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.
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