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

1. pp 65/plastin家族cDNA、重组蛋白及抗体的制备：将构建的cDNA插入pET载体中转染大肠杆菌。用重组蛋白免疫家兔制备抗L-plastin的多克隆抗体。我们还克隆了T-plastin基因。LPS诱导的磷酸化pp 65的丝氨酸激酶的表征/L-plastin：LPS刺激的磷酸化pp 65的丝氨酸激酶已经通过使用肽底物表征。体内激酶测定显示，在脂多糖处理的巨噬细胞的胞质溶胶提取物中，pp 65-激酶被刺激约3倍。酶活性不依赖于Ca ~（2+）或cAMP，也不受酪蛋白激酶II的强抑制剂肝素的抑制。LPS处理的巨噬细胞提取物中的pp 65-激酶活性在Mono Q柱上快速层析后得以保留。这些结果表明，可溶性丝氨酸激酶被LPS迅速激活，其特性使其与文献中先前描述的蛋白激酶不同. pp 65/L-plastin在抵抗沙门氏菌感染中的作用：Jones，S. L.，等人最近阐明，LPS诱导的pp 65/L-plastin的丝氨酸-5的磷酸化（最初由我们确定）通过Mac-1粘附分子增强了巨噬细胞的粘附。我们发现Mac-1+细胞在感染脾中增加约25倍于正常脾，并且细胞中pp 65激酶的活性也增加。脾肿大被认为是重要的保护宿主免受沙门氏菌感染。提示pp 65/L-plastin-Mac-1系统通过调节粘附分子的活性，使巨噬细胞迅速聚集到淋巴器官，在机体抗感染中起着关键作用。

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.