Tissue Culture and Molecular Biology

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Numerous epidemiological studies indicate that moderate alcohol and red wine consumption reduces the risk for coronary heart disease and the mortality associated with myocardial infarction. Our preliminary studies have demonstrated that the cardioprotective effects of alcoholic beverages result from both alcohol and non-alcoholic components (red wine polyphenols). However, the exact molecular mechanisms of how alcohol and polyphenols result in cardioprotection have not been fully elucidated. The overall programmatic theme of this Program Project Grant application is to further identify/define the molecular regulatory mechanisms that underlie this low ethanol- or polyphenol-induced cardioprotection. The overall goal of Tissue Culture and Plasmid Preparation Core Unit (Core B) is to better ensure high quality research, consistency, facilitate the optimal functioning and collaborative interaction between individual Program Project Grant research projects and investigators. Core B will provide Program Project investigators with cultured mammalian cell types and relevant plasmid DNA for their individual research projects in a highly cost effective, reproducible and efficient manner. These core unit support functions will alleviate Program Project Grant investigators of the logistic responsibilities, labor-intensive culture/maintenance of mammalian cell types and expansion and purification of the relevant plasmid DNA required by the each project. Core Unit B will routinely provide cultured human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), human coronary endothelial cells and cultured mouse endothelial cells to Projects 1 ("Ethanol and Polyphenolic Induced Endothelial Fibrinolysis", Booyse, years 1- 5), 2 ("Regulation of PAI-1 Gene Expression by Ethanol and Polyphenols", Grenett, years 1-5) and 3; ("Cardiovascular Protection by Ethanol and Polyphenols", Parks, years 1-5); human aortic endothelial cells (HAECs) to project 3 (years 1-5) and adult mouse heart muscle cells to Project 4 ("Mitochondria and Cardioprotection by Ethanol and Polyphenols", Darley-Usmar, years 1-5). Core B will also routinely expand and purify relevant plasmid DNA to provide Program Project Grant projects (Projects 1 [years 1-5]; Project 2 [years 1-5]; Project 3 [years 1-5] and Project 4 [years 1-2]) for in-situ hybridizations (t-PA, u-PA, PAI-1, u-PAR, Ann-II, ocenolase eNOS, iNOS, CuZnSOD, MnSOD); Real-Time PCR (PAs, PARs, PAL1, vitronectin, eNOS, iNOS, MnSOD, CuZnSOD); nuclear run-on assays (u-PAR, Ann II, _ enolase, e-NOS, iNOS, MnSOD, CuZnSOD); luciferase constructs for transient transfection assays (t-PA, u-PA, PAI-1); luciferase mutagenesis constructs (PAI-1) and expression vectors for transcription factors.