ZINC AND THE SYNTHESIS OF ZINC BINDING PROTEINS

该项目的总体长期目标仍然是 旨在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。该项目有三个 具体的目标，这是相互关联的。目的I.金属硫蛋白(MT)：三明治 人MT基因表达的ELISA法和竞争性RT-PCR法 已经被开发出来了。使用从淋巴细胞、单核细胞、血液中提取的总RNA 用于RT-PCR的干血斑卡上的细胞和用于ELISA的红细胞裂解物， 受控的锌消耗和补充研究将确定MT的表达 在这种情况下。具有已知基因序列的人类cDNA阵列将 用于筛选其他锌调节基因，使用来自 控制摄入研究中受试者的淋巴细胞和单核细胞。 与人类单核细胞系(THP-1)的比较实验将用于 锌耗竭和内毒素激活实验筛选锌调控基因 通过基因芯片分析，观察了MT在细胞内定位的变化 这些条件。MT基因敲除小鼠将用于继续发挥功能 MT作为细胞保护系统的组成部分和作为锌供体的研究 用于CD4/CDB受体和蛋白酪氨酸激酶p56Ick复合体的形成。目标 富含半胱氨酸的肠道蛋白(CRIP)：培育一种CRIP基因敲除株 用于与先前开发的CRIP过表达的小鼠的比较研究 转基因菌株。表征研究将包括FACS分析， 脂多糖刺激后细胞因子的变化及基因芯片分析。继续 Th1/Th2细胞因子差异与CRIP功能关系的研究 与锌缺乏有关的小鼠和人CRIP之前将使用酶联免疫吸附试验 和CRIP-KO和CRIP-TG两种小鼠品系。要检查的研究 计划在细胞因子刺激期间对细胞CRIP进行定位 免疫沉淀法和/或免疫沉淀法寻找CRIP蛋白伴侣的研究 双杂交筛选方法。目的III.锌调控基因：锌转运蛋白1、2、 和4在胎儿发育过程中受膳食锌调控的基因表达，以及 通过生理介质如内毒素和IL-6与糖尿病进行研究 Northern分析、Western分析和免疫组织化学。锌调控 利用差异技术研究大鼠肠道和小鼠胸腺中的基因 MRNA3显示。这些研究将扩大，利用小鼠缺锌 用基因芯片技术研究锌在脾和胰腺的调控基因 分析。

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.