PROCESSES OF METAL SELECTION BY METAL ACCUMULATING CELLS

拟议项目旨在确定镉暴露是否会导致 再分配的内在存储的铜在细胞内。 是 假设暴露于镉诱导的金属硫蛋白（MT） 作为金属如Cu的热力学和动力学陷阱， 对蛋白质具有更高的亲和力和结合常数。 隔绝 铜从细胞内蛋白质和酶需要它的存在 对正常功能的变构或催化作用可能导致 最终用于细胞病理学。 因此， 注意到镉的影响可能是由于细胞缺乏铜，而不是 而不是过量的镉。 两个无脊椎动物门的初步数据是 以支持这一假设。 实验提出了进一步评估这一命题都在 体外使用培养的哺乳动物细胞和体内使用无脊椎动物 软体动物Littorina littorea作为模式生物。 本研究的五个具体目标是确定：i）MT 诱导响应镉暴露的结果，伴随着增加 在铜的细胞/有机体负担; ii）MT诱导改变 亚细胞分布的铜，锌和其他生理上重要的 金属; iii）观察到的铜变化是由于金属的重新分配造成的 内部存储或外部铜从周围的积累 培养基，iv）含Cu酶的功能受损（例如， 细胞色素C氧化酶活性）作为镉暴露的结果：v） 镉的细胞毒性作用可通过铜的补充而得到弥补; 电感耦合等离子体质谱的两种新应用 （ICP-MS）已经被开发来研究这些解释性问题。 首先，将ICP-MS直接耦合到高效液相色谱仪， 色谱系统，以允许同时分离和元素 MT和其他细胞溶质金属结合配体的分析。 第二、 已经开发了使用稳定同位素63 Cu和65 Cu的方案， 允许进行铜代谢的长期动力学研究。 的 不断完善和发展这些技术， 能力无疑将促进门克的研究， 威尔逊氏病和其他由异常 铜代谢 最后，除了MT的研究，细胞内， 代谢中含有矿化的orth/焦磷酸盐夹杂物 CD也将被研究。 最近的证据表明， 沉积物可能是封存和沉积的重要场所 累积的Cd。 综合起来，这些研究将提供关于 必需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 如果我们要理解 金属发挥其毒理作用。 建议的研究计划是特别适合研究生 和本科少数民族学生的参与，并提供广泛的 接触了一些现代技术，这些技术通常用于 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、血浆光谱和蛋白质 分离纯化。 因此，该项目提供了一个 学生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.