The overall long-term goal of this project continues to be directed at understanding the involvement of zinc binding proteins in the absorption, metabolism and biological functions of zinc. Past research from this project has shown through metabolic studies and kinetic analysis that cytokine (interleukin 1) and hormonal regulation of zinc metabolism is mediated through changes in metallothionein gene expression. Metallothionein expression is also directly proportional to dietary zinc intake, probably via a mechanism involving direct interaction between zinc and a nuclear factor. Metallothionein in erythrocytes, having been produced in bone marrow during maturation of these cells, appears to be an index of dietary zinc intake in humans, and thus may act as an indicator of zinc status. The saturable phase of transmucosal zinc movement during absorption has been shown to correlate to binding by a protein we have identified as cysteine-rich intestinal protein (CRIP). Building upon this past information, the next project period has three major Aims.
Aim I is directed a metallothionein. We will further explore erythrocyte metallothionein in male and female human subjects to study turnover of the protein and the response to different levels of supplemental zinc. This will use the ELISA for human metallothionein. Nuclear proteins that bind both zinc and metal regulatory element (MRE) sequences will be further investigated with the goal of purifying a protein(s) that can be shown to interact with dietary zinc to initiate transcription of MRE regulated genes.
Aim II will focus on CRIP gene regulation and studies on the relationship of CRIP to absorption of zinc and, possibly, other nutrient metals. Developmental and hormonal regulation of CRIP mRNA during the postnatal period will receive particular attention, as will regulation during intestinal inflammation and in isolated intestinal cells. Initially, glucocorticoid hormone and thyroxine will receive attention as factors in regulation of neonatal CRIP. Large scale purification and characterization of CRIP will be undertaken. This will allow polyclonal antibody production. It will also allow for in vitro studies to examine the metal binding characteristics of CRIP. The involvement of CRIP in zinc transport will be studied in rat pups during postnatal development and into adulthood, during pregnancy and in monolayer cell cultures of enterocyte-like cells.
Aim III will focus on identification of genes responsive to dietary zinc. We will use differential hybridization of cDNA libraries using first strand-cDNA and subtracted probes. Poly(A)+mRNA from zinc deficient and pair-fed zinc adequate rats will be used to identify those cDNAs that are induced by zinc and these proteins will be identified from known databases where possible. Dietary regulation of genes with MRE promoter sequences will also be examined.
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