The objectives of this Physician Scientist Grant Application are for the candidate to (1) obtain an in depth basic science learning experience emphasizing a new discipline (Molecular Biology) while (2) conducting research under the direct supervision of a sponsor with extensive experience in this discipline. Subsequently, the candidate will apply these research tools to his current research interest in Phase II under the continuing supervision of his sponsor in conjunction with the advisory committee. The Phase I project will focus on characterization of murine acidic fibroblast growth factor gene.
The specific aims are: (1) to determine the gene structure of mouse aFGF. The research plan involves isolation and characterization through sequencing of mouse genomic DNA clones for aFGF. Identification of the transcription initiation site(s) will be done by primer extension and ribonuclease protection assay. Analysis of potential differences in gene expression in young and old mice will proceed by examining aFGF production in young and old cells by Northern blotting and hybridization. Determination of regulatory mechanisms of the gene encoding aFGF, will involve identifying the functional promoter region by CAT assay, deletional mapping and site directed mutagenesis to identify critical nucleotides in the promoter/enhancer region. Characterization of the mouse gene is necessary in order to: [1] better study biologic questions pertaining to inflammation and tumorigenesis; and [2] initiate studies of site-directed gene targeting in mouse systems. During phase II, studies of this and other growth factors will be applied to a murine model of autoimmune disease, the MRL lpr/lpr mouse. We hypothesize that in lupus a disturbance in the production of cytokines (HBGFs), IL-1, and PDGF) and/or in the responsiveness of macrophages to cytokines results in anomalous immunological function. It is suggested further that by systematically analyzing macrophages for production of these cytokines, we hope to better understand the role they may have in the expression of autoimmunity. Using murine models of lupus, these studies seek to screen for the production of aFGF, bFGF, IL-1 and PDGF in macrophages from autoimmune and normal mice; to biochemically characterize the proposed cytokines in interest; to assess the biological characteristics of the protein(s) of interest i.e.; mitogenic activity, chemotactic activity; to examine the effects of culture with these cytokines on plasma membrane reorganization, prostanoid and leukotriene production; and to examine the regulatory mechanisms controlling the production of these factors. Examining these factors will allow us to better understand the role they have in promoting inflammation in this setting.
