PROCESSES OF METAL SELECTION BY METAL ACCUMULATING CELLS

拟议中的项目旨在确定接触CD是否会导致 细胞内铜的本征存储的重新分布。它是 Cd暴露诱导的金属硫蛋白(MT)作用假说 作为铜等金属的热力学和动力学陷阱，这些金属显示出 对蛋白质有更高的亲和力和结合常数。自动减支 从需要铜存在的胞内蛋白质和酶中提取铜 无论是变构的还是催化的，都可能导致 最终在细胞病理学上。因此，毒物学 镉的影响可能是由于铜的细胞缺陷引起的 而不是过量的CD。来自两个无脊椎动物门的初步数据是 以支持这一假说。 为了进一步评估这一命题，我们提出了两个实验 使用培养的哺乳动物细胞进行体外实验和使用无脊椎动物进行体内实验 软体动物Littorina littorea作为模式生物。 这项研究的五个具体目标是确定：i)MT 对Cd暴露的诱导反应会导致伴随的增加 在铜的细胞/组织负荷中；ii)MT诱导改变 铜、锌等生理活性物质的亚细胞分布 金属；三)观察到的铜的变化是由于 来自周围环境的本征储存或外在铜的积累 4)含铜的酶功能受损(如： 细胞色素C氧化酶活性)镉暴露的结果：v) Cd的细胞毒作用可通过添加铜来修复； 电感耦合等离子体质谱的两个新应用 (电感耦合等离子体质谱)是为了研究这些调查问题而发展起来的。 首先，将电感耦合等离子体质谱直接耦合到高性能液体 允许同时分离和元素的层析系统 MT和其他胞质金属结合配体的分析。第二， 使用稳定同位素~(63)Cu和~(65)Cu的方案已被开发为 允许进行铜代谢的长期动力学研究。这个 继续完善和发展这些技术 能力无疑将有助于学习门克和 由异常引起的威尔逊病和其他遗传性疾病 铜的代谢。 最后，除了对MT的研究外，细胞内的作用， 矿化正/焦磷酸盐在代谢中的包裹体 也将对Cd进行研究。最近的证据表明，这些 矿床可能是封存和沉积的重要场所。 累积的Cd。 总而言之，这些研究将提供有关 必需金属和非必需金属之间的竞争性相互作用 细胞内金属结合配体。欣赏这些类型的 相互作用是重要的，如果我们要了解 金属发挥其毒理作用。 建议的研究计划特别适合两位毕业生 和本科生少数族裔学生的参与，并提供广泛的 接触到许多现代技术，这些技术通常在 生物医学科学包括无菌和无金属清洁技术， 细胞培养、电子显微镜、血浆光谱学和蛋白质 分离提纯。因此，该项目提供了一个 为学生提供接触生物医学的绝佳机会 研究经验。

The proposed project aims to determine if Cd exposure causes a redistribution of intrinsic stores of Cu within the cell. It is hypothesized that metallothionein (MT), induced by exposure to Cd, acts as a thermodynamic and kinetic trap for metals such as Cu which show a higher affinity and binding constant for the protein. The sequestration of Cu from intracellular proteins and enzymes requiring its presence either allosterically or catalytically for normal functioning may result ultimately in cellular pathologies. Consequently, the toxicological effects noted with Cd may arise from cellular deficiencies in Cu rather than excesses of Cd. Preliminary data from two invertebrate phyla are provided in support of this hypothesis. Experiments are proposed to evaluate this proposition further both in vitro using cultured mammalian cells and in vivo using the invertebrate mollusc Littorina littorea as a model organism. The five specific aims of the study are to determine whether : i) MT induction in response to Cd exposure results in a concomitant increase in the cellular/organismal burden of Cu; ii) MT induction alters the subcellular distribution of Cu, Zn and other physiologically important metals; iii) the observed changes in Cu are due to a redistribution of intrinsic stores or the accumulation of extrinsic Cu from the surrounding media, iv) there is impaired functioning of Cu containing enzymes (eg. cytochrome C oxidase activity) as a result of Cd exposure: v) the cytotoxic effects of Cd can be remedied by administering Cu; Two novel applications of inductively coupled plasma mass spectroscopy (ICP-MS) have been developed to study these investigatory questions. Firstly, an ICP-MS has been coupled directly to a high performance liquid chromatography system to allow the simultaneous separation and elemental analysis of MT and other cytosolic metal binding ligands. Secondly, protocols using stable isotopic 63Cu and 65 Cu have been developed to permit long term kinetic studies of Cu metabolism to be undertaken. The continued refinement and development of techniques with these capabilities will undoubtedly facilitate the study of Menke's and Wilson's disease and other genetic disorders which result from abnormal Cu metabolism. Finally, in addition to the studies of MT, the role of intracellular, mineralized orth/pyrophosphate containing inclusions in the metabolism of Cd will also be studies. Recent evidence has shown that these deposits may be an important site for the sequestration and deposition of accumulated Cd. In combination, these studies will provide important information on the competitive interactions between essential and non-essential metals for intracellular metal-binding ligands. An appreciation of these types of interaction are important if we are to understand the mechanisms by which metals exert their toxicological action. The proposed research program is particularly suitable for both graduate and undergraduate minority student involvement and provides broad exposure to a number of modern techniques that are commonly employed in the biomedical sciences including aseptic and metal-free clean technique, cell culture, electron microscopy, plasma spectroscopy and protein separation and purification. This project therefore provides an excellent opportunity for students to be exposed to the biomedical research experience.