ARGININE SYNTHESIS AND UTILIZATION IN BRAIN

精氨酸在脑中的合成和利用

• 批准号: 2268556
• 负责人: Marian Jackson
• 金额: $ 9.52万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2268556
• 关键词: DNA methylation; amine oxidoreductase; aminoacid biosynthesis; arginine; astrocytes; brain; brain metabolism; chromatin; complementary DNA; cyclic AMP; gene expression; genetic promoter element; genetic regulatory element; genetic transcription; glucocorticoids; growth /development; immunocytochemistry; insulin; kidney; laboratory mouse; laboratory rat; liver; messenger RNA; molecular cloning; neurogenesis; neurons; nitric oxide; nuclear runoff assay; nucleic acid sequence; recombinant DNA; tissue /cell culture; transfection

Argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) are enzymes of the urea cycle whose heritable deficiencies result in severe disease including mental retardation. The combined actions of these two enzymes leads to synthesis of arginine from citrulline + aspartate. While the complete urea cycle is not present in the brain, AS and AL are expressed in brain tissue. The role(s) of arginine in brain metabolism and neurological function are not clear. Nitric oxide synthase (NOS), catalyzes the conversion of arginine to nitric oxide + citrulline. Recent evidence indicates that nitric oxide (NO) is a novel second messenger molecule which is important in signal transduction processes in vascular endothelium and neural tissue. Regulation of nitric oxide production in vascular endothelium is pertinent to understanding cerebrovascular diseases such as stroke and migraine. Glutamate receptor stimulation is coupled to NO production. The synthesis of NO in response to stimulation of N-methyl-D-aspartate receptors may play a role in synaptic plasticity and long term potentiation. It Is our hypothesis that neural AS and AL play critical roles In nitric oxide-dependent processes by converting citrulline back to arginine for further nitric oxide synthesis. The long term goals of this research are to understand the role(s) of arginine metabolism in the brain and the mechanisms by which it is regulated during development and maturation of the nervous system. In the proposed studies we will determine the distribution of AS, AL and NOS in different regions and cell types of mouse brain using a combination of enzyme assays and immunocytochemical localization techniques. These studies will provide correlations of arginine metabolism with brain functions and will indicate whether the three enzymes are present in distinct compartments. AL expression is detected earlier in fetal brain development than is AS. In contrast, AS and AL are coordinately induced in developing liver and kidney. We will use molecular genetic approaches to characterize the molecular mechanisms which lead to discrete localization of AS and AL in different cell types at different times during development. These studies will compare and contrast the chromatin structure for the AS and AL genes in brain, liver and kidney. We will also determine whether AL is transcribed from an alternative, brain-specific promoter in the central nervous system. Glucocorticoids, cAMP and insulin are known to modulate the transcription and expression of AS and AL in liver and kidney. We will determine the effects of these effectors on AL expression in cultured brain cells. These studies will correlate enzyme activity with steady state mRNA levels and will determine whether regulation is exerted at the level of transcription. Ultimately, we will use recombinant minigenes transfected into cultured brain cells to characterize the sequences required for establishing the temporal and spatial patterns of AL expression observed in the brain.

精氨酸琥珀酸合成酶(AS)和精氨酸琥珀酸裂解酶(AL)是 尿素循环中的酶，其遗传缺陷导致严重的 包括智力低下在内的疾病。这两者的共同作用 酶能使瓜氨酸+天冬氨酸合成精氨酸。 虽然大脑中不存在完整的尿素循环，但AS和AL是 在脑组织中表达。精氨酸在脑代谢中的作用(S) 神经功能尚不清楚。一氧化氮合酶(NOS) 催化精氨酸转化为一氧化氮+瓜氨酸。 最近的证据表明，一氧化氮(NO)是一种新的第二种 信使分子在细胞内信号转导过程中的重要作用 血管内皮细胞和神经组织。一氧化氮的调节 血管内皮细胞的产生与理解有关 脑血管疾病，如中风和偏头痛。谷氨酸受体 刺激与不生产结合在一起。作为回应的NO的合成 刺激N-甲基-D-天冬氨酸受体可能在 突触可塑性和长时程增强。这是我们的假设 神经性AS和AL在一氧化氮依赖中的关键作用 将瓜氨酸转化为精氨酸以进一步硝化的过程 氧化物合成。这项研究的长期目标是了解 精氨酸代谢在脑中的作用及其机制(S) 它在神经的发育和成熟过程中受到调节 系统。在拟议的研究中，我们将确定 AS、AL和NOS在不同区域和不同细胞类型的小鼠脑中的表达 酶检测与免疫细胞化学定位相结合 技巧。这些研究将提供精氨酸的相关性。 新陈代谢与大脑功能有关，并将表明这三种 酶存在于不同的隔室中。检测到AL表达 胎脑发育早于AS。相比之下，AS和AL 在肝脏和肾脏的发育中协同诱导。我们将使用 用分子遗传学方法研究分子机制 这导致AS和AL在不同细胞类型中的离散定位 在发育过程中的不同时期。这些研究将比较和 脑、肝AS和AL基因染色质结构的比较 还有肾脏。我们还将确定AL是否从一个 中枢神经系统中大脑特有的替代性启动子。 已知糖皮质激素、cAMP和胰岛素调节转录。 AS和AL在肝、肾中的表达。我们将确定 这些效应物对培养脑细胞AL表达的影响。 这些研究将把酶活性与稳定状态的信使核糖核酸联系起来。 并将决定是否在以下级别实施监管 抄写。最终，我们将使用转基因的重组迷你基因 转化为培养的脑细胞，以确定所需的序列 建立AL表达的时间和空间模式 在大脑里。