CORE--MOLECULAR BIOLOGY, BIOCHEMISTRY AND HISTOLOGY

核心--分子生物学、生物化学和组织学

• 批准号: 6606560
• 负责人: SHYAMAL K. ROY
• 金额: $ 14.25万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606560
• 关键词: biomedical facility; immunocytochemistry; in situ hybridization; molecular biology; nitric oxide synthase; polymerase chain reaction; protein localization; western blottings

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.

该核心单位的目标是为研究项目提供互补的分子、细胞、生物化学和科学支持，方法是利用免疫组织化学和原位杂交定位组织中的蛋白质和mRNA;通过免疫沉淀和Western免疫印迹定量检测一氧化氮合酶亚型;通过使用RNA的定量逆转录-聚合酶链反应扩增（RT-PCR）监测组织如脑、颈动脉体等中神经元和内皮NOS mRNA的定量变化，通过使用NO分析仪定量一氧化氮（NO）的产生;通过组织化学和生物化学测定法测量活性NOS酶;通过放射免疫测定法测量血管紧张素II的血浆水平和血管紧张素II受体的组织水平;以及通过测定血浆样品中的儿茶酚胺水平。 在本PPG的许多列出的项目中记录了NO、血管紧张素II和儿茶酚胺在控制心血管动力学方面的作用。 然而，涉及这些因素的潜在细胞和分子机制知之甚少。Dr. S.K.Roy将作为核心单元的负责人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。 他将在必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于分析我们的结果至关重要。 涉及测量NO产生的项目将由William G.梅翰 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。 所涉及的技术专业知识和设备不能使用本PPG中研究者的任何一个实验室的设施复制。 由于这些技术将被几乎所有的PPG研究者使用，因此核心单元将是满足他们需求的理想选择;从而消除设备和人力的重大重复。