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...

Rat basophilic leukemia (RBL-2H3) cells, which exhibit Ca2+-dependent secretion of granules when stimulated with antigen or the Ca2+-ionophore A23187, were observed under a video-enhanced light/fluorescence microscope. Exocytotic events of individual granules were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in the time course. The earlier one was inhibited by selective inhibitors of protein kinase C (Ro31-8425, Ro31-8220, and chelerythrine) and the other was inhibited by an inhibitor of phosphatidate hydrolase, propranolol. Exocytosis by antigen stimulation, however, showed only one peak, which was inhibited by the selective inhibitors of protein kinase C, but not by propranolol. These results indicate that at least two distinct components of exocytosis exist in RBL-2H3 cells.

The Type III secretion system is essential for intracellular replication of Edwardsiella tarda in phagocytes of fish and mammals. We identified the secreted proteins of the Type III secretion system by comparing the wild-type strain and the Type III mutant mET1229. The wild-type strain secreted 55, 25, and 22 kDa proteins into the culture supernatant, whereas the Type III mutant did not. These proteins were identified as EseB, EseC, and EseD and are similar in sequence to Salmonella SseB, SseC, and SseD that function as a translocon. The EseB, EseC, and EseD knockout mutants did not replicate in murine macrophages, suggesting that these proteins are essential for intracellular replication of E. tarda. Highest secretion of EseBCD proteins was observed when bacterial cells were cultured in neutral and alkaline pHs but not in acidic pH. When the pH of the phagosomes was examined using an acidotropic probe, the phagosomes containing the wild-type strain showed neutral pH, whereas those containing the Type III mutant exhibited acidic pH. These results suggest that the Type III-dependent interference with formation of the acidic environment in phagosomes is essential for intracellular replication of bacteria in murine macrophages. KEY WORDS: Edwardsiella tarda · EseBCD proteins · Translocon · Phagosomal pH · Macrophage Resale or republication not permitted without written consent of the publisherDis Aquat Org 84: [115][116][117][118][119][120][121] 2009 macrophages is important for efficient intracellular growth of bacteria in the macrophages (Okuda et al. 2006). However, only scant information is available on the pathogenicity of E. tarda.Many gram-negative pathogenic bacteria use a conserved protein secretion machinery termed 'Type III secretion system' (TTSS) to transport virulence factors and cause disease. The TTSS apparatus consists of approximately 20 to 25 proteins. The unique feature of the TTSS is a needle-like structure through which particular proteins (termed 'effectors') are injected into host cells. Effectors play an important role in the pathogenic relationship between host and bacterium. Injection of effectors into host cells occurs through pores (termed 'translocons)' formed in the host cell membrane by the TTSS using Type III-secreted proteins (Ghosh 2004). The TTSS encoded within Salmonella pathogenicity island 2 (SPI2) is essential for its intracellular accumulation in macrophages. SseB, SseC, and SseD proteins secreted from the TTSS encoded within SPI2 are reported to function as translocons (Nikolaus et al. 2001). Using TnphoA transposon tagging and the proteomics approach, a TTSS was found in Edwardsiella tarda that was similar to that encoded within SPI2 of Salmonella (Srinivasa Rao et al. 2003, Tan et al. 2005. Three TTSS proteins of E. tarda, homologous to SseBCD were identified and these proteins were designated as EseB, EseC, and EseD (Srinivasa Rao et al. 2004, Tan et al. 2005. We reported that, similar to Salmonella, the TTSS encoded within E. tarda is required for its intra...

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