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(来自应用程序的逐字)：而一氧化氮(NO)是 Me被认为与心脏移植排斥反应有关，其确切机制是 我不明白。我们将研究细胞内的分子机制 过量NO在心脏移植物收缩运动障碍中的作用 包括氧化应激和亚硝化应激的范例。我们将研究 诱导型一氧化氮合酶基因表达的调控 氧化剂敏感型核因子-kB转录因子及其关键候选基因的检测 胞浆和线粒体蛋白是NO的分子靶标。这些蛋白质 包括肌红蛋白和乌头酸酶。这些蛋白质的体外亚硝化反应是 已知可抑制酶活性和02结合，从而干扰 这种对氧气要求很高的器官对氧气的高效利用。 假设：[诱导型一氧化氮合酶来源的一氧化氮针对某些细胞蛋白 心脏移植物内促进心肌收缩的亚硝酸化 功能障碍。此外，活性氧在iNOS基因调控中起着重要作用。 氧化敏感型基因在转录水平上的表达 转录因子(核因子-KB)。]申请者将使用高级、 最先进的分子和生物物理技术，包括：转录 诱导型一氧化氮合酶基因的因子调控(凝胶移位分析；Northern和Western 分析)；原位声学显微测量；亚硝基蛋白质和功能的测量 (电子顺磁共振、电子顺磁共振和免疫沉淀)； 细胞凋亡检测；活性氧定量； 水杨酸捕捉法)；iNOS基因敲除和SOD1或SOD2转基因评估 老鼠。申请人将证明下列同种异体心脏 移植：目标1：诱导型一氧化氮合酶导致铁-亚硝基复合体 同种异体移植中心肌内形成与收缩功能障碍 心脏移植。]；目标2：[某些候选蛋白质是分子 同种异体移植中过量NO亚硝化的靶点。]；目标3： [iNOS基因缺失可阻止亚硝基蛋白的形成并延长移植物 生死存亡。]；目标4：[同种异体移植期间iNOS基因表达 通过激活核因子-kB来调节。]；目标5：[抗氧化剂抑制核因子-kB的激活 核因子-KB、诱导型一氧化氮合酶的诱导、亚硝基复合体的形成和增强 收缩功能。]；目标6：[抗氧化剂转基因过表达抑制 核因子-kB的激活，诱导型一氧化氮合酶的诱导，亚硝基复合体的形成和 在同种异体心脏移植中增强收缩功能。]这些 研究将提供一种新的机制来解释该病的发病机制。 心脏移植排斥反应中的运动障碍和移植物失败。