Molecular mechanism and prevention of drug dependence

药物依赖的分子机制及预防

基本信息

批准号：
05557009
负责人：
MIKI Naomasa
金额：
$ 8.32万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

Chronic administration of morphine to NG108-15 cells or mice decreased the binding activity of a nuclear factor (ssCRE-BP) tosingle-stranded oligo-DNA of cAMP-response element (ssCRE). we have purified ssCRE-BP from mouse cerebella and isolated the cDNA clone. We found that ssCRE-BP from mouse brain was identical to Pura, which is a binding protein for single-stranded purine-rich DNA sequences that was originally isolated as a replication factor. ssCRE-BP was abundant in the cerebral cortex and cerebellum, but very low levels were detected in other tissues. Examination of the effect of chronic morphine treatment on the amount of ssCRE-BP in mouse brain showed that ssCRE-BP was not influenced by chronic morphine treatment significantly in western blot analysis. Although the DNA binding activities of purified ssCRE-BP or its GST-fusion protein were very low, they were markedly enhanced by the addition of an activator that was about 100kDa, trypsin sensitive and heat stable. The activator can be substituted with casein, which increased the affinity of ssCRE-BP for DNA by one order. A series of deletion mutants were prepared for determining the DNA binding-and activator-interacting domains and both of them were found to reside in AA 50-215 of ssCRE-BP.Although ssCRE-BP was abundant in the brain, the activator was ubiquitous. Characterization of the activator may provide insight into the physiological roles of ssCRE-BP which is involved in the development of morphine dependence and tolerance.

对NG108-15细胞或小鼠的长期给予吗啡降低了cAMP反应元件（SSCRE）的核因子（SSCRE-BP）的Tosementle-strandle-strandle-ligo-DNA的结合活性。我们已经从小鼠小脑中纯化了SSCRE-BP，并分离了cDNA克隆。我们发现，来自小鼠脑的SSCRE-BP与Pura相同，Pura是一种结合蛋白，用于单链富含嘌呤的DNA序列，最初被分离为复制因子。 Sscre-BP在大脑皮层和小脑中很丰富，但在其他组织中检测到非常低的水平。检查慢性吗啡治疗对小鼠脑中SSCRE-BP量的影响表明，在蛋白质印迹分析中，SSCRE-BP不受慢性吗啡治疗的影响。尽管纯化的SSCRE-BP或其GST融合蛋白的DNA结合活性非常低，但通过添加约100kDa，胰蛋白酶敏感和热量稳定的激活剂可显着增强它们。激活剂可以用酪蛋白取代，酪蛋白可以将SSCRE-BP对DNA的亲和力增加一个阶。准备了一系列的缺失突变体来确定DNA结合和激活剂相互作用结构域，并且发现它们都驻留在SSCRE-BP的AA 50-215中。尽管SSCRE-BP在大脑中很丰富，但激活剂无处不在。激活剂的表征可以提供对SSCRE-BP的生理作用的洞察力，该生理作用与吗啡依赖性和耐受性的发展有关。