CORE--MOLECULAR BIOLOGY, BIOCHEMISTRY AND HISTOLOGY

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The goal of this core unit is to provide complementary molecular, cellular, biochemical and scientific support for the research projects by localizing protein and mRNA in tissues using immunohistochemistry and in situ hybridization; by quantitative detection of nitric oxide synthase (NOS) isoforms by immunoprecipitation and Western immunoblotting; by monitoring quantitative changes in neuronal and endothelial NOS mRNA in tissues, such as brain, carotid body, etc. using quantitative reverse transcription-polymerase chain reaction amplification (RT-PCR) of RNA, by quantitating nitric oxide (NO) production using an NO analyzer; by measuring active NOS enzyme by histochemistry and biochemical assay; by measuring the plasma levels of angiontensin II by radioimmunoassay and tissue levels of angiotensin II receptors; and by determining catecholamine levels in plasma samples. The of NO, angiotensin II and catecholamines in controlling cardiovascular dynamics have been documented in many of the listed projects in this PPG. However, the underlying cellular and molecular mechanisms involving these factors are poorly understood. 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 enzyme assays, RIA, radioreceptor assay, Immunohistochemistry, Western immunoblotting, in situ hybridization and RT-PCR. He will make recommendations and improvements of protocols whenever needed. His expertise in these techniques is critical for analysis of our results. The projects that involved measuring NO production will be directed by Dr. William G. Mayhan. Projects involving NOS enzyme histochemistry and catecholamine measurement will be directed by Dr. Kaushik P. Patel. The technical expertise and the equipment involved cannot be reproduced using facilities in any one of the laboratories of the investigators in this PPG. Because these techniques will be used by almost all PPG investigators, a core unit will be ideal to meet their needs; thus eliminating major duplication in equipment and manpower.