Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus

Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50–100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes.     

4,6-Dimethoxy-1,3,5-triazine oligoxyloglucans: Novel one-step preparable substrates for studying action of endo-β-1,4-glucanase III from Trichoderma reesei

Two kinds of 4,6-dimethoxy-1,3,5-triazine (DMT) oligoxyloglucans, DMT-β-XXXG and DMT-β-XLLG, have been synthesized via one-step procedure starting from the corresponding unprotected oligoxyloglucans in water. The resulting DMT derivatives were found to be hydrolyzed by endo-β-1,4-d-glucanase III from Trichoderma reesei (EGIII) and utilized as substrates for determination of the kinetic parameters of EGIII. The present DMT-method would be a convenient analytical tool for studying the action of glycosyl hydrolases due to the extremely simple synthetic process of DMT-glycosides without using protecting groups.     

Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores

Here we describe the functional relationship between YabG and transglutaminase (Tgl), enzymes that modify the spore coat proteins of Bacillus subtilis. In wild-type spores at 37 degrees C, Tgl mediates the crosslinking of GerQ into higher molecular mass forms; however, some GerQ multimers are found in tgl mutant spores, indicating that Tgl is not essential. Immunoblotting showed that spores isolated from a yabG mutant after sporulation at 37 degrees C contain only very low levels of GerQ multimers. Heat treatment for 20 min at 60 degrees C, which maximally activates the enzymatic activity of Tgl, caused crosslinking of GerQ in isolated yabG spores but not in tgl/yabG double-mutant spores. In addition, the germination frequency of the tgl/yabG spores in the presence of l-alanine with or without heat activation at 60 degrees C was lower than that of wild-type spores. These findings suggest that Tgl cooperates with YabG to mediate the temperature-dependent modification of the coat proteins, a process associated with spore germination in B. subtilis.     

Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, independent of vacuolation

Helicobacter pylori vacuolating cytotoxin, VacA, which causes vacuolation of gastric epithelial cells and other types of cultured cells, is known to stimulate apoptosis via a mitochondria-dependent pathway. In the present study, we examined the mechanisms of VacA-induced mitochondrial damage. Intracellular VacA localization was monitored by immunostaining and confocal microscopy; in AZ-521 cells in which cytochrome c release was stimulated, most of VacA was localized to vacuoles rather than mitochondria. VacA reduced the membrane potential of isolated mitochondria without inducing cytochrome c release, suggesting that it did not act directly to induce cytochrome c release from mitochondria and that in intact cells, VacA-induced cytochrome c release involved apoptosis-related factor(s), such as a proapoptotic Bcl-2 family protein. In agreement, flow cyto-metric analyses using antibodies specific for activated Bax revealed that intracellular Bax was activated by VacA in a concentration- and time-dependent manner. Using active form-specific antibodies, we also observed that the Bcl-2 family protein, Bak, was activated. By confocal microscopy, Bax and Bak were activated in AZ-521 cells in which cyto-chrome c release was induced by VacA. In addition, small interfering RNA-induced silencing of the bax gene resulted in reduction of VacA-stimulated cytochrome c release, consistent with a contribution of VacA-induced Bax activation to cytochrome c release. NH4Cl enhanced both VacA-induced vacuolation and Bax activation, whereas Bax activation was not inhibited by bafilomycin A1, which inhibited vacuolation caused by VacA. These results suggest that VacA acts through different signaling pathways to induce apoptosis via Bax activation, independent of vacuolation.     

Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell

Plant cells have no beta1,4-galactosylated and sialylated glycan, which plays important roles in biological functions in animal cells. Previously, we generated transgenic tobacco BY2 suspension-cultured cells that produced human beta1,4-galactosyltransferase [N.Q. Palacpac, S. Yoshida, H. Sakai, Y. Kimura, K. Fujiyama, T. Yoshida, T. Seki, Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation pattern, Proc. Natl. Acad. Sci. USA 96 (1999) 4692-4697]. In this study, we introduced two critical genes encoding human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter into tobacco suspension-cultured cell to pave a route for sialic biosynthetic pathway. The recombinant human proteins showed their biological activities. These results show that the plant cell can be a useful bioreactor for the production of mammalian glycoproteins.     

All-trans retinoic acid increases expression of aquaporin-5 and plasma membrane water permeability via transactivation of Sp1 in mouse lung epithelial cells

Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in lacrimal glands, salivary glands, and distal lung. Several studies using AQP5 knockout mice have revealed that AQP5 plays an important role in maintaining water homeostasis in the lung. We report here that all-trans retinoic acid (atRA) increases plasma membrane water permeability, AQP5 mRNA and protein expression, and AQP5 promoter activity in MLE-12 cells. The promoter activation induced by atRA was diminished by mutation at the Sp1/Sp3 binding element (SBE), suggesting that the SBE mediates the effects of atRA. In addition, atRA increased the binding of Sp1 to the SBE without changing the levels of Sp1 in the nucleus. Taken together, our data indicate that atRA increases AQP5 expression through transactivation of Sp1, leading to an increase in plasma membrane water permeability.     

Potential role of leptin in endochondral ossification.

Leptin, a 16-kD circulating hormone secreted mainly by white adipose tissue, is a product of the obese (ob) gene. Leptin acts on human marrow stromal cells to enhance differentiation into osteoblasts and inhibit differentiation into adipocytes. Leptin also inhibits bone formation through a hypothalamic relay. To obtain a better understanding of the potential role of leptin in bone formation, the localization of leptin in endochondral ossification was examined immunohistochemically. High expression of leptin was identified in hypertrophic chondrocytes in the vicinity of capillary blood vessels invading hypertrophic cartilage and in a number of osteoblasts of the primary spongiosa beneath the growth plate. The hypertrophic chondrocytes far from the blood vessels were negative for leptin. Moreover, we detected the production and secretion of leptin by a mouse osteoblast cell line (MC3T3-E1) and a mouse chondrocyte cell line (MCC-5) by RT-PCR, immunocytochemistry, and Western blotting. Leptin enhanced the proliferation, migration, tube formation, and matrix metalloproteinase-2 (MMP-2) activity of human endothelial cells (HUVECs) in vitro. These findings suggest the possibility that leptin exerts its influence on endochondral ossification by regulating angiogenesis.     

Structural and Functional Analyses of Photosynthetic Regulatory Genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus

Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans, species that demonstrate weak or no oxygen repression of photosystem synthesis. This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from Rhodobacter capsulatus and Rhodobacter sphaeroides. These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system. The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif. The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen. A Rhodovulum sulfidophilum mutant lacking regA or regB was constructed. These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in Rhodobacter species. Rhodobacter capsulatus regA- or regB-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with reg genes from Rhodovulum sulfidophilum and Roseobacter denitrificans. These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.