Bladder microenvironment actuated proteomotors with ammonia amplification for enhanced cancer treatment

Enzyme-driven micro/nanomotors consuming in situ chemical fuels have attracted lots of attention for biomedical applications. However, motor systems composed by organism-derived organics that maximize the therapeutic efficacy of enzymatic products remain challenging. Herein, swimming proteomotors based on biocompatible urease and human serum albumin are constructed for enhanced antitumor therapy via active motion and ammonia amplification. By decomposing urea into carbon dioxide and ammonia, the designed proteomotors are endowed with self-propulsive capability, which leads to improved internalization and enhanced penetration in vitro. As a glutamine synthetase inhibitor, the loaded l-methionine sulfoximine further prevents the conversion of toxic ammonia into non-toxic glutamine in both tumor and stromal cells, resulting in local ammonia amplification. After intravesical instillation, the proteomotors achieve longer bladder retention and thus significantly inhibit the growth of orthotopic bladder tumor in vivo without adverse effects. We envision that the as-developed swimming proteomotors with amplification of the product toxicity may be a potential platform for active cancer treatment.

Cross-linking mass spectrometry reveals structural insights of the glutamine synthetase from Leishmania braziliensis

BACKGROUND Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania braziliensis, commonly found in Brazil and associated with cutaneous and visceral forms of this disease. Like other organisms, L. braziliensis has an enzyme called glutamine synthetase (LbGS) that acts on the synthesis of glutamine from glutamate. This enzyme plays an essential role in the metabolism of these parasites and can be a potential therapeutic target for treating this disease. OBJECTIVES Investigate LbGS structure and generate structural models of the protein. METHODS We use the method of crosslinking mass spectrometry (XLMS) and generate structural models in silico using I-TASSER. FINDINGS 42 XLs peptides were identified, of which 37 are explained in a monomeric model with the other five indicating LbGS dimerization and pentamers interaction region. The comparison of 3D models generated in the presence and absence of XLMS restrictions probed the benefits of modeling with XLMS highlighting the inappropriate folding due to the absence of spatial restrictions. MAIN CONCLUSIONS In conclusion, we disclose the conservation of the active site and interface regions, but also unique features of LbGS showing the potential of XLMS to probe structural information and explore new drugs.

Control of Conyza spp. with sequential application of glufosinate in soybean pre-sowing / Controle de Conyza spp. com aplicação sequencial de glufosinate em pré-semeadura da soja

ABSTRACT: Conyza spp. is among the main weeds reported worldwide. Due to its aggressiveness, such as high seed production and dispersion, and the growing reports of biotypes resistant to glyphosate, paraquat, and other herbicides, different control practices are required. Thus, this study aimed to evaluate the efficacy of herbicides with sequential application of glufosinate in soybean pre-sowing for control of Conyza spp. with indicative of resistant to paraquat. The study was carried out in the field, at Assis Chateaubriand and Palotina, state of Paraná, Brazil, in the 2018/19 season. The experiments were conducted in a randomized block design with four replications. The treatments consisted in application of glyphosate, 2,4-D, saflufenacil, glufosinate, saflufenacil/imazethapyr, diclosulam, paraquat/diuron, paraquat and imazethapyr/flumioxazin, at different combinations, in soybean pre-sowing. Control of Conyza spp., crop injury to soybean plants and variables related to agronomic performance (plant height and yield) were evaluated. All treatments were selective for soybean, which showed stronger crop injury in the presence of diclosulam herbicide, but this did not compromise soybean agronomic performance. In general, control levels were high for the treatments used. Except for paraquat treatments, in the area with the highest frequency of Conyza spp. with indicative of resistant to paraquat, and imazethapyr/flumioxazin treatment in both areas. These control results emphasized the importance of glufosinate in this management system and showed promising results for saflufenacil/imazethapyr.

RESUMO: A planta daninha Conyza spp. está entre as principais encontradas em todo o mundo. Devido a sua agressividade, como elevada produção e dispersão de sementes e os crescentes relatos de biótipos resistentes ao glyphosate, paraquat e outros herbicidas, faz-se necessário o uso de diferentes ferramentas de manejo. Assim, o objetivo deste estudo foi avaliar a eficácia de herbicidas com aplicação sequencial de glufosinate em pré-semeadura da soja, no controle de Conyza spp. com indicativo de resistência ao paraquat. O estudo foi realizado em campo nos municípios de Assis Chateaubriand e Palotina, estado do Paraná (PR), Brasil, na safra 2018/19. O delineamento experimental utilizado foi o de blocos casualizados, com quatro repetições. Os tratamentos foram compostos pela aplicação de glyphosate, 2,4-D, saflufenacil, glufosinate, saflufenacil/imazethapyr, diclosulam, paraquat/diuron, paraquat e imazethapyr/flumioxazin, em diferentes combinações em pré-semeadura da soja. Foram avaliados controle de Conyza spp., sintomas de injúria nas plantas de soja e variáveis relacionadas ao desempenho agronômico (altura de plantas e produtividade). Todos os tratamentos mostraram-se seletivos a soja. Visualizou-se maiores sintomas de injúria quando se tinha a presença do herbicida diclosulam, mas não resultou em danos ao desempenho agronômico da soja. De forma geral os níveis de controle foram altos para os tratamentos utilizados, em exceção para os tratamentos com paraquat na área que apresentava maior frequência de Conyza spp. com indicativo de resistência a este herbicida, e para o tratamento com imazethapyr/flumioxazin nas duas áreas. Estes resultados de controle enfatizam a importância do glufosinate neste sistema de manejo e demostram resultados promissores para saflufenacil/imazethapyr.

Changes induced by co-inoculation in nitrogen-carbon metabolism in cowpea under salinity stress

ABSTRACT To mitigate the deleterious effects of abiotic stress, the use of plant growth-promoting bacteria along with diazotrophic bacteria has been increasing. The objectives of this study were to investigate the key enzymes related to nitrogen and carbon metabolism in the biological nitrogen fixation process and to elucidate the activities of these enzymes by the synergistic interaction between Bradyrhizobium and plant growth-promoting bacteria in the absence and presence of salt stress. Cowpea plants were cultivated under axenic conditions, inoculated with Bradyrhizobium and co-inoculated with Bradyrhizobium sp. and Actinomadura sp., Bradyrhizobium sp. and Bacillus sp., Bradyrhizobium sp. and Paenibacillus graminis, and Bradyrhizobium sp. and Streptomycessp.; the plants were also maintained in the absence (control) and presence of salt stress (50 mmolL-1 NaCl). Salinity reduced the amino acids, free ammonia, ureides, proteins and total nitrogen content in nodules and increased the levels of sucrose and soluble sugars. The co-inoculations responded differently to the activity of glutamine synthetase enzymes under salt stress, as well as glutamate synthase, glutamate dehydrogenase aminating, and acid invertase in the control and salt stress. Considering the development conditions of this experiment, co-inoculation with Bradyrhizobium sp. and Bacillus sp. in cowpea provided better symbiotic performance, mitigating the deleterious effects of salt stress.

Expression characteristics of glutamine synthetase of wheat in Escherichia coli / 生物工程学报

Glutamine synthetase is a key enzyme in plant nitrogen assimilation. To study the structure of wheat glutamine synthetase isoenzymes, GS1, GSr, GSe, GS2 and GS2p of wheat were cloned into pET-21a, and the expression condition was optimized. Although wheat glutamine synthetase isoenzymes had 70%-80% amino acid sequence homology, the isoforms expressed with different characteristics. Induced at 30 °C, the most expression level of GSr, GSe and GS2 was after 3 h, and of GS1 was at the 7 h whereas no GS2p was expressed, and the GS isoenzymes showed different expression level, with the order of GS1 (22%)>GSr (15%)>GS2 (12%)>GSe (5%). GSe expressed as soluble protein, and GS1 expressed mainly as soluble protein whereas GSr and GS2 expressed as insoluble proteins. Induced at 30 °C for 3 h, mRNA transcript levels of GS isoforms were different, with the order of GSr (7.59)>GS2 (1.84)>GS2p (1.66)>GSe (1.46)>GS1 (1.00). The levels of mRNA transcription were not consistent with the level of the protein translation. The analysis of mRNA secondary structure showed the free energy of translation initiation region of glutamine synthetase isoforms was different, with the order of GS1 (14.4)<GSr (17.2)<GS2 (22.6) <GSe (25.4) <GS2p (31.6), the smaller freed energy, the more unstable mRNA secondary structure of translation initiation region and the higher level of protein expression. Soluble expression condition of glutamine synthetase isozymes was also different, with GS1, GSr, GSe and GS2 induced at 30 °C for 5 h, 16 °C for 15 h, 37 °C for 5 h, and 25 °C for 7 h respectively. The soluble protein showed different expression level with GS1 (20%)>GSr (13%)>GS2 (10%)>GSe (7%), and different activities with GS1>GSe>GS2, and the activity of GSr was not detected. The gene sequence of glutamine synthetase isoenzymes determines the amount, status and activity of proteins expressed in prokaryotic cells.

Diagnostic value of 13 N-ammonia PET/CT imaging targeting glutamine synthetase expression in pa-tients with prostate cancer / 中华核医学与分子影像杂志

Objective To investigate the expression of glutamine synthetase ( GS ) in prostate cancer and the utility of 13 N?NH3 PET/CT in detecting prostate cancer. Methods The uptake ratio of 13 N?NH3 and the expression of GS in PC3 and DU145 cells were measured by Western blot and PCR methods. A total of 34 patients with suspected prostate cancer underwent 13 N?NH3 PET/CT imaging and prostate biopsy. Immunohistochemistry staining of GS was performed and Gleason scores of tumors were evaluated. One?way analysis of variance, the least significant difference?t test and Spearman correlation analysis were used to an?alyze data. Results The uptake of 13 N?ammonia in PC3 and DU145 cells elevated along with the decrease of glutamine in medium. The expression of GS mRNA and protein also increased when glutamine was de?creased. In biopsy samples, the mean GS expression scores of prostate cancer, benign prostatic hyperplasia (BPH) and prostatitis were 7.76±2.57, 3.98±2.60, 3.34±0.36, respectively (F=36.85, t1=7.97, t2=4?45, all P<0.05), which had a weak correlation with Gleason scores (rs=0.52, P<0.05). In 34 patients, the mean SUVmax of prostate cancer segments (1.56±0.58 and 1.14±0.22;F=5.966, t1=2.63, t2=2.65, all P<0.05). There was a weak correlation between GS expression scores and the uptake of 13 N?ammonia in prostate cancer (rs=0.47, P<0.05). Conclusions Up?regulated expression of GS is common in prostate cancer cells. GS is the main reason for the uptake of 13 N?ammonia, which is a useful tracer for prostate cancer imaging.

Efficacy of Zinc Supplementation as an Adjunct to Anti Tubercular Drug Therapy.

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.

Animal cell screening system based on GS efficient expression vector and its application / 军事医学

Objective To obtain highly expressing cell lines by inserting the glutamine synthetase (GS) screening system and replacing the promoter of the vector.Methods The mutation of the point BamHⅠwas induced to build a new vector pIRES2-EGFP.The marker gene GS was inserted by AseⅠ and NheⅠ, and the promoter hCMV was replaced by PacⅠand NheⅠ.The new vector pHGS1.0 and the vector pIRES2-enhanced screen fluorescein protein( EGFP)-B were inserted by the recombinant protein TEM8 ( 1-227 )-VEGFR1 domain2-IgG2 ( TV-IgG2 ) gene to analyze the advantages of the expression.Results The glutamine synsthetase is successfully inserted, the human cytomegalovirus replaced, and recombinant protein is increased 5-fold by human immunoglobulin quantification kit.Conclusion The GS system is a highly protein expressing system.

beta-Catenin Activated Hepatocellular Adenoma: A Report of Three Cases in Korea

Hepatocellular adenoma (HCA) is an uncommon benign hepatic tumor, and the use of oral contraceptives is known to contribute to the development of HCA. Recently, a genotype and phenotype classification system for HCA was suggested, and malignant transformation to hepatocellular carcinoma (HCC) was shown to be strongly associated with activating mutations in beta-catenin. Here, we report three cases of HCA in Korean patients: 7-cm, inflammatory and beta-catenin-activated HCA with HCC transformation in a 46-year-old man; 13-cm, beta-catenin-activated HCA with cytological atypia in a 23-year-old woman; and 10-cm, pigmented, inflammatory and beta-catenin-activated HCA in a 36-year-old man. All cases exhibited the nuclear expression of beta-catenin and diffuse cytoplasmic expression of glutamine synthetase upon immunohistochemical staining. All tumors were completely resected, and the patients were followed for 3 to 6 years with no evidence of local recurrence or metastasis.

Glial cell activity is maintained during prolonged inflammatory challenge in rats

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.

INTERACCIÓN DE LA GLUTAMINA SINTETASA (GS) Y EL PÉPTIDO β -AMILOIDE COMO UNA ESTRATEGIA DE PURIFICACIÓN [title language="en"]INTERACTION OF GLUTAMINE SYNTHETASE (GS) AND AMYLOID β-PEPTIDE AS A PURIFICATION STRATEGY / INTERAÇÃO DA GLUTAMINA SINTETASE (GS) E PÉPTIDO β AMILÓIDE COMO UMA ESTRATÉGIA DE PURIFICAÇÃO

La enfermedad de Alzheimer (EA) es la forma de demencia más común en la edad adulta. Se manifiesta con la pérdida progresiva de la memoria a medida que las neuronas en la corteza cerebral y el hipocampo mueren. En todas las formas de EA se evidencia aumento de la expresión de diferentes proteínas, así como la presencia de agregados insolubles de péptido-β-amiloide (PBA). La glutamina sintetasa (GS) es una enzima clave en el metabolismo del glutamato y en la detoxificación de amonio (NH4+). Previamente se ha reportado una posible interacción GS-PBA que puede estar asociada con EA. En este trabajo se realizó la purificación de la enzima cerebral de rata a partir de un extracto sometido a precipitación fraccionada con (NH4)2SO4 del 20-60 % de saturación y posteriormente a través de cromatografías sucesivas de filtración en gel, intercambio iónico y afinidad. El peso molecular del complejo fue calculado en 137 kDa por el orden de elución en la columna de filtración. Se identificó interacción de la enzima con PBA 1-40, lográndose la purificación de una sola banda de 45 kDa, correspondiente a la forma monomérica de la GS. En este trabajo se presenta un nuevo método de purificación de la enzima y se demuestra la interacción de GS con el PBA. Se propone que esta interacción GS-PBA puede ser uno de los procesos que se presentan en la enfermedad al explicar la reducción de la actividad de la enzima en paciente con EA, ya que podría alterar el ciclo glutamato-glutamina y generar cambios en el entorno celular que favorecen excitotoxicidad por glutamato típica de los procesos de neurodegeneración.

Alzheimer's disease (AD) is the most common form of dementia in adulthood; it is manifested by the progressive loss of memory since neurons in both cerebral cortex and hippocampus die. In all the forms of AD is observed the increased expression of different proteins, as well as the presence of insoluble aggregates of β-amyloid peptide (BAP). Glutamine synthetase (GS) is a key enzyme in the metabolism of glutamate and in the detoxification of ammonium (NH4+). A possible interaction GS-PBA has been previously reported and it can be associated with AD. In this work we performed the purification of the enzyme from rat brain extract subjected to fractional precipitation 20-60 % saturation with (NH4)2SO4, and thereafter through successive chromatographies of gel filtration, ion exchange and affinity. The molecular weight of the complex was calculated at 137 kDa by the order of elution in the column filtration. The interaction of the enzyme with 1-40 PBA was identified, achieving the purification of a single band of 45 kDa corresponding to the monomeric form of the GS. In this paper we present a new method of the enzyme purification and we demonstrated the interaction of GS with the PBA. We propose this interaction GS-PBA can be one of the processes that occur in the disease and it could explain the reduction in enzyme activity in patients with AD, since it might alter the glutamate-glutamine cycle and generate changes in the cellular environment which favor glutamate excitotoxicity typical of neurodegeneration processes.

A doença do Alzhéimer (DA) é a forma mais comum de demência na idade adulta, que se manifesta pela perda progressiva da memória, já que os neurónios em córtex cerebral e hipocampo morrem. Em todas as formas de AD é observado o aumento da expressão de proteínas diferentes, bem como a presença de agregados insolúveis de β-amilóide péptido (BAP). Glutamina sintetase (GS) é uma enzima chave no metabolismo do glutamato e na desintoxicação de amónio (NH4+). Uma possível interacção GS-PBA foi relatada anteriormente e pode ser associada com o AD. Neste trabalho, foi realizada a purificação da enzima a partir do extrato do cérebro de rato e foi submetido a precipitação fraccionada com (NH4)2SO4 de 20- 60 % de saturação e, subsequentemente, através de cromatografias sucessivas de filtração em em gel, permuta iónica e de afinidade. O peso molecular do complexo foi calculado em 137 kDa por ordem de eluição na filtração de coluna. A interacção da enzima com 1-40 PBA foi identificada, alcançando a purificação de uma única banda de 45 kDa correspondente à forma monomérica do GS. Neste artigo, apresentamos um novo método de purificação da enzima e demonstramos a interação da GS com o PBA. Propomos que esta interacção GS-PBA pode ser um dos processos que ocorrem na doença e pode explicar a redução na actividade da enzima nos pacientes com o AD, uma vez que poderia alterar o ciclo de glutamatoglutamina e gerar alterações no ambiente celular que favorecem a excitotoxicidade típica do glutamato nos processos de neurodegeneração.

Effects of Subchronic Arsenic Exposure on Glutamate-glutamine Cycle in Mice Brain / 环境与健康杂志

Objective To explore the effects of subchronic arsenic exposure through drinking water on glutamate-glutamine cycle in the brain of mice. Methods The female Kunming mice were exposed to arsenite ( iAsⅢ ) by drinking water at the levels of 25, 50 and 100 mg/L respectively for 6 consecutive weeks. The blood and brain were taken, the concentration of inorganic arsenic (iAs), monomethylarsenic acid (MMA), dimethylarsenic acid (DMA) and the activity of glutamine synthetase (GS), phosphate activated glutaminase (PAG), superoxide dimutase (SOD) and the concentrations of glutamate (Glu), lipidperoxide(LPO) were determined. Results The concentrations of iAs, MMA and DMA in the blood and brains increased as the iAsⅢ concentrations in drinking water increased. The activity of GS, PAG and the concentrations of Glu in the arsenic exposed mice increased compared with the control. The activity of GS in 50 mg/L group, the activity of PAG in 25 and 50 mg/L groups, the concentration of Glu in 100 mg/L group showed a significant difference compared with the control. The activity of PAG in 25 mg/L group was significantly higher than that in 100 mg/L group. The activity of SOD in exposed groups was higher than that in the control, the concentration of LPO in exposed groups did not show a significant difference compared with the control. Conclusion Arsenic can enter the brain and organic arsenic is dominant both in the blood and brain, however, the composition of arsenic speciation is different in the blood and brain. DMA, as a main arsenide, distributed in the brain. Arsenic exposure can change the activity of GS and PAG which can influence the concentration of Glu. Moreover, arsenic exposure can increase the superoxide anion and make the activity of SOD increase compensatively.

Glutamine Synthetase Induced Spinal Seizures in Rats

Glutamine synthetase (GS) is a key enzyme in the regulation of glutamate neurotransmission in the central nervous system. It is responsible for converting glutamate to glutamine, consuming one ATP and NH3 in the process. Glutamate is neurotoxic when it accumulates in extracellular fluids. We investigated the effects of GS in both a spinal cord injury (SCI) model and normal rats. 0.1-ml of low (2-microM) and high (55-microM) concentrations of GS were applied, intrathecally, to the spinal cord of rats under pentobarbital anesthesia. Immediately after an intrathecal injection into the L1-L3 space, the rats developed convulsive movements. These movements initially consisted of myoclonic twitches of the paravertebral muscles close to the injection site, repeated tonic and clonic contractions and extensions of the hind limbs (hind limb seizures) that spread to the fore limbs, and finally rotational axial movements of the body. An EMG of the paravertebral muscles, fore and hind limbs, showed the extent of the muscle activities. GS (2-microM) caused spinal seizures in the rats after the SCI, and GS (6-microM) produced seizures in the uninjured anesthetized rats. Denatured GS (70 degrees C, 1 hour) also produced spinal seizures, although higher concentrations were required. We suggest that GS may be directly blocking the release of GABA, or the receptors, in the spinal cord.

Effects of dexamethasone and DHEA on the responses of rat cerebral cortical astrocytes to lipopolysaccharide and antimycin A

As part of a study on the effects of dexamethasone and dehydroepiandrosterone (DHEA) on the biological roles of astrocytes in brain injury, this study evaluated the effects of dexamethasone and DHEA on the responses of primary cultured rat cortical astrocytes to lipopolysaccharide (LPS) and antimycin A. Dexamethasone decreased spontaneous release of LDH from astrocytes, and the dexamethasone effect was inhibited by DHEA. However, the inhibitory effect of DHEA on the dexamethasone-induced decrease of LDH release was not shown in astrocytes treated with LPS, and antimycin A-induced LDH release was not affected by dexamethasone or DHEA. Unlike dexamethasone, DHEA increased MTT value of astrocytes and also attenuated the antimycin A-induced decrease of MTT value. Glutamine synthetase activity of astrocytes was not affected by DHEA or LPS but increased by dexamethasone, and the dexamethasone-dependent increase was attenuated by DHEA. However, antimycin A markedly decreased glutamine synthetase activity, and the antimycin A effect was not affected by dexamethasone or DHEA. Basal release of (3H)arachidonic acid from astrocytes was moderately increased by LPS and markedly by antimycin A. Dexamethasone inhibited the basal and LPS-dependent releases of (3H)arachidonic acid, but neither dexamethasone nor DHEA affected antimycin A-induced (3H)arachidonic acid release. Basal IL-6 release from astrocytes was not affected by dexamethasone or DHEA but markedly increased by LPS and antimycin A. LPS-induced IL-6 release was attenuated by dexamethasone but was little affected by DHEA, and antimycin A-induced IL-6 release was attenuated by DHEA as well as dexamethasone. At the concentration of dexamethasone and DHEA which does not affect basal NO release from astrocytes, they moderately inhibited LPS-induced NO release but little affected antimycin A-induced decrease of NO release. Taken together, these results suggest that dexamethasone and DHEA, in somewhat different manners, modulate the astrocyte reactivity in brain injuries inhibitorily.

Role of glutamine synthetase in citric acid fermentation by Aspergillus niger.

The activity of glutamine synthetase from Aspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0-6·5, when the pH of the external medium was varied between 2·3-7·0. Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn2+ ions partially protected the enzyme against this inactivation. Mn2+- dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg2+- supported activity. While the concentration of Mg2+ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn2+ was effective at 4 mM. Higher concentrations of Mn2+ were inhibitory. The inhibition of both Mn2+ and Mg2+-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the endproducts of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibited Aspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn2+ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis.

The response of Rhizobium meliloti to L-methionine DL-sulphoximine.

A strain of Rhizobium meliloti has been shown to be capable of growth in the presence of methionine sulphoximine concentrations at least two orders of magnitude higher than that required for the complete inhibition of glutamine synthetase activity. Neither the specific growth rate, nor the nutritional requirements of the organism were affected by methionine sulphoximine in the medium. Rhizobium meliloti appeared to assimilate ammonia via the glutamate dehydrogenase pathway during growth in the presence of methionine sulphoximine. This suggests that Rhizobium meliloti may have some regulatory mechanism controlling ammonia assimilation that is not present in other enterobacteria possessing similar enzymatic machinery.

Studies on Aspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues.

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grown Aspergillus niger was increased 3-5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH4 + , and further, the enzyme is repressed by increasing concentrations of NH4 +. In contrast to other micro-organisms, the Aspergillus niger enzyme was neither specifically inactivated by NH4 + or L-glutamine nor regulated by covalent modification. Glutamine synthetase from Aspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn2+ or Mg2+-dependent synthetase and Mn2+-dependent transferase activity. Aspergillus niger glutamine synthetase was completely inactivated by two mol of phenylglyoxal and one mol of N-ethylmaleimide with second order rate constants of 3·8 M–1 min–1 and 760 M–1 min–1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH4 +, Mn2+ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.

Studies on glutamine synthetase. Purification of the enzyme from mung bean (Phaseolus aureus) seedlings and modulation of the enzyme-antibody reaction by the substrates.

Glutamine synthetase (L-glutamate : ammonia ligase, EC 6.3.1.2) from Phaseolus aureus (mung bean) seedlings was purified to homogeneity by ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and affinity chromatography on histidine-Sepharose. The enzyme had a molecular weight of 775,000 ± 25,000. The enzyme consisted of identical subunits with an approximate subunit molecular weight of 50,000. Hyperbolic saturation curves were obtained with the substrates, glutamate, ATP and hydroxylamine. Antibody, raised in the rabbit, against mung bean glutamine synthetase, completely inhibited the activity of the enzyme. Preincubation of the enzyme with glutamate and ATP, prior to the addition of the antibody, partially protected the enzyme against inhibition. The Km values of this enzyme-antibody complex and the native enzyme were identical (glutamate, 2.5mM; ATP, 1 mM; hydroxylamine, 0·5 mM). The Km values of the partially inhibited enzyme (the enzyme pretreated with antibody prior to the addition of substrates) were 2-fold higher than those of the native enzyme. These results suggested that the substrate-induced conformational changes in the enzyme were responsible for the protection against inhibition of the enzyme activity by the antibody.

EFFECT OF GLUTAMINE SUPPLEMENTATION ON GLUTAMINE METABOLISM IN EXERCISING MICE / 营养学报

Objective: To approach the effects of glutamine supplementation on plasma and muscle levels of glutamine, and the activities of tissue glutamine synthetase and glutaminase after high intensity exercise. Methods: BAL/C mice were randomly divided into high intensity exercise(2% body weight load) and control groups to swim 2 hours per day for 8 weeks. Results: To compare with control group, high intensity exercise reduced glutamine levels in plasma and muscle by 74% (P