ARGININE TRANSPORT AND NITRIC OXIDE PRODUCTION

精氨酸转运和一氧化氮产生

• 批准号: 2101977
• 负责人: JAMES CUNNINGHAM
• 金额: $ 12.93万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2101977
• 关键词: Xenopus oocyte; aminoacid transport; antisense nucleic acid; arginine; cellular immunity; complementary DNA; complementary RNA; gene deletion mutation; gene expression; genetic regulatory element; genetic transcription; interferon gamma; interleukin 1; intracellular; laboratory mouse; laboratory rat; leukocyte activation /transformation; leukocyte oxidative burst; lipopolysaccharides; macrophage; membrane transport proteins; molecular cloning; nitric oxide; nucleic acid sequence; protein structure function; structural genes; tissue /cell culture; vascular smooth muscle

Activated macrophages demonstrate arginine-dependent cytotoxicity for tumor cells and intracellular pathogens mediated by the free radical, nitric oxide (NO). These cells increase arginine uptake, the substrate for synthesis of NO, when activated by cytokines. Preliminary studies have identified coordinate expression of CAT-2B, a new member of a family of cationic amino acid transporters (CAT), including arginine, and of nitric oxide synthase (NOS) in the gamma interferon (IFN-gamma) and lipopolysaccharide (LPS)-activated macrophage cell line, RAW 264.7. This proposal summarizes experiments designed to investigate the importance of CAT-2B in providing arginine for synthesis of NO in macrophages and other tissues that produce NO in response to cytokines. Specific Aim #1: Examine the structure/function of CAT-2B A. Obtain cDNA encoding CAT-2B from a library of RAW 264.7 cells activated by IFN-gamma/LPS. B. Perform a survey of amino acid uptake in frog oocytes injected with cRNA encoding CAT-2B. C. Compare the properties of CAT-2B to previously identified transporters, CAT-2A and CAT-1. Specific Aim #2: Investigate the dependence of nitric oxide production on CAT-2B expression. A. Correlate CAT-2B and NOS expression induced by cytokines in macrophages and in other tissues. B. Determine if NO production is dependent upon CAT-2B expression in reconstruction experiments performed in oocytes and mammalian cell lines. C. Determine if CAT-2B expression is coordinate with NOS expression or secondary to intracellular arginine depletion. D. Examine the feasibility of deleting the CAT-2B-specific exon from the mouse genome with the long term goal of determining the susceptibility to infection and tumor formation of a CAT-2B deficient mouse.

活化的巨噬细胞表现出对肿瘤细胞的依赖性细胞毒性， 肿瘤细胞和胞内病原体介导的自由基， 一氧化氮（NO）。 这些细胞增加精氨酸的摄取， 当被细胞因子激活时，用于合成NO。 初步研究 已经鉴定了CAT-2B的协调表达，CAT-2B是一个家族的新成员， 阳离子氨基酸转运蛋白（CAT），包括精氨酸，和 γ干扰素（IFN-γ）中的一氧化氮合酶（NOS）， 脂多糖（LPS）激活的巨噬细胞系，RAW 264.7。 这 提案总结了旨在调查重要性的实验 CAT-2B在巨噬细胞中为NO的合成提供精氨酸， 其他组织响应细胞因子产生NO。 具体目标#1：检查CAT-2B的结构/功能 A.从RAW 264.7细胞文库中获得编码CAT-2B的cDNA 由IFN-γ/LPS激活。 B。对注射了以下物质的青蛙卵母细胞的氨基酸摄取进行调查： 编码CAT-2B的cRNA。 C.将CAT-2B的特性与先前确定的特性进行比较 CAT-2A和CAT-1。 具体目标#2：研究一氧化氮产生的依赖性 CAT-2B的表达。 A.细胞因子诱导的CAT-2B和NOS表达的相关性 巨噬细胞和其他组织中。 B。确定NO的产生是否依赖于CAT-2B的表达， 在卵母细胞和哺乳动物细胞系中进行的重建实验。 C.确定CAT-2B表达是否与NOS表达协调， 继发于细胞内精氨酸耗竭。 D.检查从基因组中删除CAT-2B特异性外显子的可行性。 小鼠基因组，其长期目标是确定 CAT-2B缺陷小鼠的感染和肿瘤形成。