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Biomedical subjects

K Omoe

Publications and source records attributed to K Omoe.

At least 19 recordsLinked to original sources
PubMed2004

Polymerase chain reaction with a primer pair in the 16S-23S rRNA spacer region for detection of Mycoplasma pulmonis in clinical isolates.

To develop a diagnostic tool to identify Mycoplasma pulmonis (M. pulmonis) in clinical isolates, we developed a polymerase chain reaction (PCR) assay using primers specific for the 16S-23S rRNA intergenic spacer region (SR) of M. pulmonis. One pair of PCR primers reacted specifically with two reference strains of M. pulmonis tested and seven samples isolated from naturally infected rats. The primer pair did not produce PCR products of the correct size from any other rodent or human mycoplasmas or cellular DNA from rodent lungs. Specificity of the PCR assay was confirmed by Southern blotting with probe specific for the SR of M. pulmonis. The PCR assay for detection of M. pulmonis established in this study is suitable for diagnosis of M. pulmonis infection in clinical cases.

Animals↗
PubMed2004

Characterization of lactate dehydrogenase-elevating virus ORF6 protein expressed by recombinant baculoviruses.

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity and can replicate only in a subset of mouse primary macrophages in vitro. Because it is difficult to grow and purify sufficient quantities of LDV virions from the primary macrophages, it has been difficult to further characterize LDV envelope proteins. A few expression systems have been reported for structural analysis of the nonglycosylated envelope protein M/VP-2, however, very few studies of the antigenicity of M/VP-2 have been reported. We cloned and expressed the ORF6 gene, which encodes the M/VP-2, as a fusion protein with a polyhistidine metal-binding tag (6 x His-tag) in Autographa californica nuclear polyhedrosis virus (baculovirus) under the control of the polyhedrin promoter. In Western blotting analysis, the expressed protein was similar in size to the native M/VP-2 plus 6 x His-tag. The usefulness of the baculovirus-expressed LDV ORF6 protein for analysis of the immunogenicity of LDV M/VP-2 was discussed.

Animals↗
PubMed2004

Production of virus-specific antiserum corresponding to sequences in the lactate dehydrogenase-elevating virus (LDV) ORF6 protein.

The elucidation of the antigenic structure of the envelope proteins of Arteriviridae which includes lactate dehydrogenase-elevating virus (LDV) will provide further understanding of a mechanism of strict host cell specificity. To analyze the linkage between LDV envelope proteins, M/VP-2 and VP-3, which may play an important role in viral infectivity, we generated specific antibody against M/VP-2 that has not been reported in previous studies. A synthetic polypeptide corresponding to the C-terminal region of LDV strain C (LDV-C) ORF6, which encodes M/VP-2, was chemically synthesized and coupled to keyhole limpet hemocyanin (KLH). The peptide was immunogenic in rabbits and induced antibody specific for viral protein. Western blotting and immunofluorescence analysis of virion M/VP-2 in infected macrophages showed that the antibody was able to react specifically with authentic virion protein. The immunoreactive antibody against LDV M/VP-2 described in this study will be useful for further studies of the specific roles of the envelope proteins in arterivirus assembly and infectivity.

Animals↗
PubMed2004

Analysis of protein expression by mammalian cell lines stably expressing lactate dehydrogenase-elevating virus ORF 5 and ORF 6 proteins.

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity. Because only a subset of mouse primary macrophages have been identified that can support LDV replication in vitro, the precise molecular mechanism of viral entry and replication remains unclear. To analyze the LDV envelope proteins, which probably mediate viral attachment to the host cell, we developed a mammalian system for stable co-expression of LDV open reading frame (ORF) 5- and ORF 6-encoded proteins (ORF 5 and ORF 6 proteins), which correspond to envelope VP-3 and M/VP-2, respectively, and compared these expressed proteins to the native ones. Western blotting analysis combined with N-glycanase digestion revealed that ORF 5 and ORF 6 proteins were similar in size to native VP-3 and M/VP-2, and that ORF 5 protein was N-glycosylated, like the native VP-3. Immunofluorescence microscopy revealed that both ORF 5 and ORF 6 proteins were distributed throughout the cytoplasm and were colocalized in most cells. Moreover, ORF 5 protein was localized both in the perinuclear region and the Golgi complex and transported to the cell surface. This mammalian expression system in which the exogenously expressed proteins closely resemble the native proteins will provide the experimental basis for further studies of the interactions between LDV envelope proteins and host cells.

Animals↗
PubMed2004

Duration and magnitude of faecal shedding of Shiga toxin-producing Escherichia coli from naturally infected cattle.

To clarify the epidemiological relationship between cattle and human infections of Shiga toxin-producing Escherichia coli (STEC), we studied the duration and magnitude of the excretion of STEC O157 and STEC O26 with rectal faeces from naturally infected cattle at a breeding farm in the Tohoku area of Japan, using microbiological methods. The prevalence of STEC O157 was 3.5% (11/324), whereas that of STEC O26 was 7.9% (14/178). Faecal shedding of STEC O157 persisted for < 1 week to 10 weeks, whereas STEC O26 persisted from < 1 week to < 3 weeks. The magnitude of faecal shedding (per 10 g) ranged from 4 to > 110,000 c.f.u. for STEC O157 and from 3 to 2400 c.f.u. for STEC O26. All isolates of both STEC serotypes contained the stx1 or stx2 genes. Pulsed-field electrophoretic analysis of both STEC serotypes identified predominantly STEC O157 type III and STEC O26 type I in isolates, suggesting that a single STEC strain may be mutated in the intestinal tract of calves. These results indicate that STEC O157 is secreted for longer periods and in higher numbers than STEC O26 from healthy calves with natural infections, suggesting that STEC O157 may have more opportunities than STEC O26 to induce human disease.

Animals↗
PubMed2001

Analysis of the epitopes on staphylococcal enterotoxin A responsible for emetic activity.

To identify which region of staphylococcal enterotoxin A (SEA) is responsible for the emetic activity, twelve synthetic peptides corresponding to the entire SEA amino acid sequence and their respective anti-peptide antibodies were prepared and tested. The anti-peptide antibodies were tested for neutralization of SEA-induced emesis in Suncus murinus (Shrew mouse). The results indicate that SEA-induced emesis was neutralized by the mixture of three anti-peptide antibodies to A-7 (corresponding to amino acid residues 121-140), A-8 (141-160) and A-9 (160-180). These findings suggest that the regions corresponding to residues 121-180 may be the epitopes responsible for the emetic activity of SEA.

Animals↗
PubMed2000

Frequency of Shiga toxin-producing Escherichia coli in cattle at a breeding farm and at a slaughterhouse in Japan.

To investigate the frequency of Shiga toxin-producing Escherichia coli (STEC) infected calves at a breeding farm and cattle at a slaughterhouse in Tohoku area of Japan, the polymerase chain reaction (PCR) was used for detection of genes for Shiga toxin(s). The fecal samples from a total of 204 calves and 306 cattle were examined. The prevalence rates in calves less than 2 months of age, cattle 2-8 months of age, and adults greater than 1 year of age were 39.4, 78.9, and 40.8%, respectively. Detection frequency of STEC in the fecal specimens from calves aged 0-8 months was not different among the breeds of cattle (Holstein: H, Japanese black cattle: B, and F1: HxB). On the other hand, for calves over 12 months of age, the frequency of STEC in Japanese black cattle and F1 were significantly higher than in Holstein cattle. Serogroups of STEC usually identified in human cases of food poisoning (O157, O26, and O111) were not frequently found in the feces of the cattle.

Abattoirs↗
PubMed2000

Evolutionary characterization of the six internal genes of H5N1 human influenza A virus.

The entire nucleotide sequences of all six internal genes of six human H5N1 influenza A viruses isolated in Hong Kong in 1997 were analysed in detail from a phylogenetic point of view and compared with the evolutionary patterns of the haemagglutinin and neuraminidase genes. Despite being isolated within a single year in the same geographical location, human H5N1 viruses were characterized by a variety of amino acid substitutions in the ribonucleoprotein complex [PB2, PB1, PA and nucleoprotein (NP)] as well as the matrix (M) proteins 1 and 2 and nonstructural (NS) proteins 1 and 2. The presence of previously reported amino acid sequences specific for human strains was confirmed in the PB2, PA, NP and M2 proteins. Nucleotide and amino acid sequence identities of the six internal genes of H5N1 viruses examined here were separated into at least two variant groups. In agreement with the above result, phylogenetic trees of the six internal genes of human H5N1 viruses were generally composed of two minor clades. Additionally, variable dendrogram topologies suggested that reassortment among viruses contributed further to the genetic variability of these viruses. As a result, it became clear that human H5N1 viruses are characterized by divergent gene constellations, suggesting the possible occurrence of genetic reassortment between viruses of the two evolutionary lineages.

DNA-Directed RNA Polymerases↗
PubMed2000

In vitro growth profiles of respiratory syncytial virus in the presence of influenza virus.

To elucidate epidemiological interference between respiratory syncytial (RSV) and influenza viruses, the influence of influenza A (HlN1) virus on the growth of RSV was examined. Although RSV grew in MDCK cells, coinfection with influenza A virus led to a reduction of progeny RSV. The degree of growth interference depended on the time of infection with influenza A virus post infection (p.i.) with RSV. In fact, infection with influenza A virus 12 hrs p.i. with RSV did not influence growth of the latter virus. On the contrary, growth suppression of influenza A virus by RSV was observed when the coinfection began at the later stages of RSV infection. Suppression of the growth of RSV by influenza A infection was further demonstrated at the level of viral protein synthesis. An indirect immunofluorescence (IF) test revealed that a large proportion of infected cells synthesized both RSV and influenza A virus antigens. Scanning electron microscopic (SEM) examination demonstrated that influenza A and RSV virions possessing surface antigens specific for each virus were selectively released from dually infected cells. In the present study, we proved for the first time that the growth of RSV is blocked by competitive infection with influenza A virus in a susceptible cell population, competitive protein synthesis and selective budding of RSV and influenza viruses from the same infected cells.

Animals↗
PubMed1999

Studies on the functional site on staphylococcal enterotoxin A responsible for production of murine gamma interferon.

To identify the functional region(s) associated with induction of gamma interferon on the staphylococcal enterotoxin A molecule, native staphylococcal enterotoxin A molecules and 12 various synthetic peptides corresponding to different regions of entire staphylococcal enterotoxin A were compared to induce gamma interferon production in murine spleen cells. The native staphylococcal enterotoxin A molecule induced gamma interferon production, whereas all of the 12 synthetic peptides did not. Pre-treatment of the murine spleen cells with synthetic peptide A-9 (corresponding to amino acid residues 161-180) significantly inhibited the staphylococcal enterotoxin A-induced gamma interferon production, whereas those with other synthetic peptides did not. When native staphylococcal enterotoxin A was pre-treated with either anti-staphylococcal enterotoxin A serum or anti-peptide sera, anti-staphylococcal enterotoxin A serum and antisera to peptides A-1 (1-20), A-7 (121-140), A-8 (141-160), A-9 (161-180) and A-10 (181-200) inhibited the staphylococcal enterotoxin A-induced gamma interferon production. From these findings, the amino acid residues 161-180 on the staphylococcal enterotoxin A molecule may be an essential region for murine gamma interferon production. Furthermore, the neutralizing epitopes may be also located on regions of amino acid residues 1-20, 121-140, 141-160 and 181-200 on the staphylococcal enterotoxin A molecule.

Amino Acid Sequence↗
PubMed1999

Emesis in the shrew mouse (Suncus murinus) induced by peroral and intraperitoneal administration of staphylococcal enterotoxin A.

Peroral and intraperitoneal administration of staphylococcal enterotoxin A (SEA) to Suncus murinus elicited an emetic response. The 50% emetic dose of SEA by peroral administration was found to be 32 microg per kg of body weight, whereas that by intraperitoneal administration was 3 microg per kg. Multiple emetic responses occurred 70 to 108 min after peroral administration of an emetic dose of SEA. Similar responses occurred 65 to 102 min after intraperitoneal injection of an emetic dose of SEA. No significant difference in vomiting was observed between male and female animals. Anti-SEA serum neutralized SEA-induced emesis in S. murinus. These findings indicate that S. murinus may serve as a suitable animal model to study the enterotoxigenicity of SEA.

Administration, Oral↗
PubMed1999

Evaluation of immune responses to inactivated influenza vaccines prepared in embryonated chicken eggs and MDCK cells in a mouse model.

This study was initiated with the isolation of influenza A and B viruses from clinical throat swabs in both fertile chicken eggs (egg) and MDCK cells, which were used in subsequent vaccine production in the above two hosts. On the basis of haemagglutination-inhibiting (HI) tests, immune mouse sera from mice vaccinated with MDCK cell-derived vaccines revealed antigenic similarities among H3N2 or B viruses isolated in MDCK cells or eggs. Similarly, antiserum prepared by immunization with egg-derived H3N2 vaccine showed equivalent antigenicity between homologous and heterologous (MDCK cell-derived) viruses. In contrast, antigenicity of egg-derived B vaccines was differed somewhat from that of MDCK cell-derived vaccines, suggesting the occurrence of antigenic change due to passaging in eggs. The time-course of immune responses based on HI titres indicated that MDCK cell-derived vaccines elicited extremely high antibody levels. Also, it was evident that antibody production by MDCK cell-grown H3N2 vaccine was very similar to that of vaccine prepared from egg-grown viruses. These results were comparable to those of plaque neutralization tests, although antigenic differences between egg- and MDCK cell-derived challenge viruses were confirmed in the test with antiserum to MDCK cell-derived vaccine. Consistent with HI-antibody production, the immunogenicity of MDCK cell-derived B vaccine appeared to be low by plaque neutralization test, while immune responses in mice which received egg-derived vaccines were significantly higher than that of the former. Furthermore, immune responses confirmed in mice immunized with B virus vaccines prepared in eggs revealed slight antigenic differences between two viruses derived from their respective hosts. Nevertheless, through evaluation of immune responses, MDCK cell-derived influenza vaccines may be useful when weak immunogenicity of B virus vaccine is improved.

Animals↗
PubMed1998

Phylogenetic analysis of the entire genome of influenza A (H3N2) viruses from Japan: evidence for genetic reassortment of the six internal genes.

Nucleotide sequences of all eight RNA segments of 10 human H3N2 influenza viruses isolated during a 5-year period from 1993 to 1997 were determined and analyzed phylogenetically in order to define the evolutionary pathways of all genes in a parallel fashion. It was evident that the hemagglutinin and neuraminidase genes of these viruses evolved essentially in a single lineage and that amino acid changes accumulated sequentially with respect to time. In contrast, amino acid differences in the internal proteins were erratic and did not accumulate over time. Parallel analysis of the phylogenetic patterns of all genes revealed that the evolutionary pathways of the six internal genes were not linked to the surface glycoproteins. Genes coding for the basic polymerase-1, nucleoprotein, and matrix proteins of 1997 isolates were closest phylogenetically to those of earlier isolates of 1993 and 1994. Furthermore, all six internal genes of four viruses isolated in the 1995 epidemic season consistently divided into two distinct branch clusters, and two 1995 isolates contained PB2 genes apparently originating from those of viruses before 1993. It was apparent that the lack of correlation between the topologies of the phylogenetic trees of the genes coding for the surface glycoproteins and internal proteins was a reflection of genetic reassortment among human H3N2 viruses. This is the first evidence demonstrating the occurrence of genetic reassortment involving the internal genes of human H3N2 viruses. Furthermore, internal protein variability coincided with marked increases in the activity of H3N2 viruses in 1995 and 1997.

Amino Acid Sequence↗
PubMed1996

Relationship between the monosomy X phenotype and Y-linked ribosomal protein S4 (Rps4) in several species of mammals: a molecular evolutionary analysis of Rps4 homologs.

Two isoforms of the human ribosomal protein S4 gene, RPS4X and RPS4Y, are located on the X and Y chromosomes. It has been postulated that haploinsufficiency of these genes may contribute to Turner syndrome. We show here that several animal species that show the Turner-like phenotype on monosomy X have no Y-linked Rps4 homolog. There may be another gene(s) that contributes to abnormal phenotypes of monosomy X. Molecular evolutionary analysis shows that the Y-linked and RPS4X-related homologs diverged prior to the radiation of placental mammals and evolved independently. Furthermore, the functional constraints against the RPS4X-related homologs are much stronger than those against the Y-linked homologs.

Animals↗
PubMed1996

K-ras gene mutation in sputum samples containing atypical cells and adenocarcinoma cells in the lung.

We analyzed the presence or absence of K-ras mutations in sputum samples with atypical cells by colony hybridization. The frequency of K-ras mutations in 104 patients with adenocarcinoma of the lung was also examined using dot blotting. No mutations in K-ras codon 12 were detected in the 12 sputum samples examined, which included three specimens from patients who later developed lung adenocarcinoma. Because of the paucity of sputum samples, it was difficult to conclude whether K-ras gene mutations are useful for molecular screening for lung cancer at the present time. Secondly, we detected 12 cases [12%; 10 males (19%) and two females (4%)] with a K-ras mutation among 104 patients with lung adenocarcinoma. Although the smoking rate of patients (89%) was higher than the general Japanese male population (approximately 60%), indicating that smoking may be related to the occurrence of adenocarcinoma of the lung, it was unclear whether smoking was related to K-ras mutation from our results. Other factors may contribute to K-ras mutation.

Adenocarcinoma↗
PubMed1995

Viability, development and incidence of chromosome anomalies of preimplantation embryos from XO mice.

We examined the viability and developmental status of XO embryos at preimplantation stage (day 3 of gestation) by assessing blastocyst formation and counting cell number using our XO mouse colony. We also examined the incidence of chromosome anomalies. Embryos from XO mice (XY, XX and XO) developed more slowly (in cell numbers and blastocoele formation) than those from XX mice (XY and XX). XO embryos also tended to develop more slowly than XX embryos in the XO group. Although litter size at the preimplantation stage of gestation was almost twice as large (12.7) as those at mid-gestation (7.6) and near-term (7.2) in this colony, the adjusted XY:XX:XO ratio (2.8:2.0:1.0) did not differ greatly. This indicates that almost half of the embryos must have been eliminated during the first half of gestation in the XO group, probably regardless of sex chromosome complements. Thus, we consider that maternal XO sex chromosome constitution is disadvantageous for the intrauterine development of the embryo during the early period of gestation. This may be related to precocious aging of XO mice. Further, we confirmed that a high incidence of abnormal karyotypes occurs in embryos from our XO mouse colony.

Animals↗
PubMed1994

Increased incidence of unpartnered single chromatids in metaphase II oocytes in 39,X(XO) mice.

Since rare cases of sex chromosome anomalies such as XXX and XXY were observed in the offspring of our XO breeder mice, we performed a cytogenetic analysis of metaphase II oocytes of XO mice to determine whether any changes in chromosomal configurations occur. We found a significantly increased incidence of unpartnered single chromatids in metaphase II oocytes of XO mice. Such single chromatids may contribute to embryonic aneuploidy. In addition, the tendency of the X-chromosome to segregate non-randomly to the oocyte rather than to the polar body was confirmed.

Animals↗