Role of pp65/plastin in infection, immunity and oncogenesis.

pp65/plastin 在感染、免疫和肿瘤发生中的作用。

基本信息

批准号：
10670261
负责人：
SHINOMIYA Hiroto
金额：
$ 1.73万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

1. Preparation of cDNA and recombinant protein of pp65/plastin family and antibodies againt them : Recombinant L-plastin was produced in E. coli transfected with the cDNA construct in pET vector. Polyclonal antibodyies againt L-plastin were prepared by immunizing rabbits with the recombinant protein. We also cloned the T-plastin gene.2. Characterization of an LPS-induced serine kinase that phosphorylates pp65/L-plastin : An LPS-stimulated serine kinase that phosphorylates pp65 has been characterized by using peptide substrates. In vivo kinase assay has revealed that the pp65-kinase was stimulated approximately 3-fold in cytosol extracts from LPS-treated macrophages. The enzymatic activity was not dependent on Ca2+ or cAMP, and not inhibited by heparine, a strong inhibitor for casein kinase II. The pp65-kinase activity in extracts from LPS-treated macrophages was preserved after rapid chromatography on a Mono Q column. These results suggest that a soluble serine kinase is rapidly activated by LPS and its properties distinguish it from protein kinases previously described in the literature.3. Role of pp65/L-plastin in the defense against Salmonella-infection : Jones, S. L., et al. recently clarified that the LPS-induced phosphorylation of the serine-5 of pp65/L-plastin, which was originally determined by us, augmented the adhesiveness of the macrophages through Mac-1 adhesion molecules. We found that Mac-1+ cells were increased in infected-spleens about 25 times as many as normal spleens, and that activities of the pp65-kinase in the cells were also increased. Splenomegaly was found to be important in the protection of the host against Salmonella-infection. Thus, it was suggested that the pp65/L-plastin-Mac-1 system play a pivotal role in the defense againt infections by rapidly accumulationg macrophages to lymphoid organs by modulating the activities of adhesion molecules.

1。在PP65/PP65家族和抗体的cDNA和重组蛋白再次制备：重组L-普拉斯汀在PET载体中用cDNA构建体转染的大肠杆菌中产生。通过用重组蛋白免疫兔子来制备多克隆抗体。我们还克隆了T-铂基因2。 LPS诱导的丝氨酸激酶的表征，该丝氨酸激酶磷酸化PP65/L-铂：LPS刺激的丝氨酸激酶的磷酸化PP65的表征是通过使用肽底物来表征的。体内激酶测定法显示，在LPS处理的巨噬细胞中，在细胞质提取物中刺激了PP65-激酶。酶促活性不依赖于Ca2+或CAMP，也不依赖于酪蛋白激酶II的强抑制剂肝素抑制。在单声道Q柱上快速色谱后，保留了来自LPS处理的巨噬细胞提取物中的PP65-激酶活性。这些结果表明，可溶性丝氨酸激酶被LPS迅速激活，其特性将其与先前在文献中描述的蛋白激酶区分开。3。 pp65/l-普拉斯汀在防御沙门氏菌感染中的作用：琼斯，S。L。等。最近澄清说，LPS诱导的PP65/L-普拉斯汀丝氨酸-5最初由我们确定的丝氨酸-5，通过MAC-1粘附分子增强了巨噬细胞的粘附性。我们发现，在受感染的肺泡中增加了MAC-1+细胞，约25倍，是正常脾的25倍，并且细胞中PP65-激酶的活性也增加了。发现脾肿大对于保护宿主免受沙门氏菌感染很重要。因此，有人提出，PP65/L-Plastin-MAC-1系统通过调节粘附分子的活性来迅速积累巨噬细胞对淋巴机器人的迅速积累，在防御再次感染中起关键作用。