SUPPRESSION OF PROTEIN SYNTHESIS IN REPERFUSED BRAIN

再灌注脑中蛋白质合成的抑制

• 批准号: 6639463
• 负责人: GARY S KRAUSE
• 金额: $ 40.29万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6639463
• 关键词: DNA binding protein; affinity chromatography; antisense nucleic acid; apoptosis; brain injury; brain metabolism; cerebral ischemia /hypoxia; enzyme activity; enzyme inhibitors; gel mobility shift assay; genetic translation; glycosylation; immunocytochemistry; laboratory rat; neuroprotectants; nucleic acid sequence; phosphorylation; polymerase chain reaction; protein biosynthesis; protein kinase; protein localization; reperfusion; tissue /cell culture; translation factor

DESCRIPTION (Adapted from applicant's abstract): Brain ischemia and reperfusion associated with cardiac arrest and resuscitation allows less than 10 percent of these patients to resume their normal lives: in addition, stroke is the leading cause of serious permanent disability. Our long-term goal is to gain sufficient understanding of the injury mechanisms to allow clinically effective therapy. The investigators have found that in vulnerable cortical and hippocampal neurons the marked inhibition of protein synthesis during reperfusion is associated with a rapid and large increase in alpha-subunit-phosphorylated eukaryotic initiation factor 2 [eIF2alpha(P)], which inhibits the delivery of the first amino-acyl tRNA. Initially, eIF2alpha(P) is only in the cytoplasm, but at four hours reperfusion vulnerable neurons also exhibit prominent nuclear eIF2alpha(P) and morphology suggesting early apoptosis. The researchers propose that activity of a specific eIF2alpha kinase and/or deglycosylation of p67 (binds eIF2 and blocks kinase access) are responsible for eIF2alpha phosphorylation and that eIF2alpha(P) binds nuclear DNA and may alter expression. The specific aims are to (1) identify the brain kinase(s) responsible for eIF2alpha phosphorylation during reperfusion, (2) characterize the effects of ischemia and reperfusion on p67, and (3) identify the DNA sequence bound by eIF2alpha(P) and characterize the effects of this interaction. The investigators plan to isolate brain eIF2alpha kinases by affinity chromatography, microsequence them and obtain their cDNAs, utilize antibodies against them to map their brain localization, and utilize antisense oligonucleotides to test their individual responsiveness in cultured cell injury models and reperfused brains. The effects of ischemia and reperfusion on total p67, glycosylated p67, and the p67 deglycosylase will be examined by immunohistochemistry. They will utilize PCR to enrich the eIF2alpha(P)-bound fraction of DNA randomers, assess binding sequence specificity by competition titration, identify the consensus sequence in clones, identify genes containing this response element by consensus sequence-primed PCR, and produce response element-regulated reporter constructs to explore the effects of eIF2alpha(P) on expression.

描述（根据申请人的摘要改编）：与心脏骤停和复苏相关的脑缺血和再灌注允许不到10％的患者恢复其正常生活：此外，中风是严重永久残疾的主要原因。我们的长期目标是对损伤机制有足够的了解，以允许临床有效的治疗。 The investigators have found that in vulnerable cortical and hippocampal neurons the marked inhibition of protein synthesis during reperfusion is associated with a rapid and large increase in alpha-subunit-phosphorylated eukaryotic initiation factor 2 [eIF2alpha(P)], which inhibits the delivery of the first amino-acyl tRNA.最初，eIF2Alpha（P）仅在细胞质中，但是在四个小时后再灌注脆弱的神经元也表现出突出的核EIF2Alpha（P），形态表明早期凋亡。研究人员提出，p67的特定EIF2ALPHA激酶和/或脱糖基化的活性（结合EIF2和阻断激酶访问）负责EIF2Alpha磷酸化，EIF2Alpha（p）结合核DNA并可能改变表达。具体目的是（1）确定在再灌注过程中负责EIF2Alpha磷酸化的脑激酶，（2）表征缺血和再灌注对p67的影响，以及（3）识别由EIF2Alpha（P）结合的DNA序列，并表征了这种相互作用的影响。研究人员计划通过亲和力色谱分离脑EIF2Alpha激酶，微序列素质并获得其cDNA，利用针对它们的抗体来绘制其大脑定位，并利用反义寡核苷酸来测试其在培养的细胞受伤中的个体反应能力，并进行了重复使用的大脑。缺血和再灌注对总p67，糖基化的P67和P67脱胶酶基化酶的影响将通过免疫组织化学检查。他们将利用PCR来丰富DNA随机器的EIF2Alpha（P）结合部分，评估通过竞争滴定来评估结合序列特异性，识别克隆中的共有序列，鉴定通过共有序列序列序列PCR包含此响应元素的基因，并产生响应元素调节的依据元件元素对EIF2alpha的效果（PRESS）对EIF2alpha的效果（PRE）。