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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NO合成酶（iNOS）产生一氧化氮（NO）在炎症反应中起重要作用