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

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.

该核心单位的目标是通过开展各种生物化学、分子生物学和组织学技术，为四个研究项目提供互补的分子、细胞、生物化学和科学支持。这些包括nNOS、ecNOS和血管紧张素1型受体（AT 1）、超氧化物歧化酶（SOD）、NAD（P）H氧化酶亚基和NR 1受体蛋白的免疫荧光定位。我们还将通过免疫沉淀和Western免疫印迹提供nNOS、ecNOS、AT 1、NR 1、SOD和NAD（P）H氧化酶蛋白相对水平的生化测定，以及半定量逆转录-聚合酶链反应扩增（RT-PCR）检查神经元中nNOS、ecNOS、AT 1、NR 1、NR 2B、AMPAA、GABAA、GAD、p47和β-肌动蛋白稳态水平的变化，如脑、颈动脉体等，以及内皮细胞。我们将为选择性神经元组织中AT 1受体的放射自显影评价和放射免疫法测定血浆血管紧张素II水平提供支持。该核心还将为化学发光法测定NO以及NOS、SOD、NADPH氧化酶和柠檬酸合酶的活性提供支持。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得到充分证明。然而，涉及这些因素的潜在细胞和分子机制知之甚少。这一核心是项目编制小组的一个组成部分，将为所有项目提供服务。 博士S. K. Roy将作为核心单元的主任，积极参与各种方案的实验设计，这些方案使用RIA、放射受体测定、免疫组织化学、Western blot、免疫组织化学和放射免疫分析。 免疫印迹和RT-PCR。他将对协议提出建议和改进 无论何时需要。他在这些技术方面的专业知识对于及时完成研究和分析我们的结果至关重要。所涉及的技术专长和设备不能使用项目研究人员的任何一个实验室的设施进行复制。由于几乎所有PPG研究人员都将使用这些技术，因此核心单位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。事实上，在目前的资助期间，分子和生物化学核心通过分析样本以及培训对每个项目成功至关重要的各种分子技术的博士后，研究生和技术人员，帮助PPG中列出的每一位研究人员进行研究进展。因此，Core C已经证明了它在PPG中的重要性，并已成为该项目不可或缺的组成部分。如果没有核心C的协助，本提案的目标就无法实现。