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.
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