MOLECULAR TARGETS OF NITRIC OXIDE IN CARDIAC TRANSPLANTS

心脏移植中一氧化氮的分子靶标

• 批准号: 6537767
• 负责人: GALEN M PIEPER
• 金额: $ 37.5万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537767
• 关键词: apoptosis; electron spin resonance spectroscopy; enzyme induction /repression; gel electrophoresis; gene targeting; genetic transcription; genetically modified animals; heart contraction; heart transplantation; immunoprecipitation; laboratory mouse; laboratory rat; nitric oxide; nitric oxide synthase; nitroso compounds; northern blottings; nuclear factor kappa beta; oxidative stress; superoxide dismutase; transplant rejection; western blottings

DESCRIPTION (Verbatim from the application): While nitric oxide (NO) is believed to contribute to cardiac allograft rejection, me precise mechanism is not understood. We will examine the intracellular molecular mechanisms for the actions of excess NO on cardiac allograft contractile dyskinesis relating to a paradigm which includes both oxidative and nitrosative stress. We will examine the regulation of inducible NO synthase (iNOS) gene expression by the oxidant-sensitive NF-kB transcription factor and examine key candidate cytosolic and mitochondrial proteins as molecular targets of NO. These proteins include myoglobin and aconitase. In vitro nitrosylation of these proteins are known to inhibit enzyme activity and 02 binding, thus, interfering with efficient 02 utilization by this O2-demanding organ. Hypothesis: [NO derived from iNOS targets certain cellular proteins for nitrosylation within cardiac grafts that contribute to myocardial contractile dysfunction. Furthermore, reactive oxygen plays a role in regulating iNOS gene expression at the transcriptional level via activation of the oxidant-sensitive transcription factor (NF-KB).] The applicant will use advanced, state-of-the-art molecular and biophysical techniques including: transcription factor regulation of iNOS gene (gel shift assays; Northern and Western analysis); in situ sonomicrometry; measures of nitrosyl protein and function (EPR, electron paramagnetic resonance spectroscopy and immunoprecipitation); assay for apoptosis; quantitation of reactive oxygen (EPR spin trapping; salicylate trapping); evaluation with iNOS knockout and SOD1 or SOD2 transgenic mice. The applicant will show that following allogeneic cardiac transplantation: Aim #1: [Induction of iNOS leads to iron-nitrosyl complex formation within myocardium and contractile dysfunction during allogeneic cardiac transplantation.]; Aim #2: [Certain candidate proteins are molecular targets of nitrosylation by excess NO in allogeneic transplantation.]; Aim #3: [iNOS gene deletion prevents nitrosyiprotein formation and prolongs graft survival. ]; Aim #4: [iNOS gene expression during allogeneic transplantation is regulated by activation of NF-KB.]; Aim #5: [Antioxidants inhibit activation of NF-KB, induction of iNOS, formation of nitrosyl complexes and enhance contractile function.]; Aim #6: [Antioxidant transgene overexpression inhibits activation of NF-kB, induction of iNOS, formation of nitrosyl complexes and enhances contractile function during allogeneic cardiac transplantion.] These studies will provide a novel mechanism to explain the pathogenesis of dyskinesis and graft failure during cardiac transplant rejection.

描述（逐字化的应用程序）：一氧化氮（否）是 相信有助于心脏同种异体移植的拒绝，我的精确机制是 不理解。我们将检查细胞内分子机制 多余的NO在心脏同种异体移植收缩运动障碍上的作用与A 范式包括氧化和硝化应激。我们将检查 通过调节可诱导的NO合酶（INOS）基因表达的调节 对氧化剂敏感的NF-KB转录因子并检查关键候选者 胞质和线粒体蛋白作为NO的分子靶标。这些蛋白质 包括肌红蛋白和刺刺酶。这些蛋白质的体外亚硝基化是 已知可以抑制酶活性和02结合，因此会干扰 有效的02使用该O2设备器官的利用。 假设：[没有源自INOS的靶向某些细胞蛋白 心脏移植的心脏移植物中的硝基化，导致心肌收缩 功能障碍。此外，活性氧在调节iNOS基因中起作用 通过激活氧化剂敏感的转录水平表达 转录因子（NF-KB）。]申请人将使用高级 最先进的分子和生物物理技术，包括：转录 iNOS基因的因子调节（凝胶转移测定；北部和西部 分析）;原位的体学计；硝基蛋白和功能的测量 （EPR，电子顺磁共振光谱和免疫沉淀）； 凋亡的测定；反应性氧的定量（EPR自旋陷阱； 水杨酸捕集）；用iNOS敲除和SOD1或SOD2转基因评估 老鼠。申请人将表明遵循同种异体心脏 移植：目标＃1：[iNOS的诱导导致亚硝基复合物 同种异体期间心肌和收缩功能障碍内的形成 心脏移植。]；目的＃2：[某些候选蛋白是分子 同种异体移植中非过量NO的硝基化靶标。]； AIM＃3： [iNOS基因缺失可防止亚硝基动蛋白形成并延长移植物 生存。 ];目的＃4：[同种异体移植期间的iNOS基因表达是 由NF-KB的激活调节。];目的＃5：[抗氧化剂抑制激活 NF-KB，iNOS的诱导，硝基基络合物的形成并增强 收缩功能。];目标＃6：[抗氧化剂转基因过表达抑制 NF-KB的激活，iNOS的诱导，硝基基络合物的形成和 在同种异体心脏移植期间增强收缩功能。] 研究将提供一种新的机制来解释 心脏移植排斥期间的运动障碍和移植失败。