CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37°C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37°C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37°C. The depletion of plasmalemmal cholesterol with methyl ␤-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.Recent studies have revealed that the plasma membranes of cells contain microdomains with discrete molecular compositions. Rafts are sphingolipid-and cholesterol-rich membrane microdomains that are thought of as platforms for signal transduction (39, 40). Although there are still many controversies regarding how rafts exist in living cells, it is generally agreed that cholesterol is indispensable for their integrity and that the detergent-resistant membrane (DRM) fraction is the in vitro correlate of the raft. Because acyl chains of sphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins enriched in the DRM fraction are more highly saturated than those of glycerolipids in the bulk membrane, the raft domain is thought to show less fluidity than nonraft areas of the plasma membrane. However, it is difficult to capture rafts morphologically because their shape and size are likely to change dynamically (40).On the other hand, caveolae were first defined morphologically as invaginations of the plasma membrane (49). They are also susceptible to cholesterol depletion (31). Moreover, caveolin-1, -2, and -3, which were identified as major components of caveolae (31,35,44,47), are highly enriched in the DRM fraction (2,12,14,36). Several results suggest that many molecules are shared by rafts and caveolae but that at least several molecules that are enriched in the DRM fraction are not concentrated in caveolae (11). Thus, caveolae are not simply a stabilized form of rafts, but there should be a regulatory mechanism (as yet unknown) to control the molecular distribution between caveolae and rafts.It has been shown that cross-linked raft molecules, such as GPI-anchored proteins,...

Edwardsiella tarda is a pathogen with a broad host range that infects both animals and humans. Resistance to phagocytic killing may be involved in the pathogenicity of this bacterium. Here we show that intracellular replication of E. tarda in murine macrophages is dependent on the type III secretion system and induces an anti-apoptotic effect by up-regulating anti-apoptotic NF-kappaB target genes. The wild-type strain replicates within the phagosomal membrane of macrophages; whereas the type III mutant does not. Microarray analysis shows the mRNA expression level of NF-kappaB target genes (e.g. pro-inflammatory cytokines and anti-apoptotic genes) in macrophages infected with the wild-type strain were up-regulated compared to macrophages infected with the type III mutant. Up-regulation of Bcl2a1a, Bcl2a1b, cIAP-2, and TRAF1 genes induced expression of anti-apoptotic proteins to protect macrophages from apoptosis induced by staurosporine. Further, this protection was inhibited by adding kamebakaurin, an inhibitor of NF-kappaB activation and was confirmed using an NF-kappaB reporter gene assay. Up-regulation of anti-apoptotic NF-kappaB target genes is responsible for the anti-apoptotic activity of E. tarda and is required for intracellular replication in murine macrophages.

A colorless euglenoid flagellate Peranema trichophorum shows unique unidirectional gliding cell locomotion on the substratum at velocities up to 30 micro m/s by an as yet unexplained mechanism. In this study, we found that (1) treatment with NiCl(2) inhibited flagellar beating without any effect on gliding movement; (2) water currents applied to a gliding cell from opposite sides caused detachment of the cell body from the substratum. With only the anterior flagellum adhering to the substratum, gliding movement continued along the direction of the anterior flagellum; (3) gentle pipetting induced flagellar severance into various lengths. In these cells, gliding velocity was proportional to the flagellar length; and (4) Polystyrene beads were translocated along the surface of the anterior flagellum. All of these results indicate that a cell surface motility system is present on the anterior flagellum, which is responsible for cell gliding in P. trichophorum.

Monoclonal antibodies have been raised to components on the surface of glutaraldehyde-fixed zoospores and cysts of an isolate of the pathogenic fungus Phytophthora cinnamomi. Hybridoma supernatants were screened using an immunofluorescence assay, and of 35 cell lines producing antibodies that reacted with the P. cinnamomi cells, 10 have been selected and their specificities examined in detail. The monoclonal antibodies were found to possess a valuable spectrum of taxonomic specificities, and have revealed, for the first time, the presence of isolate-specific antigens on the surface of P. cinnamomi cells. The monoclonal antibodies were tested against six isolates of P. cinnamomi, six species of Phytophthora, and three species of Pythium. In addition to the isolate-specific monoclonal antibodies, species-specific and genus-specific markers which are unambiguous in tests conducted so far have been obtained. The monoclonal antibodies have also revealed the presence of spatially restricted antigens on the surface of the zoospores. Some of these segregated antigens are species-specific and others are more general, occurring in all Phytophthora and Pythium species examined. All of the monoclonal antibodies promise to be of great assistance in investigations of the biology and taxonomy of P. cinnamomi. The methods described should be readily applicable to studies of other fungal pathogens.

Two hydrophilic, low temperature-embedding resins, Lowia y 1 K4hf and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled G a onia symplicZolia agglutinin II (GSA-11) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded mate-rial than with the other. Post-furation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were idenhifed precisely. All examined lectins, soybean agglutinin (SBA), Madura pomifera agglutinin (MFA), GSA-11, and ulex empaeus agglutinin I (UEA-I), stained mucous granules and the Golgi IntroductionHydrophilic resins have been widely used in immunocytochemistry and lectin cytochemistry. Among these resins, Lowicryl K4M has been most commonly used in electron microscopic cytochemistry (1-3). Since this resin permits low-temperature embedding and postembedding cytochemical staining, one can expect satisfactory results for both preservation of biological macromolecular structures of cells and accessibility of the cytochemical reagents to the macromolecules. Although the procedure for tissue processing is simple and widely applicable, we have sometimes experienced difficulties in making ultra-thin sections of Lowicryl K4M-embedded material.In this study, we examined another hydrophilic resin, LR White (4). This resin has been demonstrated to be suitable for intestinal tissue for subsequent post-embedding lectin cytochemistry (5-7).We combined LR White with Initiator C, which was developed for Lowicryl K4M to make low-temperature embedding possible, and thus LR White is applicable to a similar embedding procedure as for Lowicryl K4M (Dr. Ekaichi, personal communication). We first 379-385, 1992)compared these two resins on lectin cytochemistry in mucous neck cells of the gastric fundic gland, pylorocytes, and Brunner's gland cells. These cells synthesize a large amount of 0-linked glycoproteins (8)(9)(10)(11) and are known to exhibit similar staining patterns among mucus-secreting cells in the gastrointestinal tract (12). In electron microscopic cytochemistry, general ultrastructure was sometimes sacrificed to obtain specific and dense labelings. Osmification may also be omitted for efficient polymerization by ultraviolet irradiation as well as for the preservation of reactivity of macromolecules with cytochemical reagents. This causes extraction of membrane phospholipids during dehydration, which results in poor membrane contrast and cell morphology. Recently, Berryman and Rodewald (13) have introduced an improved method for post-embedding immunocytochemistry, using uranyl acetate postfixation in place of osmification, and have achieved better ultrastructural preservation. Similar applications of uranyl acetate have also been made by BEnichou et al. (14) and Roth et al. (15) to improve memb...