Four tyrosine residues in phospholipase C-gamma 2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling.

Activation of phospholipase C-gamma2 (PLCgamma2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Bruton's tyrosine kinase (Btk) and Syk are required for activating PLCgamma2, the exact activation mechanism of PLCgamma2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLCgamma2 as Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLCgamma2, PLCgamma2-deficient DT40 cells were reconstituted with a series of mutant PLCgamma2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCgamma2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCgamma2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCgamma2 showed greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, which have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCgamma2.

A juvenile hormone-repressible transferrin-like protein from the bean bug, Riptortus clavatus: cDNA sequence analysis and protein identification during diapause and vitellogenesis.

We found several juvenile hormone-responsive cDNAs in the bean bug, Riptortus clavatus, by using mRNA differential display (Hirai et al., 1998). One of them, a juvenile hormone-repressible cDNA, JR-3, was cloned, sequenced, characterized and identified as a transferrin (RcTf). RcTf cDNA encoded 652 amino acids with a calculated molecular weight of 71,453 Da. The deduced amino acid sequence showed significant homology with the transferin genes of several insects, Manduca sexta (43% identity), Blaberus discoidalis (43%), Aedes aegypti (43%), Drosophila melanogaster (36%), Sarcophaga peregrina (36%) and the human (25%). Antiserum was prepared by using recombinant RcTf protein expressed in Escherichia coli as an antigen. The antiserum reacted specifically with both the recombinant protein and the native protein from the bugs, with sizes of 70 and 75 kDa, respectively. The 75 kDa protein was partially purified from hemolymph of diapausing female bugs and the first ten amino acids were found to be identical to that of RcTf cDNA, indicating that the 75 kDa protein is RcTf. The tissue distribution of RcTf in the bug was examined by Western blot analysis. In diapausing animals, RcTf was detected in the fat body, hemolymph and ovary but not in the gut. In the post-diapause stage, RcTf was also detected in eggs, in addition to the fat body and ovary. These results indicate that RcTf is incorporated into the oocytes during vitellogenesis, and suggest that it may provide iron for the developing embryos.

Antitumor effects of intraarterial infusion of tumor necrosis factor/lipiodol emulsion on hepatic tumor in rabbits.

Human recombinant tumor necrosis factor (TNF) suspended in lipiodol (TNF/lipiodol emulsion) was injected via the hepatic artery, and its antitumor effects on VX2 tumor inoculated into the liver were evaluated. In TNF/lipiodol-treated rabbits, soft-X-ray study revealed an accumulation of lipiodol in the liver tumor and the TNF concentration in the tumors was significantly higher than in rabbits treated with free TNF. 7 days after the various treatments, the tumor growth ratio evaluated macroscopically was found to be significantly lower in TNF/lipiodol emulsion-treated rabbits compared to rabbits treated with either free TNF or lipiodol (p < 0.05). Microscopically, the necrotic-area ratio of the tumors in the TNF/lipiodol emulsion-treated group was also significantly greater than in any other group (p < 0.01). Pathohistologically, liver tumors treated with TNF/lipiodol emulsion revealed massive necrosis associated with occlusive thromboangitis in the tumor vessels and fibrous capsule formation around the tumor. In these rabbits, the elevation of serum transaminase after the treatment was transient and tissue damage in the surrounding noncancerous liver tissue was minimal. These findings therefore suggest that the intraarterial infusion of TNF/lipiodol emulsion may produce prominent antitumor effects, possibly due to the retention of TNF in the tumors, which causes damage to the endothelium of the tumor vessels.

Molecular cloning of a novel Ca(2+)-binding protein (calmegin) specifically expressed during male meiotic germ cell development.

During mammalian spermatogenesis, many specific molecules are expressed. We have recently identified a 93-kDa male meiotic germ cell-specific antigen (Meg 1) exclusively expressed in germ cells from the pachytene spermatocyte to the spermatid stage using the monoclonal antibody TRA 369 (Watanabe, D., Sawada, K., Koshimizu, U., Kagawa, T., and Nishimune, Y. (1992) Mol. Reprod. Dev. 33, 307-312). In this study, we cloned a cDNA representing this antigen from a mouse testis cDNA expression library, using the monoclonal antibody TRA 369. Northern blotting showed that this transcript was 2.3 kilobases in length and was expressed only in the testis and not in other somatic tissues or in the ovary. The expression of the mRNA was first detected at the pachytene spermatocyte stage of male germ cell development, and this expression was correlated with the expression of the protein. Sequence analysis of the cDNA revealed that the predicted protein consists of 611 amino acids, including a hydrophobic NH2 terminus characteristic of a signal peptide, two sets of internal repetitive sequences (four repeats of IPDPSAVKPEDWDD and GEWXPPMIPNPXYQ), and a hydrophilic COOH terminus. The deduced amino acid sequence has 58% homology with dog calnexin (the ER membrane phosphoprotein of pancreatic cells) and significant partial homology with calreticulin (high affinity Ca(2+)-binding protein of the ER membrane) at the repetitive sequence. Furthermore, we demonstrated the 45Ca2+ binding ability of this antigen by a 45Ca2+ overlay assay, and the name calmegin is proposed for this antigen. Calmegin is a novel Ca(2+)-binding protein that is specifically expressed in spermatogenesis. The highly regulated, specific, and abundant expression of calmegin suggests that it has important roles in spermatogenesis.

A useful cholesterol solvent for medical dissolution of gallstones.

Dissolution of cholesterol gallstones by direct instillation of an agent into the biliary tract has been considered an alternate to surgical procedures. We developed an excellent direct solubilizer by combining d-limonene and medium-chain monoglyceride. This mixture enhances the advantages of each individual solvent and minimizes the disadvantages. In vitro experiments were done to determine the viscosities and the cholesterol dissolving rates of various combinations, and in vivo experiments were conducted to study their irritation effects on tissues. The optimal combination was a 3:2 mixture of d-limonene and medium-chain monoglyceride. It could quickly dissolve cholesterol gallstones with minimal or no observable side effect.