Analysis of the non-self recognition mechanism of ascidian hemocytes

1)海鞘血细胞基因表达及信号转导途径分析：血细胞聚集是海鞘损伤后的早期炎症反应。我们已经报道了单抗A74强烈抑制罗氏梭子蟹的血细胞聚集。A74抗原蛋白是一种新的膜糖蛋白，它具有两个基于酪氨酸的免疫受体激活基序(ITAM)和几个在信号转导中发挥作用的基序。为了确定H.roretzi血细胞中ITAM下游的功能蛋白，我们进行了差异显示分析，以评估血细胞聚集前后差异表达的mRNAs。分离到四个扩增片段(16A、18A、20A和20G-1)，它们在诱导血细胞聚集后表达水平升高。它们的表达被A74抗体诱导，而不被对照鼠免疫球蛋白诱导，并被…强烈抑制。克隆结果表明，18A-1克隆编码一个谷胱甘肽S转移酶omega蛋白，20G-1克隆编码一个与其他蛋白几乎没有相似性的新蛋白。我们还发现，抗TCR抗体可以特异性地诱导小鼠T细胞杂交瘤GSTO基因的表达。2)H.roretzi血浆和H.roreti血细胞C3受体的调理因子分析：H.roretzi血浆中含有两种调理因子：半乳糖特异性凝集素(Gal-lectin)和补体3。利用它们的抗体和荧光激活的细胞分类器，我们发现Gal-lectin只与SRBC结合，而C3只与酵母细胞结合。此外，我们还发现H.roretzi血细胞具有整合素α亚单位蛋白，其抗体可抑制H.roretzi血细胞C3依赖的吞噬功能。这些结果表明，H.roretzi血细胞有一种整合素型受体，其功能类似于C3受体。较少

1) Analysis of gene expression and signal transduction pathway of ascidian hemocytes : Hemocyte aggregation is an early inflammatory response upon injury in the ascidian Halocynthia roretzi. We have already reported that the monoclonal antibody A74 strongly inhibits hemocyte aggregation in H.roretzi. The A74 antigen protein is a novel membrane glycoprotein, and it has two immunoreceptor tyrosine-based activation motifs (ITAMs) and several motifs that have been proposed to play a role in signal transduction. To identify the proteins that function downstream of ITAMs in H.roretzi hemocytes, we carried out a differential display analysis to evaluate mRNAs differentially expressed before and after hemocyte aggregation. Four amplicons (16A, 18A, 20A and 20G-1), the expression levels of which increased after induction of hemocyte aggregation, were isolated. Their expressions were found to be induced by treatment with A74 antibody, but not by control mouse IgG, and were strongly inhibited by … More treatment with BAPTA-AM, Wortmannin and cyclosporin A.From the results of cDNA cloning, it was revealed that 18A-1 clone encoded a glutathione S-transferase omega (GSTO) protein, and 20G-1 clone encoded a novel protein that had little similarity to other proteins. We also found that GSTO gene expression was specifically induced in mouse T-cell hybridoma by treatment with anti-TCR antibody.2) Analyses of opsonic factors in H.roretzi plasma and complement 3 receptor of H.roreti hemocytes : H.roretzi plasma contains two opsonic factors, galactose-specific lectin (Gal-lectin) and complement 3. Using their antibodies and a fluorescence-activated-cell sorter, we found that Gal-lectin bound only to SRBC, while C3 bound only to yeast cells. Furthermore, we found that H.roretzi hemocytes had an integrin α subunit protein and that its antibody inhibited C3-dependent phagocytosis of H.roretzi hemocytes. These results suggest that H.roretzi hemocytes have an integrin-type receptor that functions as a C3 receptor. Less