CORE--MOLECULAR BIOLOGY, BIOCHEMISTRY AND HISTOLOGY

核心--分子生物学、生物化学和组织学

• 批准号: 6457669
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6457669
• 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.

这个核心单位的目标是通过免疫组织化学和原位杂交定位组织中的蛋白质和信使核糖核酸，通过免疫沉淀和免疫印迹定量检测一氧化氮合酶(NOS)亚型，通过定量反转录-聚合酶链式反应(RT-PCR)监测脑、颈动脉体等组织中神经元和内皮一氧化氮合酶mRNA的定量变化，通过使用一氧化氮(NO)分析仪定量产生一氧化氮(NO)，通过组织化学和生化分析测量一氧化氮合酶活性，为研究项目提供补充的分子、细胞、生化和科学支持。通过放射免疫法测定血浆血管紧张素II水平和组织血管紧张素II受体水平，以及通过测定血浆样本中儿茶酚胺水平。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经在本PPG列出的许多项目中得到了证明。然而，涉及这些因素的潜在细胞和分子机制却知之甚少。S.K.Roy博士将作为核心单位的主任，积极参与各种方案的实验设计，这些方案使用酶分析、放射免疫分析、放射受体分析、免疫组织化学、Western免疫印迹、原位杂交和RT-PCR。他将在必要时对协议提出建议和改进。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。不测量产量的项目将由威廉·G·梅汉博士指导。涉及一氧化氮合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调查人员的任何一个实验室的设施复制。由于几乎所有PPG调查员都将使用这些技术，因此最理想的做法是设立一个核心单位来满足他们的需要，从而消除设备和人力方面的重大重复。