Dynamics of glutamine synthetase expression in hepatic ischemiareperfusion injury: Implications for therapeutic interventions

BACKGROUND Hepatic ischemia-reperfusion injury(IRI)poses a great challenge in liver surgery and transplantation because of oxidative stress and inflammatory responses.The changes in glutamine synthetase(GS)expression during hepatic IRI remain unclear.AIM To investigate the dynamic expression of GS during hepatic IRI.METHODS Following hepatic ischemia for 1 h and reperfusion,liver tissue samples were collected at 0.5,6,and 24 hours postreperfusion for fixation,embedding,section-ing.Hematoxylin and eosin staining and GS staining were performed.RESULTS GS expression rapidly decreases in hepatocytes around the central vein after IRI,reaching its lowest point at 6 hours postreperfusion,and then gradually recovers.CONCLUSION GS is highly sensitive to IRI,highlighting its potential role as an indicator of liver injury states and a target for therapeutic intervention.

Comparative Study of Immunological Properties on Glutamine Synthetase Isozymes in Rice Plants

In rice (Oryza sativa L.) roots two glutamine synthetase (GS) isozymes, GSra and GSrb, were identified recently in the author's experiments, but the homology of both GSra and GSrb as well as their localization in the rice roots are unclear. In the present study, the purified GSra and GSrb from rice roots were used to immunize rabbits to obtain the respective antibodies. The immunodiffusion and immunoblotting experiments showed that the antibody against GSra or GSrb was specific for GS and its isozymes. The immunoprecipitation test indicated that the antibody of GSra or GSrb not only recognized its respective antigen, but also well recognized each other's antigen. GSra or GSrb antibody recognized also better cytosolic GS1 of rice leaves, but the recognization for chloroplast GS2 from rice or spinach (Spinacia oleracea Mill.) leaves was weaker. Our results indicate that GSra and GSrb from rice roots are quite similar in antigenicity and are extremely similar proteins and that both GSra and GSrb may also be a form of cytosolic GS just as the cytosolic GS1 of rice leaves.

Influences of Mo on Nitrate Reductase, Glutamine Synthetase and Nitrogen Accumulation and Utilization in Mo-Efficient and Mo-Inefficient Winter Wheat Cultivars

The objective is to study whether the accumulation and utilization of plant N are controlled by Mo status in winter wheat cultivars. Mo-efficient cultivar 97003 （eft） and Mo-inefficient cultivar 97014 （ineff） were grown in severely Mo-deficient acidic soil （Tamm-reagent-extractable Mo 0.112 mg kg^-1） with （＋Mo） and without （-Mo） the application of 0.13 mg kg^-1 Mo. The accumulation and use efficiency of plant total N were significantly higher in ＋Mo than that in -Mo and in eft than that in ineff under Mo deficiency. N use efficiency was remarkably higher in maturity but it was forwarded to jointing stage after Mo supply, thus indicating that Mo supply promoted the N use efficiency besides N uptake and eff was efficient in N uptake and utilization. The overall activity of nitrate reductase （NR, EC 1.6.6.1） was significantly higher in ＋Mo than in -Mo and ratio of ＋Mo/-Mo was even to 14.8 at filleting stage for ineff. Activity of glutamine synthetase （GS, EC 6.3.1.2） was significantly lower in ＋Mo than in -Mo. Concentration of nitrate and glutamate were also significantly lower in ＋Mo than in -Mo, thus provided evidences for enhancing N use efficiency by Mo supply. Activities of NR and GS were significantly higher and concentrations of nitrate and glutamate were significantly lower in eff than ineff under Mo deficiency, thus indicated eff was more efficient in N reduction and utilization. It is therefore concluded that Mo could promote N accumulation and utilization in winter wheat which was directly related to NR and feedback regulated by GS. Higher Mo status also results in higher accumulation and utilization of plant N in eft.

Glutamine synthetase as an early marker for hepatocellular carcinoma based on proteomic analysis of resected smal hepatocel ular carcinomas

BACKGROUND:Hepatocellular carcinoma(HCC)is a highly malignant tumor with a poor prognosis.Because small HCCs possess most of the characteristics of early HCC,we investigated small HCCs to screen potential biomarkers for early diagnosis.METHODS:Proteins were extracted from 10 sets of paired tissue samples from HBV-infected small-HCC patients.The extracted proteins were well resolved by two-dimensional electrophoresis.These HCC-associated proteins were then identified by MALDI-TOF/TOF MS following image analysis.Western blotting and immunohistochemistry were used to assess glutamine synthetase(GS)and phenazine biosynthesislike domain-containing protein(PBLD)expression in liver tissue.Enzyme-linked immunosorbent assays in 152 serum samples(from 49 healthy donors,24 patients with liver cirrhosis,and 79 with HCC)were used to further assess the significance of GS clinically.RESULTS:Fifteen up-regulated and three down-regulated proteins were identified.Western blotting confirmed GS overexpression and decreased PBLD expression in liver tissue.Immunohistochemistry showed that GS was expressed in 70.0%(84/120)of HCCs and 35.8%(43/120)of nontumor tissues;PBLD was expressed in 74.2%(89/120) of nontumor tissues and 40.8%(49/120)of HCCs.The Chi-square test showed significant expression differences between HCCs and adjacent tissues.Consistent with this,serum GS levels in HCC patients were significantly higher than those in liver cirrhosis patients and healthy donors,while the latter two groups were also significantly different.In addition, a diagnostic cutoff value of 2.6 mg/ml was used for GS;it was elevated in 19(76.0%)of 25 HCC patients with AFP≤20 ng/ml and 47(88.7%)of 53 HCC patients with AFP≤200 ng/ml.CONCLUSION:GS and PBLD are abnormally expressed in most HCCs.GS may be a novel serum marker for early HCC, especially for those patients with low AFP levels(≤200 ng/ml).

Effect of Nitrate on Activities and Transcript Levels of Nitrate Reductase and Glutamine Synthetase in Rice

Real-time polymerase chain reaction analysis was used to compare the effect of NO3^- on the activities of nitrate reductase （NR） and glutamine synthetase （GS）, and the transcript levels of two NR genes, OsNial and OsNia2, two cytosolic GS1 genes, OsGln1;1 and OsGln1;2, and one plastid GS2 gene OsGln2, in two rice （Oryza sativa L.） cultivars Nanguang （NG） and Yunjing （Y J）. Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH4^＋ nutrition. Supply of NO3^- increased NR activity in both leaves and roots. Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNial. NO3 also caused an increase in GS activity, but had a complex effect on the expression of the three GS genes. In roots, the OsGln1;1 transcript increased, but OsGln1;2 decreased. In leaves, NO3^- had no effect on the GS1 expression, but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N. These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes. In addition, the NO3-induced increase of biomass, NR activity, GS activity, and the transcript levels of NR and GS genes were proportionally higher in NG than in Y J, indicating a stronger response of NG to NO3^- nutrition than YJ.

Changes in Activities of Glutamine Synthetase during Grain Filling and Their Relation to Rice Quality

Four japonica rice varieties differed in cooking and eating qualities were used in a pot experiment to study the relationship between the activities of glutamine synthetase during grain filling and rice quality. The activities of glutamine synthetase gradually increased and then declined as a single peak curve in the course of grain filling. The 15th day after heading was a turning point, before which the enzymatic activities in the inferior rice varieties with high protein content were higher than those in the superior rice varietie with low protein content, and after which it was converse. The activity of glutamine synthetase in grain was correlated with the taste meter value, peak viscosity and breakdown negatively at the early stage of grain filling whereas positively at the middle and late stages. Moreover, it was correlated with the protein content of rice grain and setback positively at the early stage and negatively at the middle and late stages. The correlation degree varied with the course of grain filling. From 15 days to 20 days after heading was a critical stage, in which the direction of correlation between the activity of glutamine synthetase and taste meter value and RVA properties of rice changed.

Changes of Levels of Glutamine Synthetase Isoforms in Roots and Leaves in Responseto Nitrogen Fertilizer Application at Different Growth Stages in Irrigated Rice

Nitrogen is a key element to control the growth and yield of crops. Fertilizer urea nitrogen (N) 60,45, and 30 kg/hm2 was applied at three different stages, midtillering, panicle initiation, and flowering, of the growth and development of rice plants, respectively. At both midtillering and panicle initiation, the total activity of glutamine synthetase (GS) in rice roots and leaves was incrased remarkably as a result of a large amount of ammonia absorbed by roots. Native-PAGE and activity staining showed that the increase of total activity in rice roots and leaves was due to the synthesis of GSrb in roots and GS2 in leaves and that the activity of GSra in roots and GS1 in leaves remained constant. The results showed that the assimilation of external nitrogen was carried out by GSrb but not GSra in rice roots and that the activitry of GS2 was induced also by the external nitrogen, and that GSrb played main role in meeting the needs of the rapid tillering for nitrogen. At flowering, the activity of GS in rice roots and leaves did not change almost after topdressing. These rssults suggest that the change of GS activity in rice roots may use as a measure of the utilization efficiency of the fertilizer.

Effects of Ganoderma lucidum spore powder on astrocyte expression and glutamine synthetase activity in the hippocampal region of epileptic rats

BACKGROUND： Recent studies have demonstrated that astrocyte dysfunction plays a central role in inhibiting epileptic seizures and that regulation of astrocyte function may be a new target for treatment of epilepsy. OBJECTIVE： To observe the effects of Ganoderma lucidum spore powder （GLSP） on astrocyte morphology and glutamine synthetase （COS） activity in the hippocampal region of epileptic rats. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Function Laboratory, College of Basic Medicine, Jiamusi University between October and December 2006. MATERIALS： A total of 30 Sprague Dawley （SD） rats were randomized to three groups （n = 10）： control, model, and GLSP. GLSP was sourced from Jiamusi Wild Ganoderma Lucidum Planting Base and prepared to 30 g/L with physiological saline before use. Pentylenetetrazol （PTZ） （10 g/L） was provided by Sigma Company, USA. METHODS： The control group received intraperitoneal （i.p.） and intragastric （i.g.） physiological saline. Following epilepsy induction by i.p. administration of PTZ （35 mg/kg）, rats from the mode/and GLSP groups were ig injected with physiological saline and GLSP （300 mg/kg）, respectively. Each compound was administered once per day, for a total of 28 successive days. Epileptic seizure convulsions were graded 0-5. A higher grade indicated more severe epilepsy. Only those rats showing stage 2 or higher convulsions at least 5 times successively were included in further experiments. MAIN OUTCOME MEASURES： Immediately alter injection, seizure activity was monitored for 30 minutes to determine the latent period and seizure duration; simultaneously, astrocyte numbers and GS activity in the hippocampal region of rats with epilepsy were detected by immunohistochemistry. RESULTS： All 30 rats were included in the final analysis. On day 28, following PTZ administration epileptic seizures were not found in the control group. In the GLSP group, rats exhibited rhythmic head nodding or facial spasms, and the latent period was significantly longer than that of the model group （P 〈 0.05）. In the model group, the majority of rats exhibited myoclonus or generalized convulsions, and epileptic seizure duration was slightly （but not significantly） longer than that in the GLSP group （P 〉 0.05）. Within 2-3 minutes of PTZ injection, the model group exhibited grade 4 or 5 epileptic seizures, and the GLSP group mostly showed grade 2 or 3, and occasionally grade 4 or 5, epileptic seizures. All rats recovered within 30 minutes. The model group exhibited significantly increased astrocytes （P 〈 0.05）, with thicker cellular processes and a higher number of cellular processes, and significantly decreased GS activity （P 〈 0.05） tban the control group. The astrocyte count was significantly decreased but GS activity was significantly increased in the GLSP group when compared with the model group （P 〈 0.05）; however, astrocyte appearance was similar in both groups （P 〈 0.05）. CONCLUSION： GLSP can effectively inhibit astrocyte numbers and elevate GS activity in the hippocampal region of rats with epilepsy, thereby reducing epileptic seizures.

Effect of Exogenous Ammonium on GlutamineSynthetase, Glutamate Synthase, and Glutamate Dehydrogenase in the Root of Rice Seedling

Root biomass of rice seedlings was increased at lower concentration of exogenous NH 4 + , but it was decreased at higher concentration of exogenous NH 4 + . The level of free NH 4 + in the roots was accumulated gradually with the increase of NH 4 + concentration in the nutrient solution. The content of the soluble proteins was essentially constant at higher NH 4 + . The activities of glutamine synthetase (GS), NADH-dependent glutamate synthase (NADH-GOGAT), and NADH-dependent glutamate dehydrogenase (NADH-GDH) were risen with exogenous NH 4 + concentration at the lower NH 4 + concentration range. But the activities of GS and NADH-GOGAT were declined, and the level of NADH-GDH activity was kept constant under higher NH 4 + concentration. The GS/GDH ratio suggested that NH 4 + was assimilated by GS-GOGAT cycle under lower NH 4 + concentration, but NADH-GDH was more important for NH 4 + assimilation and detoxifying NH 4 + to the tissue cells at the higher NH 4 + level. According to the growth and the activity changes of these ammonium-assimilating enzymes of rice seedling roots, 10. 0 μg/mL NH 4 + -N in nutrient solution was more suitable to the rice growth.

Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase

BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxification. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS: hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and amplified. Then hGS mRNA was measured by semi-quantitative RT-PCR; hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH(4)(+) were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS: The expression of hGS mRNA in HepG2/pLNChGS cells (8.306+/-0.336) and HepG2/pLNAFhGS cells (21.358+/-1.716) was much stronger than in control cells (P<0.05), and that in HepG2/pLNAFhGS cells was markedly stronger than in HepG2/pLNChGS cells (P<0.05). The hGS enzymatic activities of HepG2/pLNChGS cells (3.279+/-0.328 U/mg prot) and HepG2/pLNAFhGS cells (4.557+/-0.253 U/mg prot) were higher than those of control cells (P<0.05), and those of HepG2/ pLNAFhGS cells were also higher than the activities of HepG2/pLNChGS cells (P<0.05). In addition, the effect of hGS introduction on HepG2 cell proliferation was not significant. The amount of glutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH(4)(+) was higher than in the two control cells (P<0.05). The amount of glutamine synthesis and cell proliferation in the higher concentrations of NH(4)(+) (5 or 10 mmol/L) in HepG2/pLNAFhGS cells increased more than those in HepG2/pLNChGS cells (P<0.05). NH(4)(+) at a high concentration (10 mmol/L) was toxic to HepG2 and HepG2/pLNCX cells, but less toxic to HepG2/pLNChGS and HepG2/pLNAFhGS cells. CONCLUSION: The constructed hepatocytes (HepG2 cells) with specific high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.

Studies on Glutamine Synthetase Activity in Sugar Beet （BetavulgarisL．） under Different Levels of Nitrogen

It was shown from the experiment that glutamine synthetase activity (GSA) in both leaf blades and roots under different nitrogen levels rose rapidly to reach its peak from seedling stage to foliage rapid growth stage and declined to its lowest level at the latter stage of root rapid growth, and then increased slightly. GSA in leaf blades had positive correlation with nitrogen level during the whole period of growth. GSA in roots showed the same tendency as it in leaf blades at the early middle stage of growth, but at the latter stage of growth, no positive correlation was established. GSA in leaf blades was the strongest compared with crowns, petioles and roots, and could represent the highest enzyme activity of the whole plant. GSA had quadratic curvilinear correlation with root yield and sugar production. GSA in leaf blades had significant positive correlation with α-NH2-N at the foliage rapid growth stage.

Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats

AIM: To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. METHODS: Male Wistar rats were divided into shamoperated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. RESULTS: We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, signif icantly and persistent increase in this excitatory neurotransmitter activity. CONCLUSION: The presence of moderate ammonia blood levels may add to the toxicity of excitotoxic glutamate in the brain, which causes alterations in brain function. Portal vein stricture that causes portal hypertension modif ies the normal function in some brain regions.

Advances in the functional study of glutamine synthetase in plant abiotic stress tolerance response

Plant glutamine synthetase(GS,EC6.3.1.2)catalyzes the synthesis of glutamine from glutamate and ammonium ions and acts as a key enzyme in the nitrogen metabolic pathway in organisms.Nitrogen is an essential element for plant growth and development and plays an important role in crop yield and quality formation.Therefore,GS is crucial in many physiological processes in plants.Currently,nitrogen regulation by GS in plants is well-studied in terms of its effect on plant growth and development.This article reviews the regulatory role of plant GS and its molecular mechanism in mitigating stress injury,such as low or high temperature,salinity,drought and oxidation.The function of plant GS in stress tolerance response is focused.The review aims to provide a reference for the utilization of plant GS in crop stress tolerance breeding.

Comparative Study of the Effects of Metformin and Garlic Extract on Hippocampal Na⁺/K⁺ATPase, Ca²⁺ATPase and Glutamine Synthetase Activities in Type II Diabetic Wistar Rat

Diabetes mellitus has not ceased to be on rise in spite of the continuous research on its management. Brain dysfunction associated with Diabetes mellitus especially Type II has been the great concern. The aim of this study was to investigate the effect of insulin sensitizing drug metformin and ethanolic extract of garlic on membrane bound enzymes Na+/K+ ATPase, Ca2+ ATPase and glutamate-glutamine cycle enzyme, Glutamine Synthetase activities in the hippocampus of streptozotocin-Nicotinamide induced Type II Diabetic rats. Twenty four male wistar rats weighted 120 - 150 g were used and divided into four groups with six rats in each group. Group A was non-diabetic (Control) and Groups B, C and D were diabetic. Group B received no treatment (DNT) while Groups C and D were treated with 1000 mg/kg of ethanolic garlic extract (EGE) and 50 mg/kg of metformin (MET) respectively orally for three weeks. All the groups were fed on standard rat chow with water ad libitum. Blood glucose was monitored weekly. Animals were sacrificed and the brains were removed and hippocampi were carefully excised and homogenate were obtained. Homogenate was analyzed for Na+/K+ ATPase, Ca2+ ATPase and Glutamine Synthetase (GS) activities. MET and EGE significantly reduced the blood glucose levels. There was a significant increase in the activities of hippocampal Na+/K+ ATPase, Ca2+ ATPase and GS in MET and EGE when compared to DNT. The results suggest that both MET and EGE increase the activities of hippocampal Na+/K+ ATPase, Ca2+ ATPase and GS which were reduced by diabetes mellitus, thus garlic and metformin administration exhibiting neuroprotective effect during hippocampal-related disorders associated with diabetes mellitus.

Optimization of crude enzyme preparation methods for analysis of glutamine synthetase activity in phytoplank-ton and field samples

Glutamine synthetase （GS） is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation methods. In this study, three enzyme preparation methods, high-speed centrifugation （HC, 〈10 000 g）, ultracentrifugation （UC, 70000 g）, and ultrafiltration （UF） with 100 kit molecular weight cutoff, were compared using two diatom species （Asterionellopsis glacialis and Thalassiosira weissflogii）, and two dinoflagellate species （Alexandriura catenella and Prorocentrum donghaiense） as experimental materials together with field samples collected from Xiamen Harbor, China. The results showed that HC is the best method to prepare crude enzymes for glutamine synthetase activity （GSA） in diatom species and diatom-dominant samples, while UF is the best method to extract GS from dinoflagellate species and dinoflagellate-dominant samples. For the HC method, the optimal centrifugal speed and time were 10 000 g and 35 min, respectively, and under these conditions, the highest GSA was obtained in all samples. This study indicates that both methods （HC and UF） overcome the limitation of centrifugal speed and could be applied to in situ GSA analysis, especially at sea.

Interaction of Glufosinate and <i>Colletotrichum truncatum</i>on Ammonia Levels and Glutamine Synthetase Activity in Hemp Sesbania

The use of microbes and microbial products as bioherbicides has been studied for several decades, and combinations of bioherbicides and herbicides have been examined to discover possible synergistic interactions to improve weed control efficacy. Bioassays were conducted to assess possible interactions of the herbicide glufosinate [2-amino-4-(hydroxymethylphosphinyl) butanoic acid] and Colletotrichum truncatum (CT), a fungal bioherbicide to control hemp sesbania (Sesbania exaltata)]. Glufosinate acts as a glutamine synthetase (GS) inhibitor that causes elevated ammonia levels, but the mode of action of CT is unknown. GS has also been implicated in plant defense in certain plant-pathogen interactions. The effects of spray applications of glufosinate (1.0 mM) orbioherbicide (8.0 × 104 conidia ml-1), applied alone or in combination were monitored (88 h time-course) on seedling growth, GS activity and ammonia levels in hypocotyl tissues under controlled environmental conditions. Growth (elongation and fresh weight) and extractable GS activity were inhibited in tissues by glufosinate and glufosinate plus CT treatments as early as 16 h, but CT treatment did not cause substantial growth reduction or GS inhibition until after ~40 h. Generally, ammonia levels in hemp sesbania tissues under these various treatments were inversely correlated with GS activity. Localization of hemp sesbania GS activity on electrophoretic gels indicated a lack of activity after 30 h in glufosinate and glufosinate plus CT-treated tissue. Untreated control tissues contained much lower ammonia levels at 24, 64, and 88 h after treatment than treatments with CT, glufosinate or their combination. CT alone caused elevated ammonia levels only after 64 - 88 h. Glufosinate incorporated in agar at 0.25 mM to 2.0 mM, caused a 10% - 45% reduction of CT colony radial growth, compared to fungal growth on agar without glufosinate, and the herbicide also inhibited sporulation of CT. Although no synergistic interactions were found in the combinations of CT and glufosinate at the concentrations used, further insight on the biochemical action of CT and its interactions with this herbicide on hemp sesbania was achieved.

Immunohistochemical Analysis of Citrulline-Nitric Oxide Cycle Enzymes and Glutamine Synthetase in Different Regions of Brain in Epilepsy Rat Model

The aim of this study was to determine the immunoreactivity of neuronal and inducible nitric oxide synthetase, argininosuccinate synthetase, argininosuccinate lyase, glutamine synthetase in different regions of brain in rats of kainic acid mediated epilepsy. Male Sprague-Dawley rats were used in this study. The acute group animals were sacrificed after 2 hours and the chronic group animals were sacrificed after 5 days of a single subcutaneous injection of kainic acid (15 mg/kg body weight). The cerebral cortex, cerebellum and brain stem slices were fixed and immunohistostained for the above enzymes. Images were captured and analyzed. In acute group, argininosuccinate synthetase and inducible nitric oxide synthetase were increased in cerebral cortex and cerebellum, neuronal nitric oxide synthetase increased in cerebral cortex and brain stem, and there was no change in argininosuccinate lyase immunoreactivity compared to control group. In chronic group, glutamine synthetase was decreased and all other enzymes immunoreactivity was increased in all the brain regions tested. This study demonstrated the up-regulation of citrul-line-nitric oxide cycle enzymes and may contribute to enhancing recycling of citrulline to arginine to support the increased production of nitric oxide in epilepsy. The decreased glutamine synthetase may increase glutamate in chronic epilepsy and may lead to neurodegeneration.

大豆GS基因家族全基因组鉴定及胁迫响应分析

【目的】谷氨酰胺合成酶(glutamine synthetase,GS)是氮代谢“GS-GOGAT循环”中的关键酶,研究GS在大豆中的家族成员以及对外界胁迫的响应情况。【方法】利用生物信息学方法,在大豆中全面鉴定GS基因,明确大豆GmGSs基因的位置与结构、蛋白的理化性质以及组织表达模式等,并对非生物胁迫的应答响应进行研究。【结果】从大豆中共鉴定出8个GS基因,位于8条染色体上,对应编码的氨基酸序列长度为356-432 aa。在10个保守基序中,8个GmGS都包含9个保守基序,GmGS7和GmGS8比其他成员多一个motif10保守基序。启动子顺式作用元件分析表明,GmGSs的启动子中包含丰富的光响应元件、激素响应元件和逆境胁迫响应元件。转录组数据分析表明GmGSs基因在所有组织中均有表达,其中GmGS3和GmGS4在各组织中表达量较高。荧光定量qPCR结果表明:在不同浓度的氯化铵处理后,大豆GS家族中GmGS4、GmGS5和GmGS7对低浓度铵盐处理后期响应最显著。且高盐胁迫处理后,GmGS5在根、茎、叶组织中表达量下降;GmGS7在根、茎组织中表达量上升。【结论】GS在大豆中共有8个成员,其中GmGS7在氯化铵处理和盐胁迫中均参与响应。

HBV相关肝细胞癌组织GLAST、GS蛋白表达及其与切除术后早期复发转移的关系

目的:检测乙型肝炎病毒(HBV)相关肝细胞癌组织兴奋性氨基酸转运体1(GLAST)、谷氨酰胺合成酶(GS)蛋白表达,并分析其与切除术后早期复发转移的关系。方法:选取本院2020年3月至2022年5月收治的125例实施切除术的HBV相关肝细胞癌患者,采用免疫组化法检测癌组织和切缘正常组织GLAST、GS蛋白表达。比较癌组织与切缘正常组织GLAST、GS蛋白阳性率;比较不同临床病理特征患者癌组织GLAST、GS蛋白阳性率;随访1年,Cox回归分析复发转移的影响因素。结果:癌组织GLAST、GS蛋白阳性率分别为28.57%、73.95%,前者低于切缘正常组织的57.98%,后者高于切缘正常组织的33.61%(P<0.05);HBV感染病程>22年患者癌组织GLAST蛋白阳性率均低于<10年、10~22年患者(P<0.05);肿瘤淋巴结转移(TNM)Ⅲ~Ⅳ期、有门静脉癌栓、未/低分化患者癌组织GLAST蛋白阳性率低于TNMⅠ~Ⅱ期、无门静脉癌栓、中/高分化患者(P<0.05),GS蛋白阳性率趋势相反;术后早期复发转移发生率为21.01%;年龄(HR=1.471,95%CI 1.086~1.993)、伴肝硬化(HR=1.728,95%CI 1.110~2.691)、GLAST蛋白阳性表达(HR=0.451,95%CI 0.224~0.910)、GS蛋白阳性表达(HR=2.255,95%CI 1.027~4.948)均是患者术后早期复发转移的影响因素(P<0.05)。结论:HBV相关肝细胞癌组织GLAST蛋白阳性率降低、GS蛋白阳性率升高,且二者均与切除术后早期复发转移有关。

Three novel alleles of OsGS1 developed by base-editing-mediated artificial evolution confer glufosinate tolerance in rice

Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.