production. The results suggest that in N₂-fixing cultures of A. cylindrica the primary NH₃ assimilating pathway involves GS, and probably glutamate synthase (GOGAT), and that the repressor of nitrogenase synthesis and heterocyst production is not NH₄⁺ but is GS, GOGAT, or a product of their reactions.     

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

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(2)O(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.     

Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco

Here, we monitor the effects of ectopic overexpression of genes for pea asparagine synthetase (AS1) in transgenic tobacco (Nicotiana tabacum). The AS genes of pea and tobacco are normally expressed only during the dark phase of the diurnal growth cycle and specifically in phloem cells. A hybrid gene was constructed in which a pea AS1 cDNA was fused to the cauliflower mosaic virus 35S promoter. The 35S-AS1 gene was therefore ectopically expressed in all cell types in transgenic tobacco and constitutively expressed at high levels in both the light and the dark. Northern analysis demonstrated that the 35S-AS1 transgene was constitutively expressed at high levels in leaves of several independent transformants. Furthermore, amino acid analysis revealed a 10- to 100-fold increase in free asparagine in leaves of transgenic 35S-AS1 plants (construct z127) compared with controls. Plant growth analyses showed increases (although statistically insignificant) in growth phenotype during the vegetative stage of growth in 35S-AS1 transgenic lines. The 35S-AS1 construct was further modified by deletion of the glutamine-binding domain of the enzyme (gln[delta]AS1; construct z167). By analogy to animal AS, we reasoned that inhibition of glutamine-dependent AS activity might enhance the ammonia-dependent AS activity. The 3- to 19-fold increase in asparagine levels in the transgenic plants expressing gln[delta]AS1 compared with wild type suggests that the novel AS holoenzyme present in the transgenic plants (gln[delta]AS1 homodimer) has enhanced ammonia-dependent activity. These data indicate that manipulation of AS expression in transgenic plants causes an increase in nitrogen assimilation into asparagine, which in turn produces effects on plant growth and asparagine biosynthesis.