SPECTROSCOPIC SPECIATION OF SULFUR IN LIVING MAMMALIAN CELLS: HIV & APOPTOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 硫是一种极其重要的元素，在所有生物体的结构、催化和新陈代谢中起着重要作用。我们建议使用硫K边X射线吸收近边光谱来监测和提高我们对哺乳动物细胞中硫代谢的理解，作为细胞发育的函数和对各种刺激的响应。特别是，细胞整体氧化还原环境的变化，以及线粒体等细胞器的变化，被认为在控制细胞的不同发育阶段--从增殖、分化、凋亡到最终死亡--具有重要意义。这一领域的研究还处于初级阶段，有关细胞硫代谢组的直接信息将非常有价值。细胞凋亡是细胞主动自杀的过程，在许多人类疾病中都非常重要，包括艾滋病毒，在艾滋病中，诱导凋亡是T细胞死亡的主要原因，在癌症中，细胞凋亡异常减少。细胞凋亡被认为是由细胞内氧化还原变化启动的，尽管缺乏直接证据证明这一点。这样的变化应该可以通过我们提出的实验来衡量，并且关于这一重要生物机制的重要信息是可以预见的。我们建议使用XAS来研究活的哺乳动物细胞--MDCK永生化细胞系--的培养，这些细胞是极化的上皮细胞。我们已经开发了处理细胞培养和记录其XAS光谱的技术和设备。我们还建立了利用硫的K边X射线吸收近边光谱来定量测定生物体系中的硫的方法。现在，我们计划使用这些工具来研究硫的生物化学作为细胞发育状态的函数，包括细胞凋亡和机械刺激。我们的实验应该为硫在这些过程中的作用提供有价值的见解，特别是在细胞凋亡的情况下，可能会产生与健康相关的重要后果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sulfur is a vitally important element, with roles in structure, catalysis and metabolism in all organisms. We propose to use sulfur K-edge X-ray absorption near-edge spectroscopy to monitor and to improve our understanding of sulfur metabolism in mammalian cells, as a function of cellular development and in response to various stimuli. In particular, changes in the overall redox environment of the cell, and in organelles such as mitochondria, are thought to be important in controlling different developmental stages of the cell, from proliferation, differentiation, apoptosis and finally to necrosis. Research in this area is in its infancy, and direct information on the sulfur metabolome of the cell would be very valuable. Apoptosis, the process by which cells actively commit suicide, is very important in numerous human diseases, including HIV, where induction of apoptosis is the primary cause of T-cell death, and in cancer, where apoptosis is abnormally reduced. Apoptosis is thought to be initiated by redox changes within cells, although direct evidence for this is lacking. Such changes should be measurable by the experiments that we propose, and vital information on this important biological mechanism can anticipated. We propose to use XAS to study cultures of living mammalian cells - the MDCK immortalized cell-line, which are polarized epithelial cells. We have already developed the techniques and equipment for handling the cell cultures and recording their XAS spectra. We have also established the use of sulfur K-edge X-ray absorption near-edge spectroscopy to quantitatively speciate the sulfur in living systems. Now we plan to use these tools to study sulfur biochemistry as a function of cellular developmental status including apoptosis and during mechanical stimulation. Our experiments should provide valuable insights into the role of sulfur in these processes, and particularly in the case of apoptosis, could have important health-related ramifications.