水稻种子萌发期间谷氨酰胺合成酶和NADH-谷氨酸合酶的变化

测定了水稻种子不同萌发时期胚乳、胚芽鞘和幼根的谷氨酰胺合成酶（GS）和依赖于NADH的谷氨酸合酶（NADH－GOGAT）活性变化。胚乳和胚芽鞘的GS活性在萌发过程中升高,幼根的GS活性则有所降低。NADH－GOGAT的活性变化趋势与GS相同。Native-PAGE活性染色表明,在萌发阶段的水稻种子胚乳和幼根里,始终只观察到一种GS活性带。但是,在水稻种子萌发3d后,在胚芽鞘中除继续检测到GS1的活性外,还可以观察到GS2的活性。蛋白质印迹显示,水稻种子胚乳中的GS（GSe)和GS1和GSra一样是一种胞质型GS。实验结果提示,这些不同组织中的GS与NADH－GOGAT构成的循环途径也许是水稻种子萌发时氨同化的主要途径。     

GS、E—cadherin和β-catenin在肝细胞癌中的表达及其临床意义

目的探讨谷氨酰胺合成酶（glutamine synthetaseGS）、E-钙粘蛋白（E—cadherin）和β-连环蛋白（β-catenin）在肝细胞癌中的表达及其与临床病理特征和预后的关系。方法采用免疫组织化学Envision法检测182例肝细胞癌和92例癌旁肝组织中GS、E-cadherin和β-catenin的表达情况,并分析其与临床病理特征和预后的关系。结果GS在肝细胞癌阳性表达率为77．5％,明显高于癌旁肝组织（4．3％）,差异显著（P〈0．05）;肝细胞癌E—cadherin和β-catenin异常表达率分别为59．3％和58．8％,亦高于癌旁肝组织（30．4％和26．1％）,差异显著（P〈0．05）。肝细胞癌中GS的表达与TNM分期、转移和术后复发显著相关（P〈0．05）;E—cadherin和β-catenin异常表达与脉管内瘤栓、TNM分期、转移和术后复发显著相关（P〈0．05）。肝细胞癌中GS表达与E-cadherin、β-catenin异常表达正相关。结论肝细胞癌中GS的高表达,与E-cadherin和β-catenin表达的下调,可能是肝细胞癌侵袭和转移的重要机制之一,联合检测GS、E-cadherin和β-catenin可能有助于判断肝细胞癌的恶性程度、转移潜能及预后分析。     

农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究

 谷氨酰胺合成酶（Glutamine synthetase,GS,E.C. 6.3.1.2）是植物氨同化过程中的关键酶,对植物的氮素吸收和代谢起着至关重要的作用。谷氨酰胺合成酶还是除草剂草胺膦(Phosphinothricin　(PPT)或Basta)的靶标酶。前期工作已从我国特有的豌豆(Pisum satium)品种中克隆了细胞质型谷氨酰胺合成酶（GS1）cDNA和叶绿体型谷氨酰胺合成酶（GS2）cDNA。为了验证谷氨酰胺合成酶的功能,构建了同时含有GS1 cDNA和GS2 cDNA的植物表达载体p2GS。以该表达载体通过农杆菌介导法,转化小麦(Triticum aestivum)的未成熟胚愈伤组织,经PPT筛选及分化再生培养,获得了抗PPT的转基因小麦植株41株。PCR和基因组Southern 杂交分析证实了GS1 和GS2基因已经整合到转基因小麦的基因组。用除草剂草胺膦Basta溶液涂抹转p2GS小麦叶片,结果证明GS转基因植株可以抗高达0.3%的 Basta溶液,而对照植株叶片逐渐变黄直至枯死。转基因小麦植株能正常结实。上述实验结果表明：1) GS基因在小麦植株中获得了有效表达,从而赋予小麦植株抗PPT特性;2) GS基因能够作为研究小麦遗传转化的筛选标记基因。     

谷氨酰胺合成酶参与Rhodopseudomonas capsulata固氮活性的氨瞬间调节

Rhodopseudomonas capsulata固氮酶活性对氨的敏感性及谷氨酰胺合成酶(GS)活性的变化在很大程度上受菌龄和氮素营养的影响。对数生长后期,固氮酶活性对氨最敏感,GS也处于高水平。限量氨(0.2mM)培养的菌体,其固氮酶活性的氨敏显著减弱,与谷氨酸(7.5 mM)培养的菌体相比,前者的GS活性较后者低50％左右。来自这两种氮源的GS本身对氨的敏感性也不一样,谷氨酸培养的其敏感性较限量氨培养的为低。此外,GS活性与氨关闭固氮酶活性的程度之间呈正相关。而与关闭的持续时间呈负相关。GS活性被抑制后,氨同化受阻,固氮酶活性的氨敏现象消失,基于上述结果,可以认为活性GS参与氨瞬间凋节光合细菌固氮酶的活性。     

急性氨氮胁迫下大弹涂鱼解毒代谢途径的研究

为探明大弹涂鱼(Boleophthalmus pectinirostris)在氨氮环境适应过程中的氨转化及代谢机制,通过氨氮(8 mmol/L NH4Cl)胁迫的方法对大弹涂鱼进行了72 h急性实验;利用酶活性测定方法检测了氨代谢相关酶:谷氨酰胺合成酶(Glutamine synthetase,GS)活性及血氨浓度;酶联免疫技术检测氨代谢协同转运蛋白:碳酸酐酶(Carbonic anhydrase,CA)、钠氢交换蛋白3(Na+/H+exchanger,NHE3)表达水平;运用qPCR技术测定急性氨氮胁迫下大弹涂鱼氨代谢相关基因:GS、CA15、NHE,以及氨转运蛋白(Ammonium transporter Rh type C-1,Rhcg1)基因mRNA的相对表达变化情况。结果表明:在急性氨氮胁迫下,大弹涂鱼血氨浓度呈先上升(12 h)后下降至平稳状态的变化趋势。肝脏GS基因表达量在12 h和48 h显著上升,酶活性在24 h显著上升。鳃中NHE3蛋白表达水平与GS活性变化趋势相同,而CA蛋白水平分别在胁迫后12 h和48 h显著上升。排氨相关基因CA15,NHE,Rhcg1的表达量在氨氮胁迫下均有不同程度的上调,其中NHE基因最早(24 h)上调,而CA15和Rhcg1在48 h显著上升,表明其可能共同参与离子氨的排泄。研究结果表明,氨氮胁迫下大弹涂鱼主要通过两种途径进行氨代谢:(1)在肝脏GS的作用下合成无毒的谷氨酰胺以避免氨在体内过量积累;(2)在鳃组织CA作用下使CO2质子化提供H+,协同NHE3,Rhcg1蛋白复合体实现氨排泄过程。     

褐飞虱体内类酵母共生菌与氨基酸营养的关系

利用全纯人工饲料饲喂技术,研究了缺失不同氨基酸对高温(35℃)处理后的缺菌褐飞虱Nilaparvata lugens Stål相对生长速度、体内共生菌数量的影响,发现10种必需氨基酸对缺菌褐飞虱生长的影响明显大于10种非必需氨基酸,饲料中必需氨基酸的缺少对褐飞虱（特别是高温处理褐飞虱）体内共生菌数量有一定的刺激作用。分析了缺菌试虫体内氨基酸组成和转氨酶活性的变化规律,发现在摄取的氨基酸营养相同的条件下（用全纯饲料D-97饲养）,高温处理试虫体内蛋白质氨基酸组成无明显变化,而游离氨基酸总量明显上升,且必需氨基酸所占比例显著下降,其中组氨酸（His）、异亮氨酸（Ile）、亮氨酸（Leu）、赖氨酸（Lys）、蛋氨酸（Met）和苯丙氨酸（Phe）摩尔百分含量均显著下降,表明必需氨基酸的相对缺乏可能是体内蛋白质合成受阻的一个重要原因,推测这可能是由于试虫体内共生菌数减少致使所合成的必需氨基酸减少而引起。处理试虫体内谷氨酰胺合成酶（GS）和丙氨酸氨基转移酶（ALT）活性明显提高,天冬氨酸氨基转移酶(AST)活性显著降低,结合游离氨基酸中谷氨酰胺（Gln）显著增多,推测类酵母共生菌可能利用谷氨酰胺等为原料进行必需氨基酸的合成。     

Wnt信号通路GS蛋白在胃癌中的表达和临床意义

目的：研究wnt信号通路的中GS蛋白（谷氨酰胺合成酶）在胃癌组织中的表达,探索其在胃癌发生、发展中的意义。方法：用免疫组织化学法测定胃癌组织（110例）、肠化生组织（30例）、不典型增生组织（20例）及慢性浅表性胃炎组织（60例）中GS蛋白表达。用快速尿素酶法与病理组织切片染色法检测上述各组织中HP感染的情况,并予统计学分析比较其差异。结果：胃癌组织GS高蛋白表达与组织分型、分化程度、淋巴结转移密切相关（P〈0．05）,与肿瘤大小、部位、TNM分期、Borrmann分型、性别、年龄等无明显相关（P〉0．05）。GS表达与HP感染密切相关。结论：GS蛋白高表达同胃癌生物学行为密切相关,在胃癌的发生、发展中起重要作用。     

光对水稻非光合组织谷氨酰胺合成酶同工酶表达的影响

以前的研究表明,高等植物叶绿体谷氨酰胺合成酶（GS2）受光调节,但叶片胞液GS（GS1）和非光合作用组织中的GS很少受光的影响,在本报道中,笔者运用GS活性染色和Western blotting研究了光对非光合作用组织水稻根GS同工酶表达的影响,在阳光的直接照射下以及在室内不同光照强度下,可以很清楚地观察到GSra和GS rb的活性带及其蛋白质带,但是,当用尼龙网档住阳光的直接照射下,GSrb的活性带和蛋白质带消失,当阳光被尼龙网遮挡住后,其光强度仍然比室内光照强度大得多,表明光照强度不是影响GSrb表达的主要因素,当分析生长在暗处以及生长在光／暗转换下的水稻幼苗根GS同工酶变化时,仍然可以观察到GSrb的在,在所有实验条件下,GSra都未发生明显变化,这些结果提示,光对GSrb表达的影响可能是由某些光谱相互作用所产生的未知因素造成的。     

蔗糖对不同氮源培养下水稻根部氨同化相关酶活性的影响

糖、有机酸以及氨基酸影响碳-氮代谢过程中的相关酶的基因表达和活性。将蔗糖分别加入到含有相同氮素浓度的（NH4）2SO4（NH4^＋）或丙氨酸（Ala）作为氮源的营养液中培养水稻,测定幼苗根的谷氨酰胺合成酶（GS）、依赖于NADH的谷氨酸合酶（NADH-GOGAT）、磷酸烯醇式丙酮酸羧化酶（PEPC）和依赖于NADP的异柠檬酸脱氢酶（NADP-ICDH）的活性。结果显示,蔗糖诱导NH4^＋氮源中幼苗根的GS、NADH-GOGAT活性,抑制Ala氮源中幼苗根的这两种酶活性,蔗糖对PEPC和NADP-ICDH活性的影响也不同;未加蔗糖时,以Ala作为氮源的幼苗根的GS、NADH-GOGAT、PEPC和NADP-ICDH的活性明显高于以NH4^＋为氮源时的活性;生物量和蛋白质水平的变化与上述参数的变化基本一致。基于Ala碳骨架的存在,这些结果表明,碳／氮平衡是影响这些酶活性差别表达的主要原因。     

南瓜种子萌发及子叶发育时谷氨酰胺合成酶和其它氨同化酶的变化

在发育的新生组织中 ,来自种子胚乳储存蛋白的降解和氨基酸分解代谢产生的氨由谷氨酰胺合成酶 ( Glutamine synthetase,GS)重新同化 ,生成的谷氨酰胺 ( Gln)被转运到正在生长着的部分。GS是高等植物氮素代谢的关键酶 [1] ,这个酶能同化不同来源的氨。 GS有多种同工酶 ,存在于植物的各种组织和器官中。它们是由一小的同源但分离的核基因家族编码的 [2 3 ] ,这些不同的 GS在植物氮素同化中起着非重叠的作用 [4] ,它们的表达受到环境、发育进程以及组织或细胞类型等许多因素的影响。在大多数已研究过的植物叶片中存在两种 GS,即胞液型GS(…     

A conserved negatively charged amino acid modulates function in human nonmuscle myosin IIA

A myosin surface loop (amino acids 391-404) is postulated to be an important actin binding site. In human beta-cardiac myosin, mutation of arginine-403 to a glutamine or a tryptophan causes hypertrophic cardiomyopathy. There is a phosphorylatable serine or threonine residue present on this loop in some lower eukaryotic myosin class I and myosin class VI molecules. Phosphorylation of the myosin I molecules at this site regulates their enzymatic activity. In almost all other myosins, the homologous residue is either a glutamine or an aspartate, suggesting that a negative charge at this location is important for activity. To study the function of this loop, we have used site-directed mutagenesis and baculovirus expression of a heavy meromyosin- (HMM-) like fragment of human nonmuscle myosin IIA. An R393Q mutation (equivalent to the R403Q mutation in human beta-cardiac muscle myosin) has essentially no effect on the actin-activated MgATPase or in vitro motility of the expressed HMM-like fragment. Three mutations, D399K, D399A, and a deletion mutation that removes residues 393-402, all decrease both the V(max) of the actin-activated MgATPase by 8-10-fold and the rate of in vitro motility by a factor of 2-3. The K(ATPase) of the actin-activated MgATPase activity and the affinity constant for binding of HMM to actin in the presence of ADP are affected by less than a factor of 2. These data support an important role for the negative charge at this location but show that it is not critical to enzymatic activity.     

Human cutaneous melanoma expresses a significant phosphate-dependent glutaminase activity: a comparison with the surrounding skin of the same patient

The protein content and the activity and type of phosphate-dependent glutaminase were determined in freshly pigmented lesions obtained from human melanoma and adjacent skin. Significant phosphate-dependent glutaminase activity was found in both the melanoma and non-pigmented adjacent skin areas. A comparison between the pigmented and adjacent skin areas suggests the occurrence of gradual metabolic changes that result in an increased protein content in the centre of the neoplasia. The presence of a kidney-type glutaminase (K(m) of 2-5 mm) indicates a high sensitivity of the melanoma to variations in glutamine plasma levels (0.6 to 1 mm). These data lead us to postulate that glutamine supply is an important factor for melanoma cell proliferation, being a source of nitrogen for DNA and RNA synthesis. The intense neovascularization observed in melanoma ensures the oxygen supply that is required for glutamine oxidation. These findings support the proposition that glutamine is an important fuel for melanoma.     

Identification of the promoter elements involved in the stimulation of ASCT2 expression by glutamine availability in HepG2 cells and the probable involvement of FXR/RXR dimers

Expression of the glutamine transport protein ASCT2 in the human hepatoma cell line HepG2 is increased when cells are cultured in the presence of glutamine and this has been shown to be due to stimulation of the ASCT2 promoter. Analysis of a number of promoter constructs localised the activation site to be between bases -653 and -543. Gel shift assays identified an IR-1 repeat within a 24bp region of this sequence which bound at least two nuclear proteins. Protein binding to this site was significantly higher in cells grown in glutamine-containing medium than when glutamine was absent. The identity of the higher molecular weight species binding to this promoter element was likely to be FXR/RXR dimers. Simultaneous overexpression of FXR and RXR increased the promoter activity in cells grown without glutamine to the same extent as did glutamine addition; the effects of glutamine and FXR/RXR expression were not additive. Mutagenesis of the FXR/RXR binding site in the promoter construct abolished glutamine and FXR/RXR stimulation. Real-time PCR showed levels of FXR mRNA were significantly increased in response to glutamine. The activity of the FXR promoter was also increased in response to glutamine. These results show that the stimulation of ASCT2 expression in response to glutamine in part involves binding of FXR/RXR to the ASCT2 promoter.     

The NF-κB member p65 controls glutamine metabolism through miR-23a

Cancer cells have elevated aerobic glycolysis that is termed the Warburg effect. But several tumor cells, including leukemic cells, also increase glutamine metabolism, which is initiated by glutaminase (GLS). The microRNA (miRNA) miR-23 targets GLS mRNA and inhibits expression of GLS protein. Here we show that in human leukemic Jurkat cells the NF-κB p65 subunit binds to miR-23a promoter and inhibits miR-23a expression. Histone deacetylase (HDAC) inhibitors release p65-induced inhibition. Jurkat cells growing in glutamine decrease proliferation due to cell accumulation in G0/G1 phase. Nevertheless, cells get used to this new source of energy by increasing GLS expression, which correlates with an increase in p65 expression and its translocation to the nucleus, leading to a higher basal NF-κB activity. Jurkat cells overexpressing p65 show increase basal GLS expression and proliferate faster than control cells in glutamine medium. Overexpressing miR-23a in leukemic cells impaired glutamine use and induces mitochondrial dysfunction leading to cell death. Therefore, p65 activation decreases miR-23a expression, which facilitates glutamine consumption allowing leukemic cells to use this alternative source of carbon and favoring their adaptation to the metabolic environment.     

Expression of four glutamine synthetase genes in the early stages of development of rainbow trout (Oncorhynchus mykiss) in relationship to nitrogen excretion

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways.     

Characterisation of G418-induced metabolic load in recombinant CHO and BHK cells: effect on the activity and expression of central metabolic enzymes

In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo ^r protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transglutaminase-2 differently regulates cartilage destruction and osteophyte formation in a surgical model of osteoarthritis

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodeling. Transglutaminases catalyze a calcium-dependent transamidation reaction that produces covalent cross-linking of available substrate glutamine residues and modifies the extracellular matrix. Increased transglutaminases-mediated activity is reported in osteoarthritis, but the relative contribution of transglutaminases-2 (TG2) is uncertain. We describe TG2 expression in human femoral osteoarthritis and in wild-type and homozygous TG2 knockout mice after surgically-induced knee joint instability. Increased TG2 levels were observed in human and wild-type murine osteoarthritic cartilage compared to the respective controls. Histomorphometrical but not X-ray investigation documented in osteoarthritic TG2 knockout mice reduced cartilage destruction and an increased osteophyte formation compared to wild-type mice. These differences were associated with increased TGFβ-1 expression. In addition to confirming its important role in osteoarthritis development, our results demonstrated that TG2 expression differently influences cartilage destruction and bone remodeling, suggesting new targeted TG2-related therapeutic strategies.     

Induction of glutamine synthetase by 8-bromo cyclic AMP in primary cultures of rat brain astrocytes

Glutamine synthetase (GS) is the key enzyme in cerebral glutamine production. Understanding the regulation of the expression of GS is important for definition of the control of glutamine metabolism in brain. Therefore, we studied the control of GS expression by 8-bromo cyclic AMP in primary cultures of astrocytes prepared from brains of neonatal rats. GS activity was increased by 8-bromo cyclic AMP in a dose- and time-dependent manner. This increase was associated with a corresponding increase in the steady-state level of GS mRNA.