CORE--MOLECULAR BIOLOGY, BIOCHEMISTRY AND HISTOLOGY

该核心单元的目标是通过使用免疫组织化学和原位杂交技术定位组织中的蛋白质和mRNA，为研究项目提供互补的分子，细胞，生化和科学支持；免疫沉淀法和免疫印迹法定量检测一氧化氮合酶（NOS）亚型；利用RNA定量逆转录-聚合酶链式反应扩增（RT-PCR）技术监测脑、颈动脉体等组织中神经元和内皮细胞NOS mRNA的定量变化，利用NO分析仪定量一氧化氮（NO）的产生；采用组织化学和生化法测定NOS活性酶；通过放射免疫法测定血浆血管紧张素II水平和血管紧张素II受体的组织水平；通过测定血浆样本中的儿茶酚胺水平。一氧化氮、血管紧张素II和儿茶酚胺在控制心血管动力学中的作用已在本PPG中列出的许多项目中得到证实。然而，涉及这些因素的潜在细胞和分子机制尚不清楚。S.K.Roy博士将作为核心单元主任积极参与各种方案的实验设计，这些方案使用酶测定、RIA、放射受体测定、免疫组织化学、Western免疫印迹、原位杂交和RT-PCR。他将在需要时提出建议和改进方案。他在这些技术方面的专业知识对分析我们的结果至关重要。涉及测量一氧化氮生产的项目将由威廉·g·梅汉博士指导。涉及NOS酶组织化学和儿茶酚胺测量的项目将由Kaushik P. Patel博士指导。所涉及的技术专长和设备不能使用本PPG任何一个调查人员实验室的设施进行复制。由于这些技术将被几乎所有的PPG研究人员使用，因此一个核心单元将是满足他们需求的理想选择；从而消除了设备和人力上的重大重复。

The goal of this core unit is to provide complementary molecular, cellular, biochemical and scientific support for the research projects by localizing protein and mRNA in tissues using immunohistochemistry and in situ hybridization; by quantitative detection of nitric oxide synthase (NOS) isoforms by immunoprecipitation and Western immunoblotting; by monitoring quantitative changes in neuronal and endothelial NOS mRNA in tissues, such as brain, carotid body, etc. using quantitative reverse transcription-polymerase chain reaction amplification (RT-PCR) of RNA, by quantitating nitric oxide (NO) production using an NO analyzer; by measuring active NOS enzyme by histochemistry and biochemical assay; by measuring the plasma levels of angiontensin II by radioimmunoassay and tissue levels of angiotensin II receptors; and by determining catecholamine levels in plasma samples. The of NO, angiotensin II and catecholamines in controlling cardiovascular dynamics have been documented in many of the listed projects in this PPG. However, the underlying cellular and molecular mechanisms involving these factors are poorly understood. Dr. S.K.Roy will be actively involved, as the director of the core unit, in the experimental design of the various protocols, which use enzyme assays, RIA, radioreceptor assay, Immunohistochemistry, Western immunoblotting, in situ hybridization and RT-PCR. He will make recommendations and improvements of protocols whenever needed. His expertise in these techniques is critical for analysis of our results. The projects that involved measuring NO production will be directed by Dr. William G. Mayhan. Projects involving NOS enzyme histochemistry and catecholamine measurement will be directed by Dr. Kaushik P. Patel. The technical expertise and the equipment involved cannot be reproduced using facilities in any one of the laboratories of the investigators in this PPG. Because these techniques will be used by almost all PPG investigators, a core unit will be ideal to meet their needs; thus eliminating major duplication in equipment and manpower.