An attenuated LC16m8 smallpox vaccine: analysis of full-genome sequence and induction of immune protection

The potential threat of smallpox bioterrorism has made urgent the development of lower-virulence vaccinia virus vaccines. An attenuated LC16m8 (m8) vaccine was developed in 1975 from the Lister strain used in the World Health Organization smallpox eradication program but was not used against endemic smallpox. Today, no vaccines can be tested with variola virus for efficacy in humans, and the mechanisms of immune protection against the major intracellular mature virion (IMV) and minor extracellular enveloped virion (EEV) populations of poxviruses are poorly understood. Here, we determined the full-genome sequences of the m8, parental LC16mO (mO), and grandparental Lister (LO) strains and analyzed their evolutionary relationships. Sequence data and PCR analysis indicated that m8 was a progeny of LO and that m8 preserved almost all of the open reading frames of vaccinia virus except for the disrupted EEV envelope gene B5R. In accordance with this genomic background, m8 induced 100% protection against a highly pathogenic vaccinia WR virus in mice by a single vaccination, despite the lack of anti-B5R and anti-EEV antibodies. The immunogenicity and priming efficacy with the m8 vaccine consisting mainly of IMV were as high as those with the intact-EEV parental mO and grandparental LO vaccines. Thus, mice vaccinated with 10(7) PFU of m8 produced low levels of anti-B5R antibodies after WR challenge, probably because of quick clearance of B5R-expressing WR EEV by strong immunity induced by the vaccination. These results suggest that priming with m8 IMV provides efficient protection despite undetectable levels of immunity against EEV.     

Human corneal epithelial cells respond to ocular-pathogenic, but not to nonpathogenic-flagellin

In this study, we investigated the expression of TLR5 in human corneal epithelial cells (CEC), and the functional outcome of TLR5 triggering by flagellins of pathogenic- and nonpathogenic bacteria. Flagellins derived from Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescense or Bacillus subtilis were used. The TLR5 protein and TLR5 specific mRNA expression was evident on human CEC. In human corneal epithelium tissues, TLR5 protein was detected at the basal and wing cells of the tissues. Ocular pathogenic bacteria, namely P. aeruginosa and S. marcescense, derived flagellin induced the significantly increased level of gene activation and IL-6 and IL-8 production. In contrast, ocular nonpathogenic S. typhimurium- and B. subtilis-derived flagellin induced neither the gene activation nor the increased production of IL-6 and IL-8 in human CEC. Human CEC would respond only to flagellin derived of ocular pathogenic bacteria, but not to those derived of ocular nonpathogenic bacteria, to generate pro-inflammatory cytokines.     

Functional Analyses of LONELY GUY Cytokinin-Activating Enzymes Reveal the Importance of the Direct Activation Pathway in Arabidopsis

Cytokinins play crucial roles in diverse aspects of plant growth and development. Spatiotemporal distribution of bioactive cytokinins is finely regulated by metabolic enzymes. LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct activation pathway in rice (Oryza sativa) shoot meristems. In this work, nine Arabidopsis thaliana LOG genes (At LOG1 to LOG9) were predicted as homologs of rice LOG. Seven At LOGs, which are localized in the cytosol and nuclei, had enzymatic activities equivalent to that of rice LOG. Conditional overexpression of At LOGs in transgenic Arabidopsis reduced the content of N⁶-(Δ²-isopentenyl)adenine (iP) riboside 5′-phosphates and increased the levels of iP and the glucosides. Multiple mutants of At LOGs showed a lower sensitivity to iP riboside in terms of lateral root formation and altered root and shoot morphology. Analyses of At LOG promoter:β-glucuronidase fusion genes revealed differential expression of LOGs in various tissues during plant development. Ectopic overexpression showed pleiotropic phenotypes, such as promotion of cell division in embryos and leaf vascular tissues, reduced apical dominance, and a delay of leaf senescence. Our results strongly suggest that the direct activation pathway via LOGs plays a pivotal role in regulating cytokinin activity during normal growth and development in Arabidopsis.     

Changes of Gap and Tight Junctions during Differentiation of Human Nasal Epithelial Cells Using Primary Human Nasal Epithelial Cells and Primary Human Nasal Fibroblast Cells in a Noncontact Coculture System

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.     

Molecular characterization of a novel salt-inducible gene for an OSBP (oxysterol-binding protein)-homologue from soybean

Oxysterol-binding protein (OSBP) and its homologues constitute a protein family in many eukaryotes from yeast to humans, which are involved in cellular lipid metabolism, vesicle transport and signal transduction. In this study, we characterized a novel salt-inducible gene for an OSBP-homologue from soybean (Glycine max [L.] Merr.). The soybean OSBP-homologous gene, denoted as G. max OSBP (GmOSBP), encoded a 789 aa putative protein with two characteristic domains; the pleckstrin homology (PH) domain and the ligand-binding (LB) domain, in the N- and C-terminus, respectively. The GmOSBP-PH domain showed localization into/around the nucleus in a transient subcellular localization assay. The phylogenetic relationship of the GmOSBP-LB domain to those in other OSBP-homologues suggested that GmOSBP might bind a lipid molecule(s) different from the ligand-candidates found for the human/yeast OSBP-homologues. The GmOSBP gene was constitutively transcribed in all of the soybean organs examined--root, stem and trifoliate leaf--at low levels and was highly induced in all these organs by high-salt stress (300 mM NaCl). Interestingly, gene expression of GmOSBP was also markedly induced in the senesced soybean cotyledon, which contains high levels of a variety of cellular lipids utilized for energy for germination and as membrane components. Therefore, we suggest that GmOSBP may be involved in some physiological reactions for stress-response and cotyledon senescence in the soybean.     

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.     

A compound possessing antitumor and interferon-inducing activities derived from the common antigen (OEP) of Pseudomonas aeruginosa.

OEP, a component consisting mainly of protein with small amounts of lipids and sugars, has been isolated from the autolysate of Pseudomonas aeruginosa and purified by physicochemical methods. It possesses remarkable biological properties, showing antitumor and interferon-inducing activities. As regards the antitumor activity of the sample, the ED50 value against ascites sarcoma-180 was 1 microgram/kg mouse/day, and its interferon-inducing activity amounted to 15 units at a concentration of 0.01 microgram/ml. Both activities increased after protease digestion, reaching about ten times those of the sample which had not undergone digestion. The protease-treated OEP contained 17% protein, 14.5% total sugars, 31% lipids, 12.5% hexosamine, 3.8% KDO, and 2.7% phosphorus. Neutral sugars consisted of 12.4% rhamnose, 2.7% mannose, 66.9% glucose, and other unidentified material. Total lipids derived from OEP consisted of 65% loosely-bound and 35% covalently-bound lipids; the former contained C14:10, C16:0, C16:1, C18:0, and C15:1 acids and the latter, beta-OH C10:0, C12:0, alpha-OH C12:0, beta-OH C12:0, C16:0, and C16:1 acids. The antitumor and interferon-inducing activities of OEP remained after the removal of loosely-bound lipids from OEP.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.