The hemolytic mechanism of thermostable direct hemolysin (TDH), a possible virulence factor of Vibrio parahaemolyticus, was studied. We demonstrated that TDH acts as a "pore-forming toxin" in temperature-dependent and -independent steps. The first temperature-dependent step requires only about 1-2 min incubation at 37 degrees C and makes a "pore" with a functional diameter of approximately 2 nm. The pore size was deduced from the molecular diameter of the colloidal inhibitory polysaccharides. The formation of the pores on TDH-treated erythrocyte membranes was also demonstrated by electron microscopic examination. The second step, which is a temperature-independent lytic step, causes the erythrocytes to swell owing to a colloidal osmotic influx of water via the "pores" into cells, resulting in erythrocyte lysis (or rupture) owing to increased intracellular pressure.

Virology 306: [244][245][246][247][248][249][250][251][252][253] 2003). Previous data also suggested that a certain protein(s) synthesized only at 41°C inhibited the association of M1 with vRNP. The potential of heat shock protein 70 (HSP70) as a candidate obstructive protein was investigated. Induction of HSP70 by prostaglandin A1 (PGA1) at 37°C caused the suppression of virus production. The nuclear export of viral proteins was inhibited by PGA1, and M1 was not associated with vRNP, indicating that HSP70 prevents M1 from binding to vRNP. An immunoprecipitation assay showed that HSP70 was bound to vRNP, suggesting that the interaction of HSP70 with vRNP is the reason for the dissociation of M1. Moreover, NS2 accumulated in the nucleoli of host cells cultured at 41°C, showing that the export of NS2 was also disturbed at 41°C. However, NS2 was exported normally from the nucleus, irrespective of PGA1 treatment at 37°C, suggesting that HSP70 does not influence NS2.Influenza virus is an enveloped RNA virus belonging to the orthomyxovirus family. The viral genome consists of eight segments of negative-sense RNA which are bound to viral RNA polymerases and nucleoprotein (NP) to form a viral ribonucleoprotein complex (vRNP). When a host cell is invaded, influenza virus delivers its vRNP into the nucleus and replicates its genome. vRNP also acts as a template for mRNAs encoding virus-specific proteins. Although the viral proteins are synthesized in the cytoplasm, NP and RNA polymerases are imported into the nucleus to form new vRNP with a replicated genome. The assembly of influenza viral components, however, occurs at the plasma membrane. Therefore, new vRNP must be exported from the nucleus into the cytoplasm for viral offspring production (16). Matrix protein 1 (M1) and nonstructural protein 2/nuclear export protein (NS2/NEP) are known to be necessary for the nuclear export of vRNP (4,19,20,21,24,30). Both proteins also migrate into the nucleus and associate with vRNP (10,19,28,32,33) for transportation via the cellular machinery for nuclear export, dependent on chromosome region maintenance 1 (CRM1) protein (9,18,21).It was previously reported that influenza virus production is suppressed at 41°C in Madin-Darby canine kidney (MDCK) cells but is normal at 37°C (24). Virus-specific proteins are synthesized and vRNP is formed even at 41°C; however, vRNP cannot be exported from the nucleus at 41°C. This failure in vRNP export is due to M1 not interacting with vRNP, demonstrating that the association of M1 with vRNP is essential for the nuclear export of vRNP. To investigate why M1 is not bound to vRNP at 41°C, viral proteins were labeled with [ 35 S]methionine at 37°C, and the M1-vRNP complex formed at 37°C was chased after the culture temperature was raised to 41°C. The temperature rise caused the release of M1 from vRNP; the M1-vRNP complex formed at 37°C was dissociated at 41°C. However, in this experiment, it was found incidentally that the dissociation of the M1-vRNP complex was inhibited if the infected cells were...

ABSTRACT. Quail embryonic pectoral myoblasts fuse with each other at 35.5°C and 41°C to essentially equal extents. Whenthe myoblasts were transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV), their fusion and biochemical processes of differentiation became temperature-sensitive: their fusion occurred at 41°C, the non-permissive temperature, but not at 35.5°C, the permissive temperature, suggesting that the fusion was regulated by the viral transforming gene. Fusion of the transformed cells proceeded more rapidly and synchronously than that of the parent cells at 41°C, and was completely suppressed at the permissive temperature, unlike that of the parent cells. These transformed cells were used to examine the relationship between myogenicdifferentiation and the tyrosine kinase activity of the src gene product. In spite of the temperature sensitivity of transformation, results showed that expressions of the src gene at 35.5°C and 41°C were similar. However, the level of tyrosine-phosphorylated protein was decreased at 41°C. Moreover, myoblast fusion could occur at 35.5°C in the presence of herbimycin A, an inhibitor of the tyrosine kinase activity of the src gene product. These results indicate that the tyrosine kinase activity of the src gene product is closely associated with regulation of myogenicdifferentiation of the cells.Myogenesis involves various biochemical and morphological differential events. These events are distinct and can be seen even in vitro using established line cells derived from myoblasts. Manyworkers have thus used myogenic cells as models in studies on the regulation of differentiation (23,32,39,40). Biochemical differentiations of myoblasts have been studied especially extensively from an early stage of muscle research. Myogenic differentiation is of interest not only with respect to characteristic biochemical events, but also with respect to cell fusion. We have been studying artificial cell fusion induced by HVJ(Sendai virus) and have examined changes of the membrane induced by the virus (18-20). For comparison with this artificial cell fusion, we examined the process of myotube formation, one of the sequential processes in muscle differentiation, to obtain more information on the phenomenonof cell fusion at molecular and cellular levels.During myogenesis, myoblasts proliferate and become aligned and committed to differentiation, and then they fuse simultaneously with each other in a socalled 'fusion burst' (39). There have been many studies on myoblast fusion by various experimental approaches (6, 8, 9, ll, 21, 22, 25, 38), butitsmechanismisstillobscure. Onereason for this seems to be that no suitable experimental system in which the fusion reaction can be controlled is available. Hitherto, the mechanismof myoblast fusion has mainly been studied using established cell lines derived from myoblasts, such as the L6 rat myoblast line, and the fusion reaction has been controlled with calcium. Calcium, however, is associated not only with various physiological react...

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The influenza virus copies its genomic RNA in the nuclei of host cells, but the viral particles are formed at the plasma membrane. Thus, the export of new genome from the nucleus into the cytoplasm is essential for viral production. Several viral proteins, such as nucleoprotein (NP) and RNA polymerases, synthesized in the cytoplasm, are imported into the nucleus, and form viral ribonucleoprotein (vRNP) with new genomic RNA. vRNP is then exported into the cytoplasm from the nucleus to produce new viral particles. M1, a viral matrix protein, is suggested to participate in the nuclear export of vRNP. It was found unexpectedly that the production of influenza virus was suppressed in MDCK cells at 41 degrees C, although viral proteins were synthesized and the cytopathic effect was observed in host cells. Indirect immunofluorescent staining with anti-NP or M1 monoclonal antibody showed that NP and M1 remained in the nuclei of infected cells at 41 degrees C, suggesting that a suppression of viral production was caused by inhibition of the nuclear export of these proteins. The cellular machinery for nuclear export depending on CRM1, which mediates the nuclear export of influenza viral RNP, functioned normally at 41 degrees C. Glycerol-density gradient centrifugation demonstrated that vRNP also formed normally at 41 degrees C. However, an examination of the interaction between vRNP and M1 by immunoprecipitation indicated that M1 did not associate with vRNP at 41 degrees C, suggesting that the association is essential for the nuclear export of vRNP. Furthermore, when infected cells incubated at 41 degrees C were cultured at 37 degrees C, the interaction between vRNP and M1 was no longer detected even at 37 degrees C. The results suggest that M1 synthesized at 41 degrees C is unable to interact with vRNP and the dissociation of M1 from vRNP is one of the reasons that the transfer of vRNP into the cytoplasm from the nucleus is prevented at 41 degrees C.

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Key words: myoblast differentiation/monoclonal antibody/myoblast fusion with HVJ (Sendai virus)/artificial myotube /kinase activity of src gene product ABSTRACT. Quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV) proliferate and do not differentiate at 35.5°C, the permissive temperature for the virus, whereas their myoblast differentiation proceeds at 41.0°C, a non-permissive temperature. In this experimental system, myogenic differentiation is controlled by src gene products. Using QM-RSV cells as an antigen, a monoclonal antibody, Mb-N3, was prepared. Expression of Mb-N3 antigen was found to increase during differentiation. Therefore, in studies on the mechanism of myogenic differentiation, we examined the expression of Mb-N3 antigen on spontaneously forming myotubes formed at 41.0°C and fused myoblasts with hemagglutinating virus of Japan (HVJ, Sendai virus) disregarding programmed processes for myogenic differentiation. When the myoblasts cultured at 35.5°C were treated with HVJ, they fused with each other. These fused myoblasts were elongated and were morphologically similar to spontaneously forming myotubes. Thus, we called fused myoblasts with HVJ "artificial myotubes. " During culture at 35.5°C, the artificial myotubes did not show increased expression of Mb-N3 antigen and increase of creatine kinase activity, which are markers of normal biochemical differentiation. When artificial myotubes were cultured at 41.0°C, expression of Mb-N3 antigen and creatine kinase activity increased. These results suggest that the expression of the antigen is regulated by kinase activity derived from src gene products even after compulsory cell fusion. Moreover, compulsory fusion does not cause myogenic differentiation and expression of Mb-N3 antigen. Thus it seems that the differentiation program must proceed in order for myogenic differentiation and expression of Mb-N3 antigen to take place.

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