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iNOS Posttranslational Regulation in Cardiac Rejection

心脏排斥反应中 iNOS 的翻译后调节

基本信息

批准号：
7579150
负责人：
GALEN M PIEPER
金额：
$ 36.78万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-15 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7579150
关键词：
3-nitrotyrosine Acute Allografting Arts Biochemical Biological Markers Cardiac Cardiac Myocytes Cell Death Cells Dimerization Electron Spin Resonance Spectroscopy Enzyme-Linked Immunosorbent Assay Filtration Fluorescence GTP Cyclohydrolase I Gene Expression Goals Graft Rejection Heart High Pressure Liquid Chromatography Human Immunohistochemistry In Vitro Inflammatory Response Injury Isogenic transplantation Lead Lipid Peroxidation Modeling Molecular Nitric Oxide Nitric Oxide Synthase Organ Transplantation Oxidative Stress Patients Play Post-Translational Regulation Process Production Proteins Public Health Rattus Regulation Reverse Transcriptase Polymerase Chain Reaction Role Solid Superoxides Techniques Transplantation Up-Regulation Western Blotting cytokine heart allograft heart function human NOS2A protein in vivo nitration overexpression oxidation prevent protein expression tetrahydrobiopterin

项目摘要

Nitric oxide (NO) production from inducible NO synthase (iNOS) plays an important role in the inflammatory response of cardiac transplant rejection. While iNOS is regulated transcriptionally, the role of posttranslational regulation of iNOS activity in acute rejection is unknown. HYPOTHESIS: Posttranslational factors alter the production of NO from iNOS and determine the downstream actions of NO in acute cardiac rejection. METHODS: Lewis (isograft) or Wistar-Furth (allograft) donor hearts will be transplanted into Lewis recipient rats. Isolated cytokine-stimulated cardiac myocytes(CM) will also be used. Gene expression will be determined by Western blot and RT-PCR. BH4 and NO synthesis will be determined by HPLC, NO analyzer or electron paramagnetic resonance (EPR) spectroscopy. Uncoupling of NO vs. superoxide production by varying co-factor synthesis (including GTP cyclohydrolase I overexpression), iNOS dimerization and nitrotyrosine formation will be examined by state-of-the-art biophysical and biochemical techniques including: get filtration with Western blot, immunohistochemistry, ELISA, EPR and fluorescence spectrosocpy, chemiluminescence. AIM#1: BH4 regulates NO production from iNOS in cytokine-activated CM and in CM after alloimmune activation. AIM #2: 6H4 regulates superoxide production from iNOS in cytokine-actived CM and in CM after alloimmune activation. AIM #3: Alteration in NO and superoxide production in cytokine-activated CM and in CM after alloimmune activation has downstream effects on biological markers of lipid peroxidation, protein oxidation and protein nitration. Findings from this study may provide unique strategies to protect against oxidate and nitrative injury in acute cardiac rejection. Public Health Statement: The loss of cardiac muscle cells is a significant problem in human cardiac transplants that may contribute to poor heart function. Our studies identify a potential molecular problem intrinsic to cardiac cells that predisposes to cell death and injury. A better understanding of this molecular process may lead to better strategies to prevent injury cardiac cells in these patients.

诱导型一氧化氮合酶 (iNOS) 产生的一氧化氮 (NO) 在炎症反应中发挥着重要作用心脏移植排斥反应。虽然 iNOS 是通过转录调节的，但急性排斥反应中 iNOS 活性的翻译后调节尚不清楚。假设：翻译后因素改变 iNOS 产生 NO 并决定急性心脏病中 NO 的下游作用拒绝。方法：Lewis（同种移植）或 Wistar-Furth（同种异体移植）供体心脏将被移植到 Lewis 体内受体大鼠。还将使用分离的细胞因子刺激的心肌细胞（CM）。基因表达将通过Western blot和RT-PCR测定。 BH4和NO合成将通过HPLC测定，NO 分析仪或电子顺磁共振（EPR）光谱。 NO 与超氧化物的解偶联通过不同的辅因子合成（包括 GTP 环化水解酶 I 过表达）、iNOS 产生二聚化和硝基酪氨酸的形成将通过最先进的生物物理和生物化学进行检查技术包括：通过蛋白质印迹、免疫组织化学、ELISA、EPR 和荧光进行过滤分光光度法、化学发光法。 AIM#1：BH4 调节细胞因子激活的 iNOS 中 NO 的产生 CM 和同种免疫激活后的 CM 中。目标#2：6H4 调节 iNOS 中超氧化物的产生细胞因子激活的 CM 和同种免疫激活后的 CM。目标#3：一氧化氮和超氧化物的改变细胞因子激活的 CM 和同种免疫激活后的 CM 中的产生具有下游影响脂质过氧化、蛋白质氧化和蛋白质硝化的生物标志物。这项研究的结果可能提供独特的策略来防止急性心脏排斥反应中的氧化和硝化损伤。公共卫生声明：心肌细胞的损失是人类心脏的一个重大问题可能导致心脏功能不良的移植。我们的研究发现了潜在的分子问题心肌细胞固有的，易导致细胞死亡和损伤。更好地了解该分子过程可能会导致更好的策略来防止这些患者的心肌细胞损伤。