Molecular Biology, Biochemistry and Histology Core

这个核心单位的目标是提供互补的分子，细胞，生物化学和科学 通过开展各种生物化学、分子生物学和生物技术研究， 组织学技术。这些包括各种蛋白质的免疫荧光定位， 仅限于nNOS、ecNOS和血管紧张素1型受体（ATiR）、超氧化物歧化酶（SOD）、 NAD（P）H氧化酶和NRI受体蛋白、ACE、ACE2和转录因子。我们还将提供 生化测定nNOS、ecNOS、ATi、NRi、SOD和NAD（P）H氧化酶的相对水平 通过免疫沉淀和Western免疫印迹以及定量真实的时间反转 转录-聚合酶链反应扩增（RT-PCR）检测稳态变化 各种组织中nNOS、ecNOS、ATiR、NRi、NR2B、AMPAA、GABAA、GAD、p47和α-肌动蛋白的水平， 该核心将提供血管紧张素II（Ang II）的血浆水平的测量， 放射免疫测定法使用核心中建立的方案。我们还将提供测量服务， 血管紧张素1 - 7肽，因为其在调节血压中的新兴作用。该测量将是 与布丽奇特·布罗斯尼汉博士（维克森林大学;见信）合作完成， 建立了协议，并同意帮助我们进行测量。核心会收集并处理等离子体 并将样本送至布罗斯尼汉博士进行分析我们认为这是最有效的方法 以获得Ang（1 - 7）的精确测量而无需显著投资。核心还将提供 支持NOS、SOD、NADPH氧化酶和柠檬酸合成酶活性的测定。核心 包括马修·C博士齐默尔曼作为合作研究员，帮助进行活性氧（ROS）分析 因为他在这方面的专业知识。一氧化氮、血管紧张素Ⅱ和活性氧在心血管疾病中的作用 所有所列项目都充分记录了动态。然而，底层的细胞和 涉及这些因素的分子机制知之甚少。该核心是 PPG将为所有项目提供服务。 博士沙伊马尔湾罗伊将积极参与，作为核心单位的主任，在设计实验 用于各种方案，其使用RIA、放射受体测定、免疫组织化学。Western免疫印迹， 和真实的RT-PCR。他将在必要时提出建议和改进方案。他 这些技术的专门知识对于及时完成研究和分析我们的结果至关重要。的 所涉技术专门知识和设备不能使用任何一个 研究人员在项目中的实验室。因为几乎所有的PPG研究人员都会使用这些 技术，一个核心单位将是理想的，以满足他们的需要，从而消除重大重复的设备和 人手齐默尔曼博士和布罗斯尼汉博士的加入加强了核心， 在拟议的研究领域的前沿问题。事实上，在目前的资助期间， 分子和生物化学核心通过分析，帮助PPG中列出的每一位研究人员进行研究 样品以及培养博士后研究员，研究生和技术人员的各种分子 生物技术对每个项目的成功至关重要。因此，Core C已经证明了它在 PPG已成为该项目不可或缺的组成部分。本提案的目标 如果没有核心C的帮助，就无法实现。

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 various proteins including but not limited to nNOS, ecNOS, and angiotensin type 1 receptors (ATiR), superoxide dismutase (SOD), subunits of NAD(P)H oxidase and NRI receptor proteins, ACE, ACE2 and transcription factors. We will also provide biochemical determination of the relative levels of nNOS, ecNOS, ATi, NRi, SOD and NAD(P)H oxidase proteins by immunoprecipitation and Western immunoblotting, as well as quantitative real time reverse transcription-polymerase chain reaction amplification (RT-PCR) examination oif changes in the steady-state levels of nNOS, ecNOS, ATiR, NRi, NR2B, AMPAA, GABAA, GAD, p47 and a-actin in various tissues such as brain, carotid body, etc. The core will provide measurement of plasma levels of Angiotensin II (Ang II) by radioimmunoassay using a protocol established in the core. We will also provide measurement service for angiotensin 1-7 peptide because of its emerging role in regulating blood pressure. This measurement will be done for a fee in collaboration with Dr. Bridget Brosnihan (Wake Forest University; see letter) who has an established protocol, and agreed to help us on this measurement. The core will collect and process the plasma as instructed and send the sample to Dr. Brosnihan for analysis. We believe that this is the most efficient way to obtain accurate measurements of Ang (1-7) without significant investment. The core will also provide support for the measurement of activities of NOS, SOD, NADPH oxidase and citrate synthase. The core has included Dr. Matthew C. Zimmerman as a co-investigator to help in reactive oxygen species (ROS) analysis because of his specific expertise in this area. The role of NO, Ang II and ROS in controlling cardiovascular dynamics has been adequately documented in all the listed projects. However, the underiying cellular and molecular mechanisms involving these factors are pooriy understood. This core is an integral component of the PPG and will provide service for all projects. Dr. Shaymal K. Roy will be actively involved, as the Director of the core unit, in designing experiments for various protocols, which use RIA, radioreceptor assay, immunohistochemistry. Western immunoblotting, and real time RT-PCR. He will make recommendations and improvements of protocols whenever needed. His expertise in these techniques is critical for the 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. The core has been strengthened by the inclusion of Drs. Zimmerman and Brosnihan to address the cutting-edge questions in the proposed area of research. In fact, during the current funding period, the molecular and biochemical core has helped every investigator listed in the PPG in their research by analyzing samples as well as training post-doctoral fellows, graduate students and technicians of various molecular biology 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.