ZINC AND THE SYTHESIS OF ZINC BINDING PROTEINS

本项目的总体长期目标继续以 了解锌结合蛋白在体内的作用 锌的吸收、代谢和生物学功能。过去的研究 通过新陈代谢研究和动力学 细胞因子(白介素1)与锌的激素调节 新陈代谢是通过金属硫蛋白基因的变化来调节的 表情。金属硫蛋白的表达也与 饮食中锌的摄入量，可能是通过一种机制，涉及直接 锌与核因子的相互作用。金属硫蛋白 红细胞，在成熟过程中在骨髓中产生 这些细胞似乎是人类膳食锌摄入量的一个指标， 因此可以作为锌状态的指示器。的可饱和相位 锌在吸收过程中的跨粘膜移动已被证明与 与一种我们已确定为富含半胱氨酸的肠道蛋白结合 蛋白质(CRIP)。在过去信息的基础上，下一个项目 Period有三个主要目标。目的I是一种金属硫蛋白。我们 将进一步探讨男女红细胞金属硫蛋白 人体研究蛋白质的周转和对 补充不同水平的锌。这将使用酶联免疫吸附试验 人类金属硫蛋白。结合锌和金属的核蛋白 调节元件(MRE)序列将与 纯化一种可以与之相互作用的蛋白质的目标(S) 日粮锌启动MRE调控基因的转录。AIM II 将重点放在CRIP基因调控与研究的关系上。 影响锌的吸收，可能还有其他有营养的金属。 CRIP mRNA在出生后发育和激素调节中的作用 期间将受到特别关注，监管期间也将受到特别关注 肠道炎症和分离的肠道细胞。最初， 糖皮质激素和甲状腺素将作为影响因素受到关注 在新生儿CRIP的调节中。大规模的净化和 将对CRIP进行定性。这将允许多克隆 抗体的产生。它还将允许体外研究以检查 CRIP的金属结合特性。CRIP参与了 将研究锌在幼鼠出生后发育过程中的转运 在成年期、怀孕期间和在单层细胞培养中 肠细胞样细胞。AIM III将专注于基因鉴定 对饮食中的锌有反应。我们将使用差异杂交技术 使用第一链cDNA文库和消减探针。 缺锌和配对喂养锌充足大鼠的Poly(A)+mRNA将被 用来识别锌和这些蛋白质诱导的cDNA 将在可能的情况下从已知数据库中确定。膳食 还将检查带有MRE启动子序列的基因的调节。

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.