BIOCHEMICAL MECHANISMS IN TOXIC CELL DEATH

细胞杀伤是缺血性疾病如心肌缺血的一个重要方面， 梗塞和中风。 在这些疾病中，动脉闭塞可防止 或减少器官的血液供应，中断氧气供应， 受影响的细胞 当替代能源耗尽时， 细胞进入不可逆损伤状态并最终死亡。 在发生显著细胞死亡之前重建血液供应 引发细胞快速死亡而不是恢复。 若干条证据 表明血液中导致这种现象的物质是Ca 2+， 其沿着陡峭的电化学梯度进入电池; 缺乏能量的细胞无法将Ca 2+泵回， 细胞内Ca 2+升高，引发细胞死亡。 升高 细胞内Ca 2+浓度可以在培养的细胞中建模， 二价阳离子载体A23187。 对此的初步研究 实验室表明，细胞内浓度升高 Ca 2+是一个活跃的过程，涉及至少3个可识别的生物化学过程。 步骤，第一个似乎是磷脂酶。 的目的 拟议的研究是了解这一过程的生物化学，即。 阐明所涉及的步骤并尽可能实际地描述 酶参与每一步。 这项研究的长期目标是 开发过程中关键步骤的抑制剂。 这些抑制剂可以 可以想象，可以开发成药物，以防止细胞死亡相关的 缺血性疾病 为研究细胞死亡的生物化学而选择的模型系统是 培养物中的永久细胞系，进行可行的突变体选择和其他 基因方法。 细胞活力将通过物理（例如， 台盼蓝排除）和遗传（例如增殖）标准。 阐明该机制的实验方法包括：（1）选择 和表征酶缺陷的突变体（变体）细胞系 （2）进一步描述受伤者的特征 通过生化标准确定的步骤中的细胞和对 药理学探针;和（3）磷脂酶的表征 与第一步相关的活动。

Cell killing is an important aspect of ischemic diseases such as myocardial infarction and stroke. In these diseases occlusion of an artery prevents or reduces blood supply to an organ, interrupting the oxygen supply to affected cells. When alternate energy sources are depleted the affected cells enter a state of irreversible injury and ultimately die. Reestablishing the blood supply before significant cell death occurs initiates rapid cell death rather than recovery. Several lines of evidence indicate that the agent in blood responsible for this phenomenon is Ca2+, which enters the cells down a steep electrochemical gradient; the energy-deprived cells are unable to pump the Ca2+ back out resulting in elevated intracellular Ca2+ which triggers cell death. Elevated intracellular Ca2+ concentrations can be modeled in cultured cells using the divalent cation ionophore A23187. Preliminary studies in this laboratory indicate that cell death triggered by elevated intracellular Ca2+ is an active process involving at least 3 identifiable biochemical steps, the first which appears to be a phospholipase. The aim of the proposed research is to understand the biochemistry of this process, i.e. to elucidate the steps involved and to characterize as far as practical the enzymes involved with each step. The long term goal of the research is to develop inhibitors of key steps in the process. These inhibitors could conceivably be developed into drugs to prevent cell death associated with ischemic diseases. The model system chosen for studying the biochemistry of cell death is a permanent cell line in culture making feasible mutant selection and other genetic approaches. Cell viability will be assessed by physical (e.g. trypan blue exclusion) and genetic (e.g. proliferation) criteria. Experimental approaches to elucidating the mechanism include (1) selection and characterization of mutant (variant) cell lines deficient in enzymes required for individual steps; (2) further characterization of the injured cells at the identified steps by biochemical criteria and response to pharmacological probes; and (3) characterization of the phospholipase activity associated with the first step.