Involvement of Ceramide in the Propagation of Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in the medical and veterinary fields. Although cholesterol has been shown to participate in both the entry and replication steps of JEV, the mechanisms of infection, including the cellular receptors of JEV, remain largely unknown. To clarify the infection mechanisms of JEV, we generated pseudotype (JEVpv) and recombinant (JEVrv) vesicular stomatitis viruses bearing the JEV envelope protein. Both JEVpv and JEVrv exhibited high infectivity for the target cells, and JEVrv was able to propagate and form foci as did authentic JEV. Anti-JEV envelope antibodies neutralized infection of the viruses. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH- and clathrin-dependent endocytic pathways. Although treatment of the particles of JEVpv, JEVrv, and JEV with cholesterol drastically reduced the infectivity as previously reported, depletion of cholesterol from the particles by treatment with methyl β-cyclodextrin enhanced infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and propagation of JEVrv, and these enhancements were inhibited by treatment with an SMase inhibitor or C₆-ceramide. These results suggest that ceramide plays crucial roles in not only entry but also egress processes of JEV, and they should assist in the clarification of JEV propagation and the development of novel therapeutics against diseases caused by infection with flaviviruses.Japanese encephalitis virus (JEV) is a small, enveloped virus belonging to the family Flaviviridae and the genus Flavivirus, which also includes Dengue virus (DENV), West Nile virus (WNV), Yellow fever virus, and Tick-borne encephalitis virus (11). JEV is the most common agent of viral encephalitis, causing approximately 50,000 cases annually, of which 15,000 will die, and up to 50% of survivors are left with severe residual neurological complications. JEV has a single-stranded positive-sense RNA genome of approximately 11 kb, encoding a single large polyprotein, which is cleaved by the host- and virus-encoded proteases into three structural proteins, capsid (C), premembrane (PrM), and envelope (E), and seven nonstructural proteins. The structural proteins are components of viral particles, and the E protein is suggested to interact with a cell surface receptor molecule(s). Although a number of cellular components, including heat shock cognate protein 70 (33), glycosaminoglycans, such as heparin or heparan sulfate (21, 41), and laminin (3), have been shown to participate in JEV infection, the precise mechanisms by which these receptor candidates participate in JEV infection remain largely unclear.In addition to the many studies identifying and characterizing receptor molecules in numerous viruses, data suggesting the involvement of membrane lipids, such as sphingolipids and cholesterol, in viral infection have also been accumulating. Lipid rafts consisting of sphingolipids and cholesterol and distributing to the outer leaflet of the cell membrane have been shown to be involved in the infection of not only many viruses but also several bacteria and parasites (24), in addition to playing roles in various functions such as lipid sorting, protein trafficking (26, 47), cell polarity, and signal transduction (38). With respect to cholesterol itself, various aspects of the life cycle of flaviviruses have been shown to involve this lipid, including the entry of DENV (34), hepatitis C virus (HCV) (16), and WNV (27), the membrane fusion of tick-borne encephalitis virus (40), and the replication of HCV (14, 17), WNV (23), and DENV (35). Recently Lee et al. (20) showed that treatment with cholesterol efficiently impairs both the entry and replication steps of JEV and DENV-2 but enhances infection with the Sindbis virus (22).On the other hand, sphingolipids, including sphingomyelins and glycosphingolipids, are ubiquitous components of eukaryotic cell membrane structures, providing integrity to cellular membranes. Ceramide is one of the intermediates of sphingolipids and plays roles in cell differentiation, regulation of apoptosis and protein secretion, induction of cellular senescence, and other processes (2). Ceramide is generated from the hydrolysis of sphingomyelin by sphingomyelinase (SMase) or from catalysis by serine-palmitoyl-coenzyme A (CoA) transferase and ceramide synthase. Ceramide spontaneously self-associates to form ceramide-enriched microdomains and then to form larger ceramide-enriched membrane platforms which serve as the spatial and temporal organization for cellular signalosomes and for regulation of protein functions (2). The ceramide-enriched platforms have also been used by many pathogens to facilitate entry and infection (2). The acid SMase is activated not only by multiple stimuli, including receptor molecules, gamma irradiation, and some chemicals, but also by infection with some bacteria or viruses (36). Rhinovirus activates the SMase for generation of ceramide and forms ceramide-enriched membrane platforms that serve in the infection of target cells (10). Sindbis virus also activates the SMase and induces apoptosis through a continuous release of ceramide (15). In contrast to these viruses, ceramide inhibits infection with HIV (7) and HCV (48). Ceramide enrichment of the plasma membrane reduces expression of HCV receptor molecules through an ATP-independent internalization and impairs entry of HCV.Pseudotype and recombinant viruses based on the vesicular stomatitis virus (VSV) bearing foreign viral envelope proteins have been shown to be powerful tools for the investigation of viral entry and the development of vaccines. These systems have been used to study infection with viruses that do not propagate readily (31, 43) or that are difficult to handle due to their high-level pathogenicity for humans (42). In addition, the systems allow us to focus on the investigation of entry mechanisms of particular viral envelope proteins by using control viruses harboring an appropriate protein on identical particles.In the present study, we generated pseudotype (JEVpv) and recombinant (JEVrv) VSVs bearing the JEV envelope protein in human cell lines and determined the involvement of sphingolipids, especially ceramide, and cholesterol in infection of human cell lines with JEV. Both JEVpv and JEVrv exhibited infection of target cells via pH- and clathrin-dependent endocytosis. Treatment of cells with cholesterol impaired infection with JEVpv and JEVrv, as previously found in JEV infection (20). In contrast, treatment of cells with SMase drastically enhanced infection with both JEVpv and JEVrv and the production of infectious JEVrv particles. These results indicate that ceramide plays crucial roles in the entry and egress of JEV.     

Pretreatment intracerebral and peripheral blood immune responses in Vietnamese adults with tuberculous meningitis: diagnostic value and relationship to disease severity and outcome

Tuberculous meningitis (TBM) is the most devastating form of tuberculosis. Both intracerebral and peripheral blood immune responses may be relevant to pathogenesis, diagnosis, and outcome. In this study, the relationship between pretreatment host response, disease phenotype, and outcome in Vietnamese adults with TBM was examined. Before treatment, peripheral blood IFN-gamma ELISPOT responses to the Mycobacterium tuberculosis Ags ESAT-6, CFP-10, and purified protein derivative (PPD) were a poor diagnostic predictor of TBM. Cerebrospinal fluid IL-6 concentrations at presentation were independently associated with severe disease presentation, suggesting an immunological correlate of neurological damage before treatment. Surprisingly however, elevated cerebrospinal fluid inflammatory cytokines were not associated with death or disability in HIV-negative TBM patients at presentation. HIV coinfection attenuated multiple cerebrospinal fluid inflammatory indices. Low cerebrospinal fluid IFN-gamma concentrations were independently associated with death in HIV-positive TBM patients, implying that IFN-gamma contributes to immunity and survival. Collectively, these results reveal the effect of HIV coinfection on the pathogenesis of TBM and suggest that intracerebral immune responses, at least in HIV-negative cases, may not be as intimately associated with disease outcome as previously thought.     

Cytopathology of extramedullary plasmacytoma of the bladder: a case report

BACKGROUND: Plasmacytoma of the bladder is an extremely rare tumor, with all information concerning this neoplasm derived from case reports. It can be a major diagnostic pitfall on both histology and urine cytology. CASE: A 95-year-old woman presented with gross hematuria and a large bladder mass detected by ultrasound. The case was initially misdiagnosed as a high grade urothelial carcinoma. Since the urine cytology did not show the classical cytologic features of urothelial carcinoma, the histologic sections were reviewed and immunohistochemical staining performed. The final diagnosis was plasmacytoma of the bladder. Subsequently the patient underwent a skeletal survey and bone scan, which did not reveal any lesion suspicious for multiple myeloma. The patient was scheduled for radiotherapy. CONCLUSION: In this case of bladder plasmacytoma, urine cytology provided a clue to the diagnosis. Urine cytology can be a diagnostic tool to help make this diagnosis in the case of poorly differentiated bladder neoplasm, especially in a patient with a known history of multiple myeloma.     

On testing the competition-colonization trade-off in a multispecies assemblage

The competition-colonization trade-off has long been considered an important mechanism explaining species coexistence in spatially structured environments, yet data supporting it remain ambiguous. Most competition-colonization research examines plants and the dispersal-linked traits of their seeds. However, colonization is more than just dispersal because rapid population growth is also an important component of colonization. We tested for the presence of competition-colonization trade-offs with a commonly used artificial assemblage consisting of protozoan and rotifer species, where colonization was the ability of a species to establish populations in patches. By ranking species according to their colonization abilities and their pairwise competitive interactions, we show that these species exhibit competition-colonization trade-offs. These results reveal that the competition-colonization trade-off exists within nonplant assemblages and that even in a laboratory setting, species are constrained to be either good competitors or colonizers but not both.     

Significant decrease in tropoelastin gene expression in fibroblasts from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene. The proliferative activity of the Costello fibroblasts was about 4-fold higher than that of the normal and pathological control ones. However, no mutations were detected in the coding region of HRAS mRNA. Transduction of the bovine tropoelastin (bTE) gene with the lentiviral vector restored the elastic fiber formation and decreased the growth rate in the Costello fibroblasts. These results strongly suggest that the defect of human tropoelastin (hTE) gene expression should induce the impaired elastogenesis and enhanced proliferation of Costello fibroblasts, and that a primary cause other than the HRAS gene mutation should contribute to the pathogenesis in the present Costello case.     

In vivo transduction of an Stx-encoding phage in ruminants

We assessed the ability of a kanamycin-marked Stx phage to move into a commensal, ovine Escherichia coli strain in the ruminant gastrointestinal tract. Transduction was detected in 19/24 sheep tested, resulting in the recovery of 47 transductants. Subtherapeutic doses of the quinolone antibiotic enrofloxacin did not increase the rate of transduction.     

Serum levels of trace elements and iron-deficiency anemia in adult Vietnamese

This study was aimed at assessing the serum levels of vitamin A, copper, zinc, selenium, and iron among adult Vietnamese with and without iron-deficiency anemia. Blood was collected from adult Vietnamese living in the midland of northern Vietnam. One hundred twenty-three subjects in the age range 20–60 yr were included in the study. Anemia, where the concentration of hemoglobin in whole blood is less than 120 g/L in females and 130 g/L in males, was found in 30% (37/123) of the study population. The levels of vitamin A and selenium in the sera of anemic subjects (n=37) were significantly lower than that in nonanemic group (n=86). On the other hand, no significant differences were observed in the concentrations of copper and zinc between the two groups. This study was the first to show serum levels of trace elements in adult Vietnamese, providing useful baseline information for further studies.     

Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II

Under strong illumination of a photosystem II (PSII) membrane, endogenous superoxide anion, hydrogen peroxide, and hydroxyl radical were successively produced. These compounds then cooperatively resulted in a release of manganese from the oxygen-evolving complex (OEC) and an inhibition of oxygen evolution activity. The OEC inactivation was initiated by an acceptor-side generated superoxide anion, and hydrogen peroxide was most probably responsible for the transportation of reactive oxygen species (ROS) across the PSII membrane from the acceptor-side to the donor-side. Besides ROS being generated in the acceptor-side induced manganese loss; there may also be a ROS-independent manganese loss in the OEC of PSII. Both superoxide anion and hydroxyl radical located inside the PSII membrane were directly identified by a spin trapping-electron spin resonance (ESR) method in combination with a lipophilic spin trap, 5-(diethoxyphosphoryl)-5-phenethyl-1-pyrroline N-oxide (DEPPEPO). The endogenous hydrogen peroxide production was examined by oxidation of thiobenzamide.     

Molecular characterization of a germ-cell-specific antigen, TEX101, from mouse testis

TEX101, a glycoprotein we recently identified, is primarily characterized as a unique germ-cell-specific marker protein that shows sexually dimorphic expression during mouse gonad development. Based on data obtained from molecular biological as well as immuno-morphological studies, we believe this molecule may play a role in the process underlying germ cell formation. However, many points remain unclear as the molecular characteristics and its physiological functions are far from being completely understood. To clarify the molecular basis of TEX101, we herein report a further biochemical characterization of the molecule using testicular Triton X-100 extracts from mice. Deglycosylation studies using endoglycohydrolases that delete N-linked oligosaccharides (OS) from the molecule show that TEX101 is highly (approximately 47%) N-glycosylated. All potential N-glycosylation sites within TEX101 are glycosylated and most of these sites are occupied by endoglycosidase F2-sensitive biantennary complex type OS units. In addition, an extremely low population among TEX101 possesses only endoglycosidase H-sensitive hybrid type OS units. In studies using phosphatidylinositol-specific phospholipase C against native testicular cells or TEX101 transfectant, the enzyme treatment caused major reduction of the TEX101 expression on the cell, suggesting that TEX101, at least in part, is expressed as a glycosylphosphatidylinositol-anchored protein. Taken together, these findings will help elucidate the molecular nature of TEX101, a marker molecule that appeared on germ cells during gametogenesis.