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LYMPHOKINE PRODUCTION--EFFECT OF CYCLOSPORINE

淋巴因子的产生--环孢菌素的作用

基本信息

批准号：
2178858
负责人：
ANGELA GRANELLI-PIPERNO
金额：
$ 19.63万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-04-01 至 1994-11-30
项目状态：
已结题

项目摘要

Lymphokines, the specialized products of stimulated T cells, mediate the growth and function of lymphocytes and other cell types. Lymphokine synthesis is controlled at the transcriptional level and is induced by stimulation with antigens or mitogens. The production of certain lymphokine mRNAs [IL-2, IFN-gamma, IL-4] but not the induction of other mRNAs [p55 IL-2 receptor, c-fos, a heat shock protein, IL-6, GM-CSF] is blocked selectively by the immunosuppressive drugs cyclosporin A [CSA] and FK-506. Several regulatory sequences have been identified in the promoter of the human IL-2 gene, using in vivo functional analysis and DNA-binding [electromobility shift assays EMSA]. The characterization of the factors that interact with these regulatory sites varies greatly: some have been purified and cloned while others are defined primarily by EMSA. The nuclear factor for activated T cells [NF-AT], which binds at position -289 to -269 in the 5' region of the IL-2 enhancer, remains uncharacterized. This gap is significant since NF-AT is the only known factor that is T cell restricted, requires "two signals" for induction, and is sensitive to CSA and FK-506. Other sites on the IL-2 enhancer, such as NF-KB and OCT-1, are activated by only one signal and are insensitive to CSA and FK-506. To pursue these and my preliminary findings we will: a) isolate, clone, and generate antibodies to the inducible, CSA:FK-506 sensitive factor that binds to oligonucleotide columns made with the wild-type but not mutant NF- AT site; b) similarly characterize a noninducible, CSA:FK-506 resistant modulator which was identified in the effluent of NF-AT columns and which markedly increases the activity of the NF-AT binding protein; and c) conduct a series of experiments to assess the role of these two factors in lymphokine gene expression. The latter experiments will describe the subcellular localization of the corresponding proteins and mRNA's following application of different T cell stimuli, the role of inhibitors and posttranslational modification such as tyrosine kinase dependent phosphorylation, the interaction of the two proteins with the NF-AT site as revealed by UV-crosslinking and in vitro transcription assays, the presence of comparable binding and modulator proteins in other CSA-sensitive and CSA-resistant genes, and the effects of immunosuppressive drugs on each of the above aspects of NF-AT activity.

受刺激的T细胞的特化产物--趋化因子介导了免疫应答。淋巴细胞和其他细胞类型的生长和功能。淋巴因子合成在转录水平上受到控制，用抗原或有丝分裂原刺激。生产某些淋巴因子mRNA [IL-2，IFN-γ，IL-4]，但不诱导其他 mRNA [p55 IL-2受体，c-fos，热休克蛋白，IL-6，GM-CSF]是被免疫抑制药物环孢菌素A [CSA]选择性阻断， FK-506 在启动子中已经鉴定了几个调控序列的人IL-2基因，使用体内功能分析和DNA结合 [电迁移率变动测定EMSA]。因素的特征与这些调控位点相互作用的基因差异很大：有些已经被纯化和克隆，而其他主要由EMSA定义。的活化T细胞核因子[NF-AT]，结合于-289位至IL-2增强子5 ′区的-269，仍然未被表征。这一差距是显著的，因为NF-AT是唯一已知的T细胞因子，受限制，需要“两个信号”感应，对CSA敏感 FK-506 IL-2增强子上的其他位点，如NF-κ B和OCT-1，仅由一个信号激活，对CSA和FK-506不敏感。到根据这些和我的初步发现，我们将：a）分离，克隆，产生针对可诱导的CSA：FK-506敏感因子的抗体，结合到用野生型而不是突变型NF-κ B制备的寡核苷酸柱上， AT位点; B）类似地表征非诱导型CSA：FK-506抗性在NF-AT柱的流出物中鉴定的调节剂，显著增加NF-AT结合蛋白的活性;和c）进行了一系列的实验，以评估这两个因素在淋巴因子基因表达。后面的实验将描述相应蛋白质和mRNA的亚细胞定位如下：应用不同的T细胞刺激，抑制剂的作用，翻译后修饰如酪氨酸激酶依赖性磷酸化，两种蛋白质与NF-AT位点的相互作用，通过紫外线交联和体外转录测定显示，在其他CSA敏感性和 CSA耐药基因，以及免疫抑制药物对每种上述NF-AT活性方面。