CORE--MOLECULAR BIOLOGY, BIOCHEMISTRY & HISTOLOGY

该核心单元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，为这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（AT1）、超氧化物歧化酶（SOD）、NAD(P)H氧化酶亚基和NR1受体蛋白的免疫荧光定位。我们还将采用免疫沉淀和Western免疫印迹法对nNOS、ecNOS、AT1、NR1、SOD和NAD(P)H氧化酶蛋白的相对水平进行生化检测，并采用半定量逆转录聚合酶链反应扩增（RT-PCR）检测nNOS、ecNOS、AT1、NR1、NR2B、AMPAA、GABAA、GAD、p47和β -actin在神经元（如脑、颈动脉体等）和内皮细胞中的稳态水平变化。我们将为选择性神经组织中AT1受体的放射自显影评估以及通过放射免疫分析法测量血浆血管紧张素II水平提供支持。该核心还将为化学发光法测定NO、NOS、SOD、NADPH氧化酶和柠檬酸合酶活性提供支持。一氧化氮、血管紧张素II和活性氧（ROS）在控制心血管动力学中的作用在所有列出的项目中都得到了充分的证明。然而，涉及这些因素的潜在细胞和分子机制尚不清楚。该核心是PPG的一个组成部分，将为所有项目提供服务。

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.