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

1.pp65/纤溶酶原家族基因和重组蛋白的制备及其抗体的制备：将构建的重组蛋白转入pET载体，在大肠杆菌中表达重组L-纤溶酶原蛋白。用重组蛋白免疫兔，制备了抗L-纤溶酶原蛋白的多克隆抗体。我们还克隆了T-纤溶酶基因。脂多糖诱导的丝氨酸激酶磷酸化pp65/L-纤溶酶：一种脂多糖刺激的磷酸化pp65的丝氨酸激酶已经通过使用多肽底物进行了表征。活体蛋白激酶分析显示，在脂多糖处理的巨噬细胞胞浆提取物中，pp65-激酶被激活了大约3倍。该酶的活性不依赖于Ca~(2+)或cAMP，也不受酪蛋白激酶II的强抑制剂肝素的抑制。经Mono Q柱层析后，巨噬细胞提取液中的pp65-激酶活性得以保存。这些结果表明，可溶性丝氨酸激酶能被脂多糖迅速激活，其性质有别于先前文献中所描述的蛋白激酶。Pp65/L-纤溶酶在沙门氏菌感染防御中的作用：琼斯，S.L.，等.最近阐明，我们最初确定的脂多糖诱导的pp65/L-纤溶酶原蛋白的丝氨酸-5的磷酸化，通过Mac-1黏附分子增强了巨噬细胞的黏附。我们发现感染脾中Mac-1+细胞的数量大约是正常脾的25倍，并且细胞中pp65-激酶的活性也增加了。研究发现，脾肿大在保护宿主免受沙门氏菌感染方面很重要。因此，我们认为pp65/L-纤溶酶-Mac-1系统通过调节黏附分子的活性，使巨噬细胞迅速聚集到淋巴器官，在防御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.