The overall long-term goal of this project continues to be directed at understanding the involvement of Zn-binding proteins in the absorption, metabolism, and biological functions of Zn. The project has three specific aims, which are interrelated.
Aim I. Metallothionein (MT): Sandwich ELISA and competitive RT-PCR methods for human MT expression experiments have been developed. Using total RNA derived from lymphocytes, monocytes, blood cells on dried blood spot cards for RT-PCR and erythrocyte lysates for ELISA, controlled Zn depletion and supplementation studies will define MT expression under these conditions. Human cDNA arrays with sequences for known genes will be used to screen for other Zn regulated genes using the RNA derived from lymphocytes and monocytes from subjects in the controlled intake studies. Comparison experiments with a human monocytic cell line (THP-1) will be used in Zn depletion and LPS activation experiments to screen for Zn regulated genes using cDNA array analysis, and the changes in the cellular location of MT under these conditions. MT knockout mice will be used for continuing functional studies on MT as a component of a cytoprotective system and MT as a Zn donor for CD4/CDB receptor and protein-tyrosine kinase p56Ick complex formation.
Aim II. Cysteine Rich Intestinal Protein (CRIP): Develop a strain of CRIP knockout mice for comparison studies with the previously developed CRIP overexpressing transgenic strain. Characterization studies will include FACS analysis, cytokine profiles after LPS stimulation and cDNA array analysis. Continuing studies on CRIP function related to Th1/Th2 cytokine differences and those related to Zn depletion will use the ELISA for murine and human CRIP previously developed, and both the CRIP-KO and CRIP-Tg murine strains. Studies to examine cellular CRIP localization during cytokine stimulation are planned, as are studies to identify a protein partner for CRIP using immunoprecipitation and/or two-hybrid screening methods.
Aim III. Zn Regulated Genes: Zn Transporter 1, 2, and 4 gene expression as regulated by dietary Zn, during fetal development, and by physiologic mediators such as LPS and IL-6 and diabetes will be studied by northern analysis, western analysis, and immunohistochemistry. Zn-regulated genes are being studied in rat intestine and mouse thymus using differential MRNA display. These studies will be expanded, using the murine Zn deficiency model, to identify Zn regulated genes in spleen and pancreas using cDNA array analysis.
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