CORE--MOLECULAR BIOLOGY, BIOCHEMISTRY & HISTOLOGY

核心--分子生物学、生物化学

• 批准号: 6928293
• 负责人: SHYAMAL K. ROY
• 金额: $ 22.44万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6928293
• 关键词: biomedical facility; immunocytochemistry; immunofluorescence technique; immunoprecipitation; in situ hybridization; molecular biology; nitric oxide synthase; polymerase chain reaction; protein localization; western blottings; biomedical facility; immunocytochemistry; immunofluorescence technique; immunoprecipitation; 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 four research projects by carrying out a variety of biochemical, molecular biological and histological techniques. These include immunofluorescence localization of nNOS, ecNOS, and angiotensin type 1 receptors (AT1), superoxide dismutase (SOD), subunits of NAD(P)H oxidase and NR1 receptor proteins. We will also provide biochemical determination of the relative levels of nNOS, ecNOS, AT1, NR1, SOD and NAD(P)H oxidase proteins by immunoprecipitation and Western immunoblotting, as well as semi-quantitative reverse transcription-polymerase chain reaction amplification (RT-PCR) examination of changes in the steady-state levels of nNOS, ecNOS, AT1, NR1, NR2B, AMPAA, GABAA, GAD, p47 and beta-actin in neuronal, such as brain, carotid body, etc, and endothelial cells. We will provide support for autoradiographic evaluation of AT1 receptors in selective neuronal tissues, and measurement of plasma levels of angiotensin II by radioimmunoassay. The core will also provide support to the measurement NO by chemiluminescence, and activities of NOS, SOD, NADPH oxidase and Citrate synthase. The role of NO, angiotensin II and reactive oxygen species (ROS) in controlling cardiovascular dynamics has been adequately documented in all the listed projects. However, the underlying cellular and molecular mechanisms involving these factors are poorly understood. This core is an integral component of the PPG and will provide service for all projects. 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 RIA, radioreceptor assay, immunohistochemistry, Western immunoblotting, and RT-PCR. He will make recommendations and improvements of protocols whenever needed. His expertise in these techniques is critical for timely completion of the study and analysis of our results. The technical expertise and the equipment involved cannot be reproduced using facilities in any one of the laboratories of the investigators in the program project. Because almost all PPG investigators will use these techniques, a core unit will be ideal to meet their needs; thus eliminating major duplication in equipment and manpower. In fact, during the current funding period, the molecular and biochemical core has helped every investigators listed in the PPG in their research progress by analyzing samples as well as training post-doctoral, graduate students and technicians of various molecular techniques critical for the success of each project. Therefore, Core C has proved its importance in the PPG and has become an indispensable and integral part of the project. The objectives of the present proposal cannot be reached without the assistance of Core C.

该核心单元的目的是通过执行各种生化，分子生物学和组织学技术来为这四个研究项目提供互补的分子，细胞，生化和科学支持。其中包括NNO，ECNOS和血管紧张素1受体（AT1），超氧化物歧化酶（SOD）的免疫荧光定位，NAD（P）H氧化酶和NR1受体蛋白的亚基。我们还将通过免疫沉淀和西方免疫印迹，以及半质量逆转录 - 转录 - - 转录 - 链链反应放大器（RT-PCR）在NN中的稳定级别，NNOS，ecnos，ecnos，ecnos，ecnos，ecnos，ecnos，ecnos，ecnos，at1，nr1和nad（p）h氧化酶蛋白的相对水平确定NNOS级别的稳定级别，均具有稳定级别的级别，并将其半定量逆转录 - 转录 - 氧化NR2B，AMPAA，GABAA，GAD，P47和β-肌动蛋白在神经元中，例如脑，颈动脉体等和内皮细胞。我们将为选择性神经元组织中AT1受体的放射自显影评估，并通过放射免疫测定法测量血管素II的血浆水平。核心还将通过化学发光和NOS，SOD，NADPH氧化酶和柠檬酸盐合酶的活性为测量NO提供支持。 NO，血管紧张素II和活性氧（ROS）在控制心血管动力学中的作用已在所有列出的项目中充分记录。但是，涉及这些因素的潜在细胞和分子机制知之甚少。该核心是PPG不可或缺的组成部分，将为所有项目提供服务。 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RIA，辐射受体测定，免疫组织化学，西方 免疫印迹和RT-PCR。他将提出建议和改进协议 只要需要。他在这些技术方面的专业知识对于及时完成研究和我们结果的分析至关重要。技术专长和所涉及的设备不能使用计划项目中调查人员的任何一个实验室中的设施复制。由于几乎所有PPG调查人员都会使用这些技术，因此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的重复重复。实际上，在当前的资助期间，分子和生化核心通过分析样本以及培训博士后，研究生和各种分子技术的技术人员，帮助每个项目的研究进度中列出了PPG中列出的每个研究人员，这对每个项目的成功至关重要。因此，Core C已证明其在PPG中的重要性，并已成为该项目必不可少的组成部分。没有CoreC的协助，将无法达成本提案的目标。