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NO合酶（INOS）产生的一氧化氮（NO）在炎症中起重要作用心脏移植排斥反应的反应。 iNOS受到转录的调节，但急性排斥反应中iNOS活性的翻译后调节尚不清楚。假设：翻译后因素改变了INOS的NO产生，并确定急性心脏中NO的下游作用拒绝。方法：刘易斯（等距移植）或wistar-furth（同种异体移植）供体心脏将被移植到刘易斯接收者老鼠。还将使用分离的细胞因子刺激的心肌细胞（CM）。基因表达会由Western blot和RT-PCR确定。 BH4且无合成将由HPLC确定，否分析仪或电子顺磁共振（EPR）光谱。与超氧化物的解偶联通过不同的共同因素合成（包括GTP环溶酶I的过表达），INOS生产二聚化和硝基酪氨酸形成将通过最先进的生物物理和生化检查技术包括：使用蛋白质印迹，免疫组织化学，ELISA，EPR和荧光进行过滤光谱，化学发光。目的＃1：BH4在细胞因子激活中不调节INOS的生产 CM和CM激活后CM。目的＃2：6H4调节Inos的超氧化物产生同种免疫激活后的细胞因子活性CM和CM。目标＃3：NO和超氧化物的变化在同种免疫激活后，细胞因子激活的CM和CM的产生对下游对脂质过氧化，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牛和硝化损伤。公共卫生声明：心脏肌肉细胞的丧失是人类心脏的重大问题可能导致心脏功能不佳的移植物。我们的研究确定了潜在的分子问题心脏细胞的内在细胞易于细胞死亡和损伤。更好地理解该分子过程可能会导致更好的策略来预防这些患者的损伤心脏细胞。