PROCESSES OF METAL SELECTION BY METAL ACCUMULATING CELLS

金属积累池的金属选择过程

• 批准号: 3734623
• 负责人: ANDREW Z MASON
• 金额: --
• 依托单位: CALIFORNIA STATE UNIVERSITY LONG BEACH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3734623
• 关键词: Mollusca; cadmium; chemical kinetics; copper; cytochrome c; cytotoxicity; enzyme activity; high performance liquid chromatography; ligands; mass spectrometry; metal complex; metal metabolism; metallothionein; stable isotope; thermodynamics

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.

拟议的项目旨在确定CD暴露是否导致 细胞内Cu内在存储的重新分布。 这是 假设通过暴露于CD诱导的金属硫蛋白（MT）作用 作为金属（例如Cu）的热力学和动力学陷阱 蛋白质的较高亲和力和结合常数。 隔离 来自细胞内蛋白质和酶的Cu，需要其存在 可以导致变构或催化作用以正常功能 最终在细胞病理中。 因此，毒理学 CD指出的效果可能是由Cu中的细胞缺陷引起的 比过量的CD。 来自两个无脊椎动物门的初步数据是 为支持这一假设提供了提供。 提出了实验以进一步评估这一主张 使用培养的哺乳动物细胞和体内使用无脊椎动物的体外体外 Mollusc littorina littorea作为模型有机体。 该研究的五个具体目的是确定是否：i）MT 响应CD暴露的诱导导致随之增加 在Cu的细胞/生物负担中； ii）MT感应改变了 Cu，Zn和其他生理上重要的亚细胞分布 金属； iii）观察到的Cu的变化是由于重新分布 内在商店或周围外部CU的积累 培养基，iv）含有酶的Cu功能受损（例如。 CD暴露导致细胞色素C氧化酶活性）：v） CD的细胞毒性作用可以通过施用Cu来补救； 电感耦合等离子体质谱的两个新型应用 （ICP-MS）已开发用于研究这些研究性问题。 首先，ICP-MS已直接耦合到高性能液体 色谱系统允许同时分离和元素 MT和其他胞质金属结合配体的分析。 第二， 使用稳定的同位素63CU和65 Cu的协议已开发为 允许进行CU代谢的长期动力学研究。 这 持续改进和开发这些技术 毫无疑问，能力将有助于研究Menke的研究 威尔逊氏病和其他由异常引起的遗传疾病 CU代谢。 最后，除了对MT的研究，细胞内的作用 矿化的矫置/焦磷酸含有代谢中的包含物 CD也将是研究。 最近的证据表明这些 沉积物可能是隔离和沉积的重要部位 累积的CD。 结合起来，这些研究将提供有关 基本金属和非必需金属之间的竞争相互作用 细胞内金属结合配体。 对这些类型的欣赏 如果我们要了解的机制，相互作用很重要 金属发挥其毒理作用。 拟议的研究计划特别适合两位研究生 和本科少数族裔学生的参与并提供广泛的 暴露于许多通常使用的现代技术 生物医学科学在内，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 分离和纯化。 因此，该项目提供了一个 让学生接触生物医学的绝佳机会 研究经验。