Nitrogen is one of the most important nutrient elements for plants and a major limiting factor in plant growth and crop productivity. Glutamine synthase (GS) is a key enzyme involved in the nitrogen assimilation and recycling in plants. So far, members of the glutamine synthase gene family have been characterized in many plants such as Arabidopsis, rice, wheat, and maize. Reports show that GS are involved in the growth and development of plants, in particular its role in seed production. However, the outcome has generally been inconsistent, which are probably derived from the transcriptional and post-translational regulation of GS genes. In this review, we outlined studies on GS gene classification, QTL mapping, the relationship between GS genes and plant growth with nitrogen and the distribution characters, the biological functions of GS genes, as well as expression control at different regulation levels. In addition, we summarized the application prospects of glutamine synthetase genes in enhancing plant growth and yield by improving the nitrogen use efficiency. The prospects were presented on the improvement of nitrogen utility efficiency in crops and plant nitrogen status diagnosis on the basis of glutamine synthase gene regulation.
Arabidopsis ; Genes, Plant ; Glutamate-Ammonia Ligase ; genetics ; Nitrogen ; metabolism ; Oryza ; Plants ; enzymology ; genetics ; Triticum ; Zea mays

The toxic effect of ammonia on rCHO-GS cell decreased obviously due to the transfection of GS system in serum-free culture. The maximum cell density, 15.6 x 10(5) cells/mL was obtained in the culture with 1.42 mmol/L ammonia. The growth of rCHO-GS cell was inhibited with an increased ammonia concentration. However, a cell density of 8.9 x 10(5) cells/mL was obtained when the concentration of ammonia was 12.65mmol/L. The intracellar metabolic pathways were affected due to the decrease of the toxic effect of ammonia on rCHO-GS cell. With the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L, the yield coefficients of cell to glucose and lactate to glucose decreased. The activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) increased by 43%, 140% and 25%, respectively, indicating that the utilization of glucose increased and the glycolysis pathway was more prone to efficient energy metabolism pathway. An increased activity of glutamate-pyruvate aminotransferase (GPT) showed that the conversation from glutamate to alpha-ketoglutarate was shifted to glutamate-pyruvate transamination pathway. The deamination pathway was inhibited due to a decreased activity of glutamate dehydrogenase. In addition, the number of cells in G0/G1 phase increased and the specific production rate of recombinant protein increased by 2.1-fold with the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L.
Ammonia ; metabolism ; toxicity ; Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Genetic Engineering ; methods ; Glutamate-Ammonia Ligase ; genetics ; metabolism ; Glutamine ; metabolism

Glutamine is an important conditionally necessary amino acid in human body. The effort is to establish a new and high efficient L-glutamine production system instead of traditional fermentaion. In this paper, high efficiency of L-glutamine production is obtained by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system. Glutamine Synthetase gene (glnA) was amplified from Bacillus subtilis genomic DNA with primers designed according to sequences reported in EMBL data bank, then it was inserted into expression vector PET28b, the sequence of glnA was proved to be the same as that reported in the data bank by DNA sequencing. After transformation of this recombinant plasmid PET28b-glnA into BL-21 (DE3) strain, Lactose and IPTG were used to induce GS expression at 37 degrees C separately. Both of them can induce GS expression efficiently. The induced protein is proved to be soluble and occupies about 80% of the total proteins by SDS-PAGE analysis. The soluble GS was purified by Ni2+ chelating sepharose colum. After purification, the purified enzyme was proved active. Results reveal that the optmum temperature of this enzyme is 60 degrees C and optmum pH is 6.5 in biosynthetic reaction by using glutamate, ammonium choloride and ATP as substrates. After induction, the enzyme activity in crude extract of BL-21/PET28b-glnA is 83 times higher than that of original BL-21 extract. Mn2+ can obviously increase the activity and stability of this enzyme. Experiments show that the transformation efficiency of glutamate to glutamine is more than 95%. Because of the high cost from ATP, a system coupling GS with yeast for ATP regenaration was established. In this system, GS utilizes ATP released by yeast fermentation to synthesize L-glutamine. Yeast was treated by 2% toluence to increase its permeability and a yeast named YC001 with high yield of glutamine by coupling with recombinant GS was obtained. The good efficiency was achieved with the presence of 250 mmol/L glucose and 200 mmol/L phosphate, the transformation efficiency of glutamate to glutamine in this system is more than 80%, the average yield of glutamine is about 22g/L. This provides the basis for future large scale production of L-glutamine.
Bacillus subtilis ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; methods ; Glutamate-Ammonia Ligase ; biosynthesis ; genetics ; Glutamic Acid ; metabolism ; Glutamine ; biosynthesis ; genetics ; Yeasts ; genetics ; metabolism

No abstract available.
Adenoma, Liver Cell/metabolism/*pathology/surgery ; Adult ; Glutamate-Ammonia Ligase/metabolism ; Humans ; Immunohistochemistry ; Liver Neoplasms/metabolism/*pathology/surgery ; Male ; Tomography, X-Ray Computed ; beta Catenin/*metabolism

A recombinant strain Escherichia coli DH5alpha(pMD19-glnA) including Bacillus subtilis glnA gene was constructed. Capillary electrophoresis and nuclear magnetic resonance were used to determine qualitatively the product of transformation by recombinant strain, and the relative level of mRNA expression of glnA was also determined by fluorescence quantitative RT-PCR. Subsequently, SDS-PAGE (polyacrylamide gel electrophoresis) was used to analysis the relative level of protein. Surprisingly, there was no increase of glutamine production in this recombinant strain, but an obvious increase in the GABA (gamma-aminobutyric acid ) production. It was showed in the experiment that protein expression of the glutamine synthetase did not increase, although glnA gene can be transcribed normally in this recombined strain. The phenomenon of exogenous glnA gene interfering metabolism of Escherichia coli was worthy of further study.
Bacillus subtilis ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Glutamate-Ammonia Ligase ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Recombination, Genetic ; gamma-Aminobutyric Acid ; biosynthesis

BACKGROUND: Glutamine synthetase (GS) and arginase 1 (Arg1) are widely used pathological markers that discriminate hepatocellular carcinoma (HCC) from intrahepatic cholangiocarcinoma; however, their clinical significance in HCC remains unclear. METHODS: We retrospectively analyzed 431 HCC patients: 251 received hepatectomy alone, and the other 180 received sorafenib as adjuvant treatment after hepatectomy. Expression of GS and Arg1 in tumor specimens was evaluated using immunostaining. mRNA sequencing and immunostaining to detect progenitor markers (cytokeratin 19 [CK19] and epithelial cell adhesion molecule [EpCAM]) and mutant TP53 were also conducted. RESULTS: Up to 72.4% (312/431) of HCC tumors were GS positive (GS+). Of the patients receiving hepatectomy alone, GS negative (GS-) patients had significantly better overall survival (OS) and recurrence-free survival (RFS) than GS+ patients; negative expression of Arg1, which is exclusively expressed in GS- hepatocytes in the healthy liver, had a negative effect on prognosis. Of the patients with a high risk of recurrence who received additional sorafenib treatment, GS- patients tended to have better RFS than GS+ patients, regardless of the expression status of Arg1. GS+ HCC tumors exhibit many features of the established proliferation molecular stratification subtype, including poor differentiation, high alpha-fetoprotein levels, increased progenitor tumor cells, TP53 mutation, and upregulation of multiple tumor-related signaling pathways. CONCLUSIONS GS- HCC patients have a better prognosis and are more likely to benefit from sorafenib treatment after hepatectomy. Immunostaining of GS may provide a simple and applicable approach for HCC molecular stratification to predict prognosis and guide targeted therapy.
Humans ; Carcinoma, Hepatocellular/metabolism* ; Sorafenib/therapeutic use* ; Liver Neoplasms/metabolism* ; Glutamate-Ammonia Ligase/metabolism* ; Hepatectomy ; Retrospective Studies ; Prognosis ; Neoplasm Recurrence, Local/surgery*

It has been postulated that central nervous system disorders characterized by convulsive seizures are caused by alterations in one or more cerebral metabolism especially in cellular energy metabolism, electrolyte metabolism and glutamate metabolism. In the present study, alterations in cerebral energy metabolism, cellular electrolyte metabolism and glutamate metabolism were studied to investigate biochemical nature of cerebral disturbances in rats injected intraperitoneally with massive doses of penicillin. Also carried out were in vitro experiments by which direct effects of penicillin on concentrations of high energy compounds and Na+, K+-ATPase activity in the brains were determined. Intraperitoneal injections of pencillin G sodium(1,000,000 I.U. per kg body weight) to rats twice daily for 3 to 5 days resulted in significant decreases in cerebral concentrations of ATP, phosphocreatine and lactate, suggesting that penicillin induces cerebral dysfunctions by inhibiting energy production. While cerebral Na+, K+-ATPase activity and brain K+ content were significantly decreased in rats with penicllin-induced cerebral dysfunctions, brain Na+ and water contents were significantly increased. Observations that, in rats with penicillin-induced cerebral dysfunctions, the fall in high-energy phosphate contents in the brain took place as rapidly as the cation shifts indicate that alterations in both energy metabolism and electrolyte metabolism in the brain may be responsible for cerebral dysfunctions induced by penicillin. These assumptions were further supported by the findings that Na+, K+-ATPase activity and high-energy phosphate contents in the isolated cerebral hemispheres were profoundly affected by the addition of penicillin to the medium in which in vitro experiment was carried out. While the activity of glutamine synthetase in the brain was increased in rats exhibiting cerebral dysfunctions induced by penicillin, the activity of glutamine dehydrogenase was significantly decreased. Significance of changes in activities of these two brain enzymes in penicillin-induced cerebral dysfunctions was not immediately understood. It may be possible, however, that penicillin may influence glutamate contents in the brain directly orindirectly through the alteration of these two brain enzyme activities so as to modify the cerebral functions.
Adenosine Triphosphate ; Animals ; Brain ; Central Nervous System Diseases ; Cerebrum ; Energy Metabolism ; Glutamate-Ammonia Ligase ; Glutamic Acid ; Glutamine ; Injections, Intraperitoneal ; Lactic Acid ; Metabolism ; Oxidoreductases ; Penicillins* ; Phosphocreatine ; Rats ; Seizures

This paper is aimed to study the effects of nitrogen form on the growth and quality of Chrysanthemums morifolium at the same nitrogen level. In order to provide references for nutrition regulation of Ch. morifolium in field production, pot experiments were carried out in the greenhouse at experimental station of Nanjing Agricultural University. Five proportions of ammonium and nitrate nitrogen were set up and a randomized block design was applied four times repeatedly. The results showed that the growth and quality of Ch. morifolium were significantly influenced by the nitrogen form. The content of chlorophyll and photosynthesis rate were the highest at the NH4(+) -N /NO3(-) -N ratio of 25:75; The activities of NR in different parts of Ch. -morifolium reached the highest at the NH4(+) - N/NO3(-) -N ratio of 0: 100. The contents of nitrate nitrogen in the root and leaves reached the highest at the NH4(+) -N/NO3(-) -N ratio of 50:50. The activities of GS, GOGAT and the content of amylum increased with the ratio of NO3(-) -N decreasing and reached it's maximum at the NH4 + -N/NO3 - -N ratio of 100: 0. The content of ammonium nitrogen were the highest at the NH4 + -N /NO3 --N ratio of 75: 25, while the content of soluble sugar reached the highest at the NH4(+)-N/NO3(-) -N ratio of 25: 75. The content of flavones, chlorogenic acid and 3,5-O-dicoffeoylqunic acid were 57.2 mg x g(-1), 0.673% and 1.838% respectively, reaching the maximum at the NH4(+) -N /NO3(-) -N ratio of 25:75; The content of luteoloside increased with the ratio of NO3(-) -N increasing and reached it's maximum at the NH4(+) -N/NO3(-) -N ratio of 0: 100. The yield of Ch. morifolium reached it's maximum at the NH4(+) -N /NO3(-) -N ratio of 25:75. Nitrogen form has some remarkable influence on the nitrogen metabolism, photosynthesis and growth, Nitrogen form conducive to the growth and quality of Ch. morifolium at the NH4(+) -N /NO3(-) -N ratio of 25: 75.
Ammonium Compounds ; metabolism ; pharmacology ; Chlorophyll ; metabolism ; Chrysanthemum ; drug effects ; growth & development ; metabolism ; Flowers ; drug effects ; growth & development ; metabolism ; Glutamate Synthase ; metabolism ; Glutamate Synthase (NADH) ; metabolism ; Glutamate-Ammonia Ligase ; Nitrates ; metabolism ; pharmacology ; Nitrogen ; metabolism ; pharmacology ; Photosynthesis ; drug effects ; Plant Leaves ; drug effects ; growth & development ; metabolism ; Plant Proteins ; metabolism ; Plant Roots ; drug effects ; growth & development ; metabolism ; Plant Stems ; drug effects ; growth & development ; metabolism

Mammalian cells are prone to apoptosis when cultured in large scale for production of biopharmaceuticals. And this will reduce production duration and result in high cost of production. Apoptosis is triggered by various factors, and delicately regulated by a set of genes. Bcl-2, a component integrated in mitochondria membrane, is an important member of these genes. By maintaining the integrity of mitochondria membrane, Bcl-2 keeps cytochrome C from releasing into cytoplasm, and thus blocks the activation of caspases, and subsequent onset of apoptosis. Over-expression of Bcl-2 has proven to be useful in blocking apoptosis in various cell lines, including CHO, hybridoma, myeloma, lymphoma and insect cells. Ammonia, a metabolite of cultured cells, however, showed apparent pro-apoptosis activity. In living cells, ammonia can be utilized by glutamine synthetase (GS) to synthesize glutamine, and thus lower the concentration of ammonia in medium, and its negative effects. Glutamine is essential to living cells. If not added into medium, glutamine can only be synthesized by GS, which makes GS a qualified selection marker. This marker can be used for gene amplification by adding into medium increased concentration of MSX, an inhibitor of GS. In this study, we over-expressed Bcl-2 using GS amplification in a recombinant CHO cell line stably expressing human interferon-beta. The modified cell line, with higher expression of Bcl-2 and lower production of ammonia, exhibited good anti-apoptosis quality and higher interferon-beta production in continuous culture.
Animals ; Apoptosis ; genetics ; physiology ; Biopharmaceutics ; CHO Cells ; cytology ; metabolism ; Cricetinae ; Cricetulus ; Glutamate-Ammonia Ligase ; genetics ; metabolism ; Interferon-beta ; metabolism ; Models, Genetic ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism

OBJECTIVETo investigate the effect of Huanglian Jiedutang (HLJDT) on hippocampal protein expressions in senescence accelerated mouse-prone/8 (SAMP8).

METHODThe 12-month-old senescence accelerated mice (SAM) were divided into three groups: SAM-resistance/1 (SAMR1), SAM-prone/8 (SAMP8) and SAMP8 treated with HLJDT. The effect of HLJDT on expressions of hippocampal proteins was analyzed by two dimensional electrophoresis (2DE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

RESULTCompared with same age SAMR1, there were 29 differential expressed hippocampal proteins in SAMP8. After treated with HLJDT, the expressions of 38 hippocampal proteins of SAMP8 were changed significantly. 12 reactive proteins of HLJDT were chosen to be identified by MALDI-TOF-MS and the results were searched in MASCOT database. Among 12 reactive proteins, the expressions of 4 hippocampal proteins which expressed differentially between SAMR1 and SAMP8 could be improved by HLJDT.

CONCLUSIONHLJDT may improve the aging of SAMP8 by regulating the expressions of proteins related with energy metabolism, signal transduction, cytoskeletal, amino acid metabolism and so on.

Animals ; Coptis ; chemistry ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Electrophoresis, Gel, Two-Dimensional ; Fructose-Bisphosphate Aldolase ; metabolism ; Glutamate-Ammonia Ligase ; metabolism ; Hippocampus ; drug effects ; metabolism ; Male ; Mice ; Plants, Medicinal ; chemistry ; Proteome ; analysis ; metabolism ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization