坐骨神经结扎后大鼠背根神经节和脊髓CGRP表达的变化

目的研究大鼠坐骨神经结扎后降钙素基因相关肽(calcitoningene-relatedpeptide,CGRP)表达变化。方法SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1、3、5、7、14、21和28d(n=8),免疫荧光(双标法)和免疫组织化学(SABC法)观察术后不同时间点CGRP和NGF在坐骨神经、背根神经节(dorsalrootganglion,DRG)和脊髓的表达变化,Westernblot结合图像分析技术对不同时间的变化进行定量测定。结果术后1d结扎远端坐骨神经内NGF大量堆积,持续到28d仍高于正常。结扎后7dDRG内CGRP阳性细胞百分率减少,持续到28d仍低于正常;结扎后14d脊髓后角CGRP下降,28d仍低于正常,各时间点脊髓前角CGRP表达未见明显变化。结论神经结扎可导致DRG和脊髓后角的CGRP表达下调,可能与靶源性的NGF来源减少有关。     

大鼠坐骨神经结扎后疼痛受体P2X3在背根神经节内的表达变化

目的:研究坐骨神经结扎损伤后疼痛受体P2X3在相应背根神经节(dorsal root ganglia,DRG)内的表达变化情况。方法:选取健康成年SD大鼠35只,建立右侧坐骨神经结扎损伤模型,采用免疫组织化学和图像分析技术检测相应L4-6DRG内P2X3的表达情况。结果:正常大鼠L4-6DRG内有大量P2X3免疫阳性神经元,坐骨神经结扎后3d P2X3表达即下调,3,7,14,21和28d其表达呈进行性下降趋势,各时间点与正常和假手术对照比较差异均有统计学意义(P＜0．05)。结论:坐骨神经结扎后P2X3在L4-6DRG内表达明显下调,提示其可能在神经源性疼痛中发挥一定的作用。     

P物质对大鼠分离的DRG细胞GABA激活电流的抑制作用

本文就用全细胞膜片箝技术,在新鲜分离的大鼠ＤＲＧ细胞上证明,在部分细胞Ｐ物质（１０＾－７－１０＾－５ｍｏｌ／Ｌ）可引起浓度依赖性的内向流（４／２６）;在多数细胞虽未检测到ＳＰ引起的膜电流,但却能对ＧＡＢＡＡ受体激活介导的膜内向流产生抑制效应（１８／２２）,并有加速去敏感的作用。本文就有关ＳＰ以ＧＡＢＡ激活电流抑制效应的可能意义进行了讨论。     

刺芹侧耳谷氨酰胺合成酶编码基因克隆和表达分析

谷氨酰胺合成酶（GS）是真菌氮素同化代谢和谷氨酸合成中的关键酶,采用3’RACE和5’RACE实验技术,克隆获得刺芹侧耳（杏鲍菇）谷氨酰胺合成酶编码基因（PE-GS）全长序列,长度为1 271bp,具有4个内含子和5个外显子,编码353个氨基酸残基。系统进化树分析表明,刺芹侧耳PE-GS与糙皮侧耳GS在分子进化关系上相近。通过real time RT-PCR方法对PE-GS基因在刺芹侧耳基质菌丝体和子实体中的表达情况进行了分析,结果表明,刺芹侧耳PE-GS基因在子实体具有较高的表达水平,这暗示刺芹侧耳PE-GS基因在子实体的氮素代谢中可能承担重要功能。     

力复霉素合成与谷氨酰胺合成酶活力的正相关性

本文报道了地中海诺卡氏菌U一32的氮代谢研究的初步结果。U一32的丙氨酸脱氢酶(ADH)和谷氨酰胺合成酶(GS)同化氨途径受培养基中氨浓度的调节。高氨时,GS的活力低,ADH活力高;低氨时,GS话力高,ADH活力低,发酵后期ADH活力近于零。     

甜菜胞质型谷氨酰胺合成酶基因组DNA的克隆

以甜菜叶片为材料,用CTAB法提取基因组DNA.以分段PCR法扩增得到了完整的甜菜胞质型谷氨酰胺合成酶(GS1)基因组DNA.采用RT-PCR法扩增此GS1基因(GS1)的cDNA序列应用于对照.获得了长度为9 606bp的完整的GS1 DNA序列和长度为1 068 bp的GSI cDNA序列.分析GS1基因组DNA序列表明,它包含13个外显子,被12个内含子分隔开.其外显子区与已公布的GS1 mRNA序列的相似性达99.5%.RT-PCR法获得的cDNA序列与已知的GS1 mRNA序列相似性达99.6%.而2次实验中GS1基因组DNA外显子区与GS1 cDNA序列的相似性达99.9%.GenBank登录号为EU370974.     

高糖环境对Mu ller细胞谷氨酸转运体及谷氨酰胺合成酶表达的影响

目的：研究高糖环境对原代培养新生7天SD乳鼠视网膜Muller细胞谷氨酸转运合成系统的影响及其可能机制。方法：新生7天SD乳鼠视网膜Muller细胞原代培养并模拟高糖环境构建乳鼠视网膜muller细胞体外高糖环境模型。处理分为3组：对照组,高糖组,高糖＋白藜芦醇干预组。培养时间为24h,通过westernblot等检测方法,对照观察各组Muller细胞谷氨酸转运体（GLAST）、谷氨酰胺合成酶（GS）的表达情况。结果：模拟高糖环境可以造成新生SD乳鼠视网膜Muller细胞谷氨酸转运体（GLAST）表达的降低（0．225foldVScontrol,P〈0．05）,并导致其表达的谷氨酰胺合成酶（GS）表达水平的显著降低（0．653foldVScontrol,P〈0．05）;而干预药物白藜芦醇作用后可明显逆转新生SD乳鼠Mu ller细胞谷氨酸转运体（GLAST）（1．133foldvSHGgroup,P〈0．05）、谷氨酰胺合成酶（GS）（1．720foldVSHGgroup,P〈0．05）等蛋白的表达水平。结论：模拟高糖环境可以影响视网膜M0ller细胞谷氨酸转运体（GLAST）、谷氨酰胺合成酶的表达,其结局可能导致视神经细胞因谷氨酸堆积而导致的兴奋性毒性,白藜芦醇能提高Mcjller细胞谷氨酸转运体（GLAST）、谷氨酰胺合成酶表达,从而保护视神经细胞。     

利用谷氨酰胺合成酶基因（GS）作扩增标记在CHO细胞中高效表达乙型…

利用谷氨酰胺合成酶基因（ＧＳ）作扩增选择标记,结合ＣＭＶ－ＩＥ启动子,在ＣＨＯ细胞中高效表达乙型肝炎的基因。初筛克卫表达水平ＲＰＨＡ检测为１：６４,经过谷氨酰胺合成酶基因的抑制剂ＭＳＸ的两轮基因扩增。ＨＢｓＡｇ的表达水平ＲＰＨＡ在１：２５６以上。方静置培养收液,ＲＩＡ检测ＨＢｓＡｇ最高产量为９．５μｇ／毫升。表达水平较以前利用ｄｈｆｒ基因扩增选择系统所得到的高表达细胞系Ｂ４３高一倍以上。利用ＧＳ基     

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。     

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。     

Glucocorticoid Stimulation of Glutamine Synthetase Production in Cultured Rat Glioma Cells

Abstract: A sensitive radioisotopic assay has been used to examine the kinetic properties and regulation of biosynthesis of glutamine synthetase in C-6 glioma cultures. The K_m values for glutamate, MgATP, and ammonium ion were 5mM, 14 mM, and 0.042 mM, respectively, when measured at the pH optimum of 7.2. There was an absolute requirement for a divalent metal ion, with 15 mM- Mg²⁺ being the preferred ion at pH 7.2. Activity was completely inhibited after 30 min with 8 mM-L-methionhe sulfoximine. The addition of 1 μM-cortisol to C-6 cultures caused a two to threefold increase in glutamine synthetase specific activity over a 96-h period, while dexamethasone at the same concentration elevated the level some 7-10-fold. This was specific for glucocorticoids, as other steroid hormones or catecholamines did not significantly affect glutamine synthetase specific activity. Cycloheximide (30 μM) or actinomycin D (0.01 μg/ml) blocked the hormone response. The continued presence of hormone was required in order to maintain an elevated enzyme level. The results suggest that glucocorticoids act to induce glutamine synthetase by stimulating new enzyme synthesis.     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.     

Regulation of Neurotransmitter Aspartate Metabolism by Glial Glutamine Synthetase

Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+-dependent, K+-stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium-dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.     

Derepression of the Glutamine Synthetase in Neuroblastoma Cells at Low Concentrations of Glutamine

Abstract: Regulation of the biosynthesis of glutamine synthetase was studied in neuroblastoma cells (Neuro-2A) by use of a recently developed, sensitive radioisotopic assay. The removal of glutamine from the culture medium of these cells for 24 h resulted in a 10-fold increase in glutamine synthetase specific activity (15-fold after 2 weeks) compared with the basal level found in cells grown in the presence of 2 m M glutamine. Following the growth of these cells for 2 weeks in the presence of various concentrations of glutamine, a negative linear correlation was observed between the specific activity of glutamine synthetase (from 1.7 to 0.14 unit/mg) and the concentration of glutamine in the growth medium (from 0.5 to 2 m M ). Cycloheximide or actinomycin D blocked the increase in glutamine synthetase activity observed in the absence of glutamine. These results suggest that the removal of glutamine led to the induction of glutamine synthetase by stimulating new enzyme synthesis. The enzyme was not degraded, but only diluted, by growth upon readdition of glutamine to the medium. The influence of glutamine depletion is also reported for C-6 glioma cells and glial cells in primary cultures.     

Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte-Enriched Cultures from Rat Brain

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.     

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections.     

Valproate Increases Glutaminase and Decreases Glutamine Synthetase Activities in Primary Cultures of Rat Brain Astrocytes

Abstract: It has been proposed that hyperammonemia may be associated with valproate therapy. As astrocytes are the primary site of ammonia detoxification in brain, the effects of valproate on glutamate and glutamine metabolism in astrocytes were studied. It is well established that, because of compartmentation of glutamine synthetase, astrocytes are the site of synthesis of glutamine from glutamate and ammonia. The reverse reaction is catalyzed by the ubiquitous enzyme glutaminase, which is present in both neurons and astrocytes. In astrocytes exposed to 1.2 mM valproate, glutaminase activity increased 80% by day 2 and remained elevated at day 4; glutamine synthetase activity was decreased 30%. Direct addition of valproate to assay tubes with enzyme extracts from untreated astrocytes had significant effects only at concentrations of 10 and 20 mM, When astrocytes were exposed for 4 days to 0.3, 0.6, or 1.2 mM valproate and subsequently incubated with l -[U-¹⁴C]glutamate, label incorporation into [¹⁴C]glutamine was decreased by 11, 25, and 48%, respectively, and is consistent with a reduction in glutamine synthetase activity. Label incorporation from l -[U-¹⁴C]glutamate into [¹⁴C]aspartate also decreased with increasing concentrations of valproate. Following a 4-day exposure to 0.6 mM valproate, the glutamine levels increased 40% and the glutamate levels 100%. These effects were not directly proportional to valproate concentration, because exposure to 1.2 mM valproate resulted in a 15% decrease in glutamine levels and a 25% increase in glutamate levels compared with control cultures. Intracellular aspartate was inversely proportional to all concentrations of extracellular valproate, decreasing 60% with exposure to 1.2 mM valproate. These results indicate that valproate increases glutaminase activity, decreases glutamine synthetase activity, and alters Krebs-cycle activity in astrocytes, suggesting a possible mechanism for hyperammonemia in brain during valproate therapy.