Zinc and the Synthesis of Zinc Binding Proteins

描述（由申请人提供）：该项目的总体长期目标仍然是了解锌结合蛋白的参与，以及那些与锌相互作用的蛋白质对膳食锌的吸收、代谢和生物功能的影响。项目目标是相互关联的。目的1。金属硫蛋白（MT）在人和鼠细胞和组织中表达的功能意义采用定量PCR （Q-PCR）技术，对SLC30和SLC39家族的hMT和特异性锌转运基因进行检测，以评估这些基因在单核细胞和T细胞中的锌反应性。通过膳食锌调节的小鼠MT的影响将被评估为亚硝化应激的一个因素。这些实验将使用MT零基因型和诱导型一氧化氮合酶零基因型，并将重点放在培养的离体肝实质细胞和一氧化氮的靶点肠细胞上。目的二世。锌反应基因的鉴定。这些研究将集中在THP1细胞作为人类单个核细胞的模型，使用螯合剂诱导的锌耗尽和补充。将比较锌反应基因与LPS激活之间的关系。类似的研究将比较锌缺乏和锌补充小鼠胸腺细胞脂多糖的锌反应基因。分析将通过微阵列进行，并通过蛋白质组学进行确认。比较MTF1和MTF2转录因子对饲粮锌的响应性。第三目标。小鼠锌转运蛋白及其对日粮锌摄入和生理介质的反应。IL-6响应性转运蛋白Zip14将在转染细胞中进一步表征，并在炎症性低锌血症中发挥作用。在急性期反应期间网状内皮系统锌转运蛋白基因的表达将被评估。胰腺腺泡细胞和Paneth细胞利用锌加工步骤进行内源性锌释放将受到密切关注。将广泛使用免疫组织化学和免疫细胞化学以及荧光显微镜下可见的锌荧光团来定位转运体。

DESCRIPTION (provided by applicant): The overall long-term goal of this project continues to be directed at understanding the involvement of zinc binding proteins and those proteins that interact with zinc for the absorption, metabolism, and biological function of dietary zinc. The project aims are interrelated. Aim 1. Functional Significance of Metallothionein (MT) Expression in Human and Murine Cells and Tissues. Using quantitative PCR (Q-PCR), assays for hMT and specific zinc transporter genes of both the SLC30 and SLC39 families will be used in controlled zinc depletion and zinc supplementation studies with human subjects to evaluate the zinc responsiveness of these genes in monocytes and T cells. The influence of murine MT, as regulated by dietary zinc, will be evaluated as a factor in nitrosative stress. These experiments will use MT null and inducible nitric oxide (NO) synthase null genotypes and focus on isolated liver parenchymal cells in culture and targets of NO, viz., intestinal cells. Aim II. Characterization of Zinc Responsive Genes. These studies will focus on THP1 cells as a model for human mononuclear cells using chelator-induced zinc depletion and supplementation. Comparisons will be made between zinc-responsive genes ¿ LPS activation. Similar studies will compare zinc-responsive genes of thymocytes ¿ LPS from zinc-deficient and zinc-supplemented mice. Analysis will be by microarrays, with confirmation by proteomics. Responsiveness of the MTF1 and MTF2 transcription factors to dietary zinc will be compared. Aim III. Murine Zinc Transporters and Their Response to Dietary Zinc Intake and Physiologic Mediators. The IL-6 responsive transporter Zip14 will be further characterized with transfected cells and for a role in inflammatory hypozincemia. Expression of zinc transporter genes of the reticuloendothelial system during the acute phase response will be evaluated. The zinc processing steps the pancreatic acinar cells and Paneth cells use for endogenous zinc release will receive close attention. Extensive use will be made of transporter localization using immunohistochemistry and immunocytochemistry and zinc fluorophores as visualized by fluorescence microscopy.