...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Infection and immunity >Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7
【24h】

Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7

机译：Nramp1在小鼠巨噬细胞系RAW264.7中的功能性表达

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes.Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-typeNramp1G169 cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1D169 allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane ofSalmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defectiveS. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.
机译：体内 Nramp1 位点的突变会导致不相关的细胞内微生物感染。 Nramp1 编码一种在巨噬细胞的内体-溶酶体区室中大量表达的完整膜蛋白，被募集到吞噬作用后的吞噬膜。 Nramp1影响吞噬体以控制微生物复制的生化特性的机制尚不清楚。为了设计Nramp1功能的体外检测方法，我们将野生型 Nramp1 G169 cDNA引入了RAW 264.7巨噬细胞（该载体具有纯合突变体 Nramp1 D169 等位基因，因此可以复制特定的细胞内寄生虫）。重组Nramp1在RAW264.7细胞的一个膜室中表达，并被募集到含有鼠伤寒沙门氏菌和小肠结肠炎耶尔森菌的吞噬膜上。 RAW264.7转染子的抗菌活性评估表明重组Nramp1蛋白的表达废除了 S的细胞内复制。鼠伤寒。复制缺陷型 S的研究。鼠伤寒突变体表明这是通过增强抑菌活性而发生的。 Nramp1表达的效果是特异的，因为（i）在过表达紧密相关的Nramp2蛋白的RAW264.7转染子中未观察到，并且（ii）控制RAW264.7细胞，Nramp1和Nramp2转染子均可以有效杀死温度-敏感的， S的复制缺陷型突变体。鼠伤寒。最后，Nramp1 RAW264.7转染子增加的抗菌活性与吞噬体酸化增加有关，吞噬体酸化是表达野生型Nramp1等位基因的主要巨噬细胞的显着特征。总之，这些结果表明 Nramp1 cDNA在RAW264.7巨噬细胞系中的转染可以用作研究Nramp1功能和作用机制以及鉴定结构与功能关系的直接方法在这种蛋白质中。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号