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.

该核心单元的目的是通过使用免疫组织化学和原位杂交在组织中定位蛋白质和mRNA来为研究项目提供互补的分子，细胞，生化和科学支持。通过定量检测一氧化氮合酶（NOS）同工型，通过免疫沉淀和西部免疫印迹；通过使用NO分析仪，使用NO分析仪，使用定量的RNA的定量逆转录 - 聚合酶链反应（RT-PCR）来监测组织中神经元和内皮NOS mRNA的定量变化，例如大脑，颈动脉体等。通过通过组织化学和生化测定测量活性NOS酶；通过通过放射免疫测定和血管紧张素II受体的组织水平测量血浆II的血浆水平；并通过确定血浆样品中的儿茶酚胺水平。 在此PPG中的许多列出项目中，已经记录了控制心血管动力学的血管紧张素II和儿茶酚胺的控制。 但是，涉及这些因素的潜在细胞和分子机制知之甚少。 S.K. Roy博士将作为核心单位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，辐射受体测定，免疫组织化学，西方免疫印迹，原位杂交和RT-PCR。 他将根据需要提出建议和改进。 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。 涉及测量无生产的项目将由威廉·G·梅汉（William G. Mayhan）博士指导。 Kaushik P. Patel博士将指导涉及NOS酶组织化学和儿茶酚胺测量的项目。 该PPG中调查人员的任何一个实验室中的设施无法使用技术专长和所涉及的设备进行复制。 由于这些技术几乎所有PPG调查人员都将使用这些技术，因此核心单元将是满足其需求的理想选择。从而消除了设备和人力的重复重复。