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Biomedical subjects

K Maeno

Publications and source records attributed to K Maeno.

At least 91 records · Page 5Linked to original sources
PubMed1980

Characterization of altered BHK cells resistant to HVJ (Sendai virus) infection.

An altered baby hamster kidney cell culture which resists the c.p.e. of HVJ (haemagglutinating virus of Japan - the Sendai strain of parainfluenza I virus) has been obtained and characterized. These cells, designated BHK-R, were originally obtained by prolonged cultivation of cells surviving HVJ infection; they have been subcultured in the presence of HVJ. No infectious virus was recovered from BHK-R cells and no evidence for the presence of HVJ antigens in the cells was demonstrated by immunofluorescent staining. When BHK-R cells were inoculated with HVJ the growth of challenge virus was suppressed and no obvious cytopathic changes were detected, while these cells normally supported the replication of mumps, influenza, Newcastle disease, vesicular stomatitis or Sindbis viruses. BHK-R cells became susceptible to HVJ infection after serial subculture in growth medium free of HVJ. It was suggested that sialic acid residues present in the surface of BHK-R cell membranes and responsible for adsorption of HVJ were split off by the action of neuraminidase of virus particles, resulting in inhibition of the attachment of challenge virus of HVJ.

Adsorption↗
PubMed1980

Growth of poliovirus in HeLa cells persistently infected with HVJ (Sendai virus).

The growth of poliovirus in a HeLa cell culture persistently infected with the hemagglutinating virus of Japan (HVJ, the Sendai strain of parainfluenza 1 virus) (HeLaHVJ) was studied. Plaques produced by poliovirus on HeLaHVJ cell monolayers were hazier, smaller and fewer than those on HeLa cells. HeLaHVJ cells were indistinguishable from normal HeLa cells with respect to adsorption rate and penetration efficiency of poliovirus. Extracellular yields of poliovirus in HeLaHVJ cells were lower, and the cytopathic changes were less than those in normal HeLa cells, while cell-associated virus growth in HeLaHVJ cells was nearly equal to that in HeLa cells. HeLaHVJ cells responded more effectively to the action of magnesium chloride, which facilitates virus release from infected cells, resulting in an cytopathic effects. No reduction in poliovirus yield could be detected in HeLa cells acutely infected with HVJ. The relationship between the inhibition of the release of poliovirus from HeLaHVJ cells and the persistent infection of the cells with HVJ is discussed.

Adsorption↗
PubMed1980

Analysis of nuclear accumulation of influenza nucleoprotein antigen in the presence of p-fluorophenylalanine.

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.

Antigens, Viral↗
PubMed1980

Enumeration of immune interferon-producing cells induced by allogeneic stimulation.

We succeeded in enumerating interferon-producing cells induced by allogeneic stimulation, and proved that they were indeed T lymphocytes. The peak level of these cells in spleen was attained on day 5 after immunication, was maintained for about 2 days, and declined thereafter. Titers of interferon produced in vitro by sensitized spleen cells were maximum on day 7. This suggests that the maturation of immune interferon-producing cells follows cell proliferation after antigen stimulation.

Animals↗
PubMed1980

Generation and maintenance of immune interferon-producing cells induced by allogeneic stimulation in mice.

When spleen cells derived from C57BL/6 mice immunized with L cells 7 days previously were cocultured with antigenic cells, immune interferon appeared in the culture fluid. We analyzed the tissue distribution of the immune interferon-producing cells (IIPC) which appeared in various lymphoid organs after allogeneic stimulation. Although fluid from cocultures of L-cell-sensitized thymocytes and L-cells could not detect interferon activity consistently, small numbers of IIPC could be detected by using the enumeration method of IIPC. The generation, maintenance, and nature of IIPC emerging in the spleen were different depending on how the host mice were immunized. Multiple antigenic stimulations were more effective and induced longer-lasting immune interferon production than a single stimulation. IIPC induced by a single stimulation appeared to be sensitive to cortisone, vinblastine, and cyclophosphamide and were relatively short lived. In contrast, IIPC induced by multiple stimulations seemed to be partially resistant to these drugs and long lived. When mice were immunized with intact L-cells, carrageenan, a known antimacrophage agent, had no effect on immune interferon production. However, when mice were immunized with solubilized L-cell antigen, this drug displayed a suppressive effect on immune interferon production.

Animals↗
PubMed1979

The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins.

The pathogenic strain Italien and the apathogenic strain Ulster of Newcastle disease virus have been compared with respect to organ tropism and spread of infection in 11-day-old chick embryos. After infection of the endodermal layer of the chorioallantoic membrane by intra-allantoic inoculation with strain Italien, high virus titres are found in all extra-embryonic membranes and fluids and in the embryo itself. Infection results in early death of the embryo. In contrast, after infection with strain Ulster by the same route of inoculation, high virus titres are found only in the allantoic sac and embryos are not killed. Inoculations with strain Italien on to the ectodermal layer through an artificial air sac results in rapid spread of infection in the chorioallantoic membrane and the embryo dies before the virus invades other tissues including the embryo. Under the same conditions of infection, strain Ulster neither spreads within chorioallantoic membrane nor does it kill the embryo. Virus spread in each germinal layer of the chorioallantoic membrane was analysed by immune fluorescence. These studies showed that endoderm as well as mesoderm and ectoderm allowed the spread of strain Italien, whereas only the endoderm is permissive for strain Ulster. These differences in host range are based upon differential activation of the virus glycoproteins by proteolytic cleavage. The glycoproteins of strain Italien are cleaved in each germinal layer, whereas those of strain Ulster are cleaved only in endoderm. These studies demonstrate that, in the system analysed here, spread of infection and organ tropism are important factors for pathogenicity and both of these factors are determined by the susceptibility of the virus glycoproteins to proteolytic cleavage.

Allantois↗
PubMed1979

Isolation and characterization of heat-resistant (HR) mutants of Newcastle disease virus.

Heat-resistant (HR) mutants (MR 70 and HR 74) of Newcastle disease virus (NDV) which exhibited significantly higher thermostability in their infectivity than wild-type virus were isolated and characterized. They differ from each other in their plaque morphology; HR 70 produces small turbid plaques, whereas those of HR 74 are large and clear. Cytopathogenicity of these mutants is much lower than that of the wild-type virus in cultured cells such as CEF, LLCMK2 and HeLa cells. Moreover, these HR mutants exhibited extended mean embryo survival times. Synthesis of cellular RNA's and proteins in cells infected with HR mutants was not significantly reduced under conditions in which synthesis of these macromolecules was strongly reduced in cells infected with wild-type virus. No significant differences were observed between HR mutants and wild-type virus in their other phenotypic characteristics such as the capacity for interferon production, growth characteristics at a low multiplicity of infection, and cleavage of viral glycoproteins in infected cells. From these findings, it was suggested that the inhibitory effect of virus infection on cellular macromolecular synthesis is a possible determinant of cytopathogenicity of NDV.

Animals↗
PubMed1978

Cross-linking of Newcastle disease virus (NDV) proteins.

The proxomity and spatial relationships of the structural proteins of Newcastle disease virus (NDV) were studied by chemical cross-linking with a series of imidoesters. When the virions were reacted by the cross-linker with a distance 6.1A or longer between the functional groups and analyzed by polyacrylamide gel electrophoresis, remarkable changes were observed in the migration patterns of the viral proteins. The most striking one was the extensive decrease in the intensity of the M protein band, and although not so strikingly, glycoprotein and nucleocapsid protein bands were reduced significantly. Instead, several protein complexes appeared at and near the top of the gels. The protein complexes formed by a reversible cross-linker, dimethyl-3,3'-dithiobispropionimidate (DTBP), were analyzed by two dimensional electrophoresis; the complexes on the first-dimension cylindrical gels were cleaved by reduction with 2-mercaptoethanol and electrophoresed laterally on the second-dimension slab gels. The results indicated that homodimers of glycoprotein, nucleocapsid protein and M protein were generated under the condition of the most gentle cross-linking employed. At the same time, however, trimer and higher homopolymers of M protein were already detectable. Under the more extensive conditions, the bulk of M protein was cross-linked to form a large protein complex with very high molecular weight. Further, small but significant amounts of glycoprotein and nucleocapsid protein were always detected in this complex. These results suggest that M protein may be present in the virion in close enough proximity to interact with each other and may further have some interactions with glycoprotein and nucleocapsid protein. On the basis of these findings possible roles of M protein in virus assembly were discussed.

Dimethyl Adipimidate↗
PubMed1977

Restitution of hemagglutinating activity to spikeless particles of HVJ (Sendai virus) by glycoprotein components of Newcastle disease virus.

Spikeless particles of HVJ (Sendai virus) lacking in hemagglutinating (HA) activity were obtained by enzymatic digestion of virions with trypsin followed by centrifugation through a sucrose gradient. When they were mixed with glycoprotein components of Newcastle disease virus (NDV) obtained by treatment of purified virions with deoxycholate (DOC), the mixture showed hemagglutination reaction, which was inhibited by anti-NDV serum, but not by anti-HVJ serum. Sedimentation profile of the HA active agents was then examined by centrifugation of the mixture of spikeless particles of HVJ (labeled with 3H-uridine) and glycoproteins of NDV (labeled with 14C-amino acid mixture). The results showed that the peak of HA activity had both of the radioactivities, and that the sedimentation rate of the HA was faster than that of spikeless HVJ but slower than that of intact HVJ. Electron micrographs of such HA active structures showed that they were morphologically closely similar to intact virion of HVJ, although they had neither hemolytic activity nor infectivity. The mixture of spikeless HVJ and glycoproteins of HVJ or NDV which were removed from virions by proteolytic enzymes, on the other hand, did not show any detectable hemagglutinating activity.

Deoxycholic Acid↗
PubMed1977

Intracellular development of membrane protein of influenza virus.

The intracellular development of membrane protein (MP) of influenza A virus was investigated by immunofluorescent staining. Monospecific antiserum was prepared by immunizing rabbits with MP eluted from SDS-polyacrylamide gels of SDS-disrupted NWS virions. In the productive infection in clone 1-5C-4 cells, MP antigen was first detected over the whole cell at 4 hr after infection, concomitantly with the appearance of hemagglutinin (HA) antigen in the cytoplasm, and bright nuclear fluorescence was then observed. Nucleoprotein (NP) antigen was detected in the nucleus prior to the appearance of fluorescence of MP antigen and thereafter the cytoplasmic fluorescence developed. Late in infection, all of these three antigens were observed predominantly in the cytoplasm with stronger fluorescence at the cell surface. Essentially similar findings were obtained in the abortive infections in L cells and BHK cells. The above results suggest that the membrane protein of influenza A virus is present in the nucleus as well as in the cytoplasm of infected cells.

Cell Line↗
PubMed1976

Surface antigens on HeLa cells persistently infected with HVJ (Sendai virus).

Surface antigens of HeLaHVJ cells, a cell line persistently infected with HVJ, were studied by fluorescent antibody staining. After absorption with concentrated HVJ virions and HeLa cells, anti-HeLaHVJ antiserum was able to demonstrate specific surface fluorescence on HeLaHVJ cells, while this serum no longer reacted with original HeLa cells nor with HVJ virions. During cytolytic infection of HeLa cells with HVJ, this specific surface antigen appeared at an early stage of infection prior to the appearance of newly synthesized HVJ viral antigens and moreover appeared in spite of the inhibition of viral protein synthesis. This antigen was detected neither on HeLa cells infected with other myxoviruses except HVJ nor on various other kinds of cells infected with HVJ. The specific surface antigen was still found on the HeLaHVJ cell surface after incubation at 38 degrees C for two days, while HVJ structural antigens on the cell surface no longer could be detected. Mild short-term treatment of HeLa cells with trypsin, neuraminidase from vibrio cholerae, phospholipase-C and hyaluronidase failed to expose specific antigen. The antigen was distinguishable from the Forssman and human blood type antigens. The mechanism of appearance of a new antigen on the surface of HeLaHVJ cells remains unclear.

Antigens↗
PubMed1975

Modification of normal cell surface by smooth membrane preparations from BHK-21 cells infected with Newcastle disease virus.

Smooth membrane fractions were prepared from the cytoplasmic extract of BHK-21 cells infected with Newcastle disease virus (NDV). These membranes exhibited high hemagglutinating, neuraminidase, and hemolytic activity but little infectivity, suggesting that they might be precursors for viral envelope. When such membranes were adsorbed to the monolayers of uninfected BHK-21 cells at 4 degrees C and then incubated at elevated temperature for a couple of hours, the cells became highly hemadsorptive even in the presence of cycloheximide. This phenomenon occurred between 15 degrees C and 25 degrees C, and was maximal at 31 degrees C, where approximately 4 times more erythrocytes were adsorbed than to the cells incubated at 4 degrees C. Immunofluorescent staining suggested that diffusion of viral antigens might occurred rapidly over the entire surface of the cells. Cell fractions containing virions induced hemadsorption in uninfected cells, too. However, induction occurred now at 31 degrees C and was maximal at 37 degrees C, and erythrocytes appeared to be adsorbed not to the entire surface of the monolayer but restricted areas of the cells. The diffusion of viral antigens on the cell surface was not so significant under these conditions. On the basis of these findings the possible role of the membranes of the smooth endoplasmic reticulum in virus replication is discussed.

Antigens, Viral↗