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1.
Chinese Medical Journal ; (24): 2066-2076, 2023.
Article in English | WPRIM | ID: wpr-1007579

ABSTRACT

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.


Subject(s)
Humans , Carcinoma, Hepatocellular/metabolism , Sorafenib/therapeutic use , Liver Neoplasms/metabolism , Glutamate-Ammonia Ligase/metabolism , Hepatectomy , Retrospective Studies , Prognosis , Neoplasm Recurrence, Local/surgery
2.
Braz. j. biol ; 82: e235927, 2022. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1249226

ABSTRACT

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.


A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.


Subject(s)
Bacterial Proteins/genetics , Azospirillum brasilense/enzymology , Azospirillum brasilense/genetics , Ammonium Compounds , Glutamate-Ammonia Ligase/genetics , Escherichia coli/genetics
3.
Braz. j. biol ; 82: 1-8, 2022. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1468474

ABSTRACT

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.


A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.


Subject(s)
Azospirillum brasilense/enzymology , Azospirillum brasilense/genetics , Escherichia coli , Nitrogen Fixation , Glutamate-Ammonia Ligase/biosynthesis
4.
International Journal of Oral Biology ; : 129-135, 2017.
Article in Korean | WPRIM | ID: wpr-201471

ABSTRACT

The present study investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain or trigeminal neuropathic pain. Experiments were carried out on male Sprague-Dawley rats weighing between 230 and 280 g. Under anesthesia, a polyethylene tube was implanted in the atlanto-occipital membrane for intracisternal administration. IL-1β-induced inflammation was employed as an orofacial acute inflammatory pain model. IL-1β (10 ng) was injected subcutaneously into one vibrissal pad. We used the trigeminal neuropathic pain animal model produced by chronic constriction injury of the infraorbital nerve. DL-threo-β -benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block the spinal glutamate transporter and the glutamine synthetase activity in astroglia. Intracisternal administration of TBOA produced mechanical allodynia in naïve rats, but it significantly attenuated mechanical allodynia in rats with interleukin (IL)-1 β-induced inflammatory pain or trigeminal neuropathic pain. In contrast, intracisternal injection of MSO produced anti-allodynic effects in rats treated with IL-1β or with infraorbital nerve injury. Intracisternal administration of MSO did not produce mechanical allodynia in naive rats. These results suggest that blockade of glutamate recycling induced pro-nociception in naïve rats, but it paradoxically resulted in anti-nociception in rats experiencing inflammatory or neuropathic pain. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic pain conditions.


Subject(s)
Animals , Humans , Male , Rats , Amino Acid Transport System X-AG , Anesthesia , Astrocytes , Chronic Pain , Constriction , Glutamate-Ammonia Ligase , Glutamic Acid , Hyperalgesia , Inflammation , Interleukins , Membranes , Methionine Sulfoximine , Models, Animal , Neuralgia , Polyethylene , Rats, Sprague-Dawley , Recycling
5.
Experimental & Molecular Medicine ; : e280-2016.
Article in English | WPRIM | ID: wpr-149851

ABSTRACT

Diabetic retinopathy (DR), one of the most serious complications of diabetes, has been associated with inflammatory processes. We have recently reported that interleukin (IL)-17A, a proinflammatory cytokine, is increased in the plasma of diabetic patients. Further investigation is required to clarify the role of IL-17A in DR. Ins2(Akita) (Akita) diabetic mice and high-glucose (HG)-treated primary Müller cells were used to mimic DR-like pathology. Diabetes induced retinal expression of IL-17A and IL-17 receptor A (IL-17RA) in Müller cells in contrast to ganglion cells. Further evidence demonstrated that retinal Müller cells cultured in vitro increased IL-17A and IL-17RA expression as well as IL-17A secretion in the HG condition. In both the HG-treated Müller cells and Akita mouse retina, the Act1/TRAF6/IKK/NF-κB signaling pathway was activated. IL-17A further enhanced inflammatory signaling activation, whereas Act1 knockdown or IKK inhibition blocked the downstream signaling activation by IL-17A. HG- and diabetes-induced Müller cell activation and dysfunction, as determined by increased glial fibrillary acidic protein, vascular endothelial growth factor and glutamate levels and decreased glutamine synthetase and excitatory amino acid transporter-1 expression, were exacerbated by IL-17A; however, they were alleviated by Act1 knockdown or IKK inhibition. In addition, IL-17A intravitreal injection aggravated diabetes-induced retinal vascular leukostasis, vascular leakage and ganglion cell apoptosis, whereas Act1 silencing or anti-IL-17A monoclonal antibody ameliorated the retinal vascular damage and neuronal cell apoptosis. These findings establish that IL-17A exacerbates DR-like pathology by the promotion of Müller cell functional impairment via Act1 signaling.


Subject(s)
Animals , Humans , Mice , Apoptosis , Diabetic Retinopathy , Excitatory Amino Acids , Ganglion Cysts , Glial Fibrillary Acidic Protein , Glutamate-Ammonia Ligase , Glutamic Acid , In Vitro Techniques , Interleukin-17 , Interleukins , Intravitreal Injections , Leukostasis , Neurons , Pathology , Plasma , Retina , Retinaldehyde , Vascular Endothelial Growth Factor A
6.
Chinese Journal of Biotechnology ; (12): 1301-1312, 2015.
Article in Chinese | WPRIM | ID: wpr-337490

ABSTRACT

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.


Subject(s)
Arabidopsis , Genes, Plant , Glutamate-Ammonia Ligase , Genetics , Nitrogen , Metabolism , Oryza , Plants , Genetics , Triticum , Zea mays
7.
Indian J Exp Biol ; 2014 Apr; 52(4): 352-358
Article in English | IMSEAR | ID: sea-150366

ABSTRACT

Effect of salinity (0, 50, 100, 250, 500 and 750 mM NaCl) was observed on some important physiological parameters of nitrogen metabolism such as nitrate uptake, intracellular and extracellular ammonium status and activities of nitrogenase, nitrate reductase, nitrite reductase and glutamine synthetase among Frankia strains differing in their salt tolerance capacity. Nitrogenase activity closely followed the growth pattern with regular decline on NaCl supplementation. All the other enzymes showed optimum activity at 100 mM and declined further. Co-regulation of the nitrate uptake system and sequential enzyme activities plays a crucial role in governing the nitrogen status of strains during salt stress. HsIi10 experiencing minimum decline in enzyme activities and best possible nitrogen regulation under NaCl replete condition showed adequate nutritional management. Among all the strains, HsIi10 proved to be salt tolerant on account of above features while the salt sensitive strain HsIi8 lacked the ability to regulate various steps of nitrogen metabolism during salinity, and thus Frankia strain HsIi10 can potentially serve as a potential biofertilizer in the saline soil.


Subject(s)
Ammonia/metabolism , Frankia/enzymology , Frankia/metabolism , Glutamate-Ammonia Ligase/metabolism , Nitrates/metabolism , Nitrogen/metabolism , Nitrogenase/metabolism , Salinity , Salt Tolerance , Sodium Chloride/metabolism
8.
Article in English | IMSEAR | ID: sea-157577

ABSTRACT

Zinc being a stronger electron acceptor than iron might replace iron from the critical thiol groups. So, Zinc supplementation in Tubercular Subjects might help the decompartmentalised state of iron in the body to revert back to normal compartmentalized state of iron. Again, Zinc inhibits the formation of superoxide radicals. Thus, Zinc supplementation might decrease the excess superoxide with simultaneous decrease in the formation of soluble oxygen made by dismutation reaction by the iron cofactored superoxide dismutase secreted extracellularly by the pathogenic M. tuberculosis. The study shows early and effective recovery with Zinc supplementation (50mgm. of elemental zinc orally / day for one month) along with anti - Tubercular drug therapy. This gets support by the significant changes in the serum level of three enzymes – Glutamine Synthetase, Superoxide Dismutase and Cholienesterase. Again, the dose of zinc supplementation instituted with a great benefit and without any toxic symptoms and signs, is below the Lowest Observed Adverse Effect Level (LOAEL) based on the superoxide dismutase activity in erythrocytes with zinc intake.


Subject(s)
Antitubercular Agents/administration & dosage , Dietary Supplements , Drug Tolerance , Cholinesterases/blood , Female , Glutamate-Ammonia Ligase/blood , Humans , Male , No-Observed-Adverse-Effect Level , Superoxide Dismutase/blood , Tuberculosis/drug therapy , Zinc/administration & dosage , Zinc/therapeutic use
9.
Journal of the Korean Neurological Association ; : 113-116, 2014.
Article in Korean | WPRIM | ID: wpr-91983

ABSTRACT

Glufosinate ammonium is the active ingredient in broad-spectrum contact herbicides such as BASTA(R) that inhibits the activity of glutamine synthetase, which is necessary for the production of the amino acid glutamine and for ammonia detoxification. Complications of glufosinate ammonium intoxication include gastrointestinal symptoms, loss of consciousness, convulsions, memory impairment, respiratory failure, and cardiovascular instability. We report herein a case of encephalopathy and reversible signal changes in the splenium of the corpus callosum, the bilateral corticospinal tracts, the hippocampi, and the cerebellar peduncles as seen in diffusion-weighted magnetic resonance imaging and fluid-attenuated inversion recovery images following BASTA(R) intoxication.


Subject(s)
Ammonia , Ammonium Compounds , Corpus Callosum , Glutamate-Ammonia Ligase , Glutamine , Herbicides , Magnetic Resonance Imaging , Memory , Pyramidal Tracts , Respiratory Insufficiency , Seizures , Unconsciousness
10.
China Journal of Chinese Materia Medica ; (24): 3263-3268, 2014.
Article in Chinese | WPRIM | ID: wpr-244584

ABSTRACT

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.


Subject(s)
Ammonium Compounds , Metabolism , Pharmacology , Chlorophyll , Metabolism , Chrysanthemum , Metabolism , Flowers , Metabolism , Glutamate Synthase , Metabolism , Glutamate Synthase (NADH) , Metabolism , Glutamate-Ammonia Ligase , Nitrates , Metabolism , Pharmacology , Nitrogen , Metabolism , Pharmacology , Photosynthesis , Plant Leaves , Metabolism , Plant Proteins , Metabolism , Plant Roots , Metabolism , Plant Stems , Metabolism
11.
Korean Journal of Pathology ; : 411-417, 2013.
Article in English | WPRIM | ID: wpr-114870

ABSTRACT

BACKGROUND: Hepatocellular adenoma (HCA) is a rare benign tumor of the liver. A subtype classification of HCA (hepatocyte nuclear factor 1alpha [HNF1alpha]-mutated, beta-catenin-mutated HCA, inflammatory HCA, and unclassified HCA) has recently been established based on a single institutional review of a HCA series by the Bordeaux group. METHODS: We used histologic and immunohistochemical parameters to classify and evaluate eight cases from our institution. We evaluated the new classification method and analyzed correlations between our results and those of other reports. RESULTS: Seven of our eight cases showed histologic and immunohistochemical results consistent with previous reports. However, one case showed overlapping histologic features, as previously described by the Bordeaux group. Four cases showed glutamine synthetase immunohistochemical staining inconsistent with their classification, indicating that glutamine synthetase staining may not be diagnostic for beta-catenin-mutated HCA. HNF1alpha-mutated HCA may be indicated by the absence of liver fatty acid binding protein expression. Detection of amyloid A may indicate inflammatory HCA. HCA with no mutation in the HNF1alpha or beta-catenin genes and no inflammatory protein expression is categorized as unclassified HCA. CONCLUSIONS: Although the new classification is now generally accepted, validation through follow-up studies is necessary.


Subject(s)
Adenoma, Liver Cell , Amyloid , beta Catenin , Fatty Acid-Binding Proteins , Glutamate-Ammonia Ligase , Hepatocyte Nuclear Factor 1-alpha , Liver , Serum Amyloid A Protein
12.
Clinical and Molecular Hepatology ; : 185-189, 2013.
Article in English | WPRIM | ID: wpr-25401
13.
Braz. j. med. biol. res ; 45(8): 784-791, Aug. 2012. ilus, tab
Article in English | LILACS | ID: lil-643651

ABSTRACT

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.


Subject(s)
Animals , Male , Rats , Calcium-Binding Proteins/drug effects , Ferritins/drug effects , Glial Fibrillary Acidic Protein/drug effects , Glutamate-Ammonia Ligase/drug effects , Hippocampus/drug effects , Hypothalamus/drug effects , Neuroglia/metabolism , Biomarkers/metabolism , Calcium-Binding Proteins/metabolism , Ferritins/metabolism , Glial Fibrillary Acidic Protein/metabolism , Glutamate-Ammonia Ligase/metabolism , Hippocampus/chemistry , Hippocampus/cytology , Hypothalamus/chemistry , Hypothalamus/cytology , Immunohistochemistry , Lipopolysaccharides , Neuroglia/drug effects , Rats, Wistar
14.
IJB-Iranian Journal of Biotechnology. 2012; 10 (1): 61-65
in English | IMEMR | ID: emr-122608

ABSTRACT

Bacillus clausii TnrA transcription factor is required for global nitrogen regulation. In order to obtain an overview of gene regulation by TnrA in B. clausii KSM-K16, the entire genome of B. clausii was screened for the consensus sequence, 5'-TGTNAN7TNACA-3' known as the TnrA box, and 13 transcription units were found containing a putative TnrA box. The TnrA targets identified in this study were tnrA, glnA, nrgA, nasFDEB, puc genes, licT, the two operons of the oligopeptide ABC transporter, lytR, transcriptional regulator of the Lrp/AsnC family, sodium-dependent transporter of SNF family, hyu genes and a biochemically uncharacterized protein


Subject(s)
Bacteriocins , Repressor Proteins , Nitrogen , Transcription Factors , Genome-Wide Association Study , Genome , Consensus Sequence , Glutamate-Ammonia Ligase , Operon , ATP-Binding Cassette Transporters , Regulatory Elements, Transcriptional
15.
Chinese Journal of Biotechnology ; (12): 626-631, 2009.
Article in Chinese | WPRIM | ID: wpr-286663

ABSTRACT

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.


Subject(s)
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
16.
Indian J Exp Biol ; 2007 Dec; 45(12): 1022-30
Article in English | IMSEAR | ID: sea-58552

ABSTRACT

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.


Subject(s)
Amino Acids/pharmacology , Genes, Dominant , Glutamate-Ammonia Ligase/genetics , Glutamine/pharmacology , Mutation , Plant Infertility/genetics , Plants, Genetically Modified/genetics , Pollen/genetics , Promoter Regions, Genetic/genetics , Nicotiana/genetics
17.
China Journal of Chinese Materia Medica ; (24): 2289-2294, 2007.
Article in Chinese | WPRIM | ID: wpr-324357

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of Huanglian Jiedutang (HLJDT) on hippocampal protein expressions in senescence accelerated mouse-prone/8 (SAMP8).</p><p><b>METHOD</b>The 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).</p><p><b>RESULT</b>Compared 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.</p><p><b>CONCLUSION</b>HLJDT 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.</p>


Subject(s)
Animals , Male , Mice , Coptis , Chemistry , Drug Combinations , Drugs, Chinese Herbal , Pharmacology , Electrophoresis, Gel, Two-Dimensional , Fructose-Bisphosphate Aldolase , Metabolism , Glutamate-Ammonia Ligase , Metabolism , Hippocampus , Metabolism , Plants, Medicinal , Chemistry , Proteome , Metabolism , Proteomics , Methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
18.
Chinese Journal of Biotechnology ; (12): 94-100, 2006.
Article in Chinese | WPRIM | ID: wpr-237018

ABSTRACT

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.


Subject(s)
Animals , Cricetinae , Ammonia , Metabolism , Toxicity , CHO Cells , Cell Culture Techniques , Methods , Cricetulus , Culture Media, Serum-Free , Genetic Engineering , Methods , Glutamate-Ammonia Ligase , Genetics , Metabolism , Glutamine , Metabolism
19.
Genet. mol. biol ; 28(3,suppl): 555-561, Nov. 2005. ilus, tab
Article in English | LILACS | ID: lil-440454

ABSTRACT

This work was aimed at locating Eucalyptus ESTs corresponding to the GS enzyme (Glutamine Synthetase, EC = 6.3.1.2) and to the D1 protein, which are directly related to resistance to herbicides that promote oxidative stress. Glutamine Synthetase corresponds to the site of action of the herbicide glufosinate. Herbicides that belong to groups such as ureas, uracils, triazines and triazinones act on the D1-Qb complex (receptor of electrons from the Photosystem II) by inactivating it. The clusters EGEQRT3302E01.g, EGEQRT3001F12.b; EGEZLV1203B04.g; EGBGFB1211H06.g and EGEZLV1205F09.g enclosed complete sequences (with 356 amino acids) of the Glutamine Synthetase enzyme. The cluster EGEQSL1054G06.g is a consensus of four reads and enclosed a complete sequence of D1 Protein (with 353 amino acids). The comparison of the sequences of Protein D1 from different species showed that the substitutions of serine (S) by glycine (G) or serine (S) by threonine (T) at the position 264 could produce plants resistant to herbicides that act on electron flow on Photosystem II. The sequence of amino acids corresponding to the cluster EGEQSL1054G06.g had a serine in position 264 indicating sensitivity of the Eucalyptus plants to herbicides that act on this site


Subject(s)
Expressed Sequence Tags , Eucalyptus/genetics , Databases, Genetic , Glutamate-Ammonia Ligase , Herbicides , Oxidative Stress
20.
Electron. j. biotechnol ; 7(3): 13-14, Dec. 2004. ilus, tab, graf
Article in English | LILACS | ID: lil-448770

ABSTRACT

The ubiquity of heavy metals in the biosphere results in the introduction of high amounts of toxic metals into the food chain from various sources. In the present study, one of the strongest nitrogen fixing cyanobacterium of the rice fields, Aulosira fertilissima, was subjected to nickel and chromium stress and the ameliorating effect of immobilization was investigated. Cell immobilization could protect the organism's growth against the toxicity of both heavy metals at LC50 as compared to lethal concentrations. The nitrate reductase activity in free cells treated with the metals was substantially inhibited but immobilized cells treated with 0.1 ppm nickel was not affected by the metal treatment. Cell immobilization also resulted in a significant protection against sub-lethal concentration of chromium but to a lesser degree than it did with sub- lethal levels of nickel. Control immobilized cells also had higher Nitrogenase activity than control free cells. Nickel and chromium addition markedly decreased the enzyme activity in free cells but immobilized cells exposed to sublethal concentrations of both metals could overcome this decrease. Glutamine synthetase showed similar response under immobilized conditions compared to free cells with both metals. The addition of algal filtrate in 3:1 ratio further increased the nitrogenase activity compared with immobilized cells treated with sublethal doses of both metals. Immobilization facilitated higher uptake of nickel as compared to chromium. The observations of the present study clearly demonstrate the protective effect of immobilization on Aulosira fertilissima against Nickel and chromium toxicity. Rice field ecosystem thus possess a bidirectional natural metal ameliorating system where Aulosira mats act as a naturally immobilized system and the decay of Aulosira along with other cyanobacteria act as natural chelators protecting the rice plants from deleterious effects of the heavy metals. Most importantly is...


Subject(s)
Cyanobacteria/metabolism , Chromium/metabolism , Nickel/metabolism , Agriculture , Cyanobacteria/enzymology , Water Pollution, Chemical/prevention & control , Chromium/toxicity , Glutamate-Ammonia Ligase/metabolism , Nitrogen Fixation , Nickel/toxicity , Nitrate Reductase/metabolism , Nitrogenase/metabolism
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