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、生化和科学 支持四个研究项目，开展各种生化、分子生物学和 组织学技术。这些包括各种蛋白质的免疫荧光定位，包括但不是 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 NNOS、ecNOS、ATI、NRI、SOD和NAD(P)H氧化酶相对水平的生化测定 通过免疫沉淀和Western免疫印迹以及定量实时逆转 转录-聚合酶链式反应扩增技术检测细胞稳态变化 NNOS、ecNOS、Atir、NRI、NR2B、AMPAA、GABAA、GAD、P47和α-肌动蛋白在不同组织中的水平 脑、颈动脉体等。核心将提供血浆血管紧张素II(Ang II)水平的测量，通过 使用在核心建立的方案的放射免疫分析。我们还将为您提供测量服务 血管紧张素1-7肽因其在调节血压方面的新兴作用而受到人们的关注。这一测量将是 与布里奇特·布罗斯尼汉博士(维克森林大学；见信)合作付费完成的 制定了协议，并同意在这一测量方面帮助我们。核心将收集和处理血浆 按照指示进行，并将样本送往Brosnihan博士进行分析。我们认为，这是最有效的方式 在不需要大量投资的情况下获得准确的Ang(1-7)测量结果。核心还将提供 支持测定一氧化氮合酶、超氧化物歧化酶、NADPH氧化酶和柠檬酸合成酶的活性。核心有 包括马修·C·齐默尔曼博士作为合作研究员，帮助进行活性氧物种(ROS)分析 因为他在这方面的专业知识。NO、Ang II和ROS在心血管调控中的作用 Dynamic在所有列出的项目中都得到了充分的记录。然而，底层的蜂窝和 涉及这些因素的分子机制还知之甚少。此核心是 PPG并将为所有项目提供服务。 Shaymal K.Roy博士将作为核心单位主任积极参与设计实验 用于各种方案，其中使用RIA、放射受体分析、免疫组织化学。蛋白免疫印迹， 和实时荧光RT-PCR。他将在必要时对协议提出建议和改进。他的 这些技术方面的专门知识对于及时完成对我们结果的研究和分析至关重要。这个 技术专长和涉及的设备不能使用以下任何一种设备的设施进行复制 计划项目中的研究人员的实验室。因为几乎所有的PPG调查人员都会使用这些 技术方面，理想的做法是设立一个核心单位，以满足他们的需要；从而消除设备和 人力。包括齐默尔曼博士和布罗尼汉博士在内的核心得到了加强，以解决 拟议研究领域的前沿问题。事实上，在本资助期内， 分子和生化核心通过分析帮助了PPG中列出的每一位研究人员的研究 样品以及培养各类分子博士后、研究生和技术人员 生物技术对每个项目的成功至关重要。因此，核心C已经证明了它在 PPG，并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，是无法到达的。

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.