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S Kyuwa

Publications and source records attributed to S Kyuwa.

At least 19 recordsLinked to original sources
PubMed2006

Identification and analysis of MHC class II DRB1 (Patr-DRB1) alleles in chimpanzees.

The MHC-DRB1 gene is known to display the most extensive allelic polymorphisms among MHC class II genes. We attempted the selective identification of chimpanzee (Pan troglodytes) DRB1 (Patr-DRB1) alleles using the polymerase chain reaction (PCR) technique in three steps: first, we performed Patr-DRB1*02 lineage-specific 8-kb PCR for *02 lineage detection in each chimpanzee; second, we performed 620-bp PCR for amplification of full-length exon 2; and finally, we carried out an insert check using the pattern of microsatellite repeat length variability. In the genomic DNA of 23 chimpanzees, nine Patr-DRB1 alleles containing two new alleles were detected. Our approach provides a relatively effective method of identifying Patr-DRB1 alleles in individual chimpanzees and should also contribute to our understanding of the features of MHC molecules in non-human primates.

Alleles↗
PubMed2005

Localization of ubiquitin carboxyl-terminal hydrolase-L1 in cynomolgus monkey placentas.

Ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) is a restrictedly expressed enzyme in neural and reproductive tissues, and it is considered to have a significant role in reproduction. In the present study, we investigated the localization of UCH-L1 in placenta of cynomolgus monkeys (Macaca fascicularis). UCH-L1 protein was detected in cytotrophoblasts of chorionic plate and villi, and decidual cells of decidua basalis in cynomolgus monkey placenta, and the amount of UCH-L1 protein in whole placenta increased as pregnancy progressed. These results supported that UCH-L1 is necessary for placental and fetal development in primate placenta. This is the first report to demonstrate the presence of UCH-L1 in primate placenta, and the cynomolgus monkey may be a useful model for the study of the functions of the ubiquitin-proteasome system in human pregnancy.

Animals↗
PubMed2005

Age-related changes of intracellular Abeta in cynomolgus monkey brains.

To confirm the intracellular accumulation of amyloid beta-protein (Abeta), we carefully performed immunohistochemistry using brains of cynomolgus monkeys of various ages. Cortical neurones and their large neurites were immunostained with antibodies against Abeta in young monkey brains. In aged monkey brains, intracellular Abeta localized within cortical neurones; no clear association was found between the presence of intracellular Abeta and senile plaques (SPs). Interestingly, we did not observe Abeta-immunoreactive cortical neurones in brains fixed with neutral buffered formalin. Western blot analyses of microsomal and nerve ending fractions derived from the brains of young to aged monkeys revealed that intracellular Abeta generation changed with age. In the microsomal fraction, the amount of Abeta42 significantly increased in brains from older monkeys (>30 years of age), and the amount of Abeta43 significantly decreased with age in the microsomal fraction. The amount of Abeta40 remained the same regardless of age. Biochemical analyses also showed that intracellular levels of each of these Abeta molecules significantly increased with age in nerve ending fractions. As we previously observed that a similar accumulation of presenilin1, beta-amyloid precursor protein (APP) and APP C-terminal fragment cleaved by beta-secretase in the nerve ending fractions obtained from brains with SPs, the accumulation of intracellular Abeta in this fraction may be closely related to formation of spontaneous SPs with age. Taken together, these results suggest that intensive investigation of age-related changes in the nerve ending will contribute to a better understanding of the pathogenesis of age-related neurodegenerative disorders such as sporadic Alzheimer's disease.

Aging↗
PubMed2005

Sequence analysis of the MHC class II DPB1 gene in chimpanzees (Pan troglodytes).

The diversity of the MHC class II region in non-human primates is a focus of biomedical research because this region plays a crucial role in the recognition of antigens in the immune system. In particular, the chimpanzee [Pan troglodytes (Patr)], which belongs to the superfamily Hominoidea, has been used as a human model for the study of diseases such as human hepatitis C virus (HCV), human hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections, to which only humans and chimpanzees are susceptible. In the present study, polymorphisms of the MHC-DPB1 gene (Patr-DPB1) in a chimpanzee colony in Japan were examined using a stepwise polymerase chain reaction (PCR) technique. In order to design a suitable primer pair which would amplify exon 2 of the Patr-DPB1 gene, a fragment of approximately 8 kb from exon 1 to exon 3 was amplified from chimpanzee genomic DNA. After designing a 500-bp primer pair at the 3' region of intron 1 and the 5' region of intron 2, analysis of DPB1 exon 2 alleles of each chimpanzee was carried out. Twenty-two chimpanzees were used in our study, and we identified seven alleles by sequence analysis on the Patr-DPB1 gene, including one new allele. The obtained nucleotide sequence patterns suggest that Patr-DPB1 alleles emerge by genetic variations such as the exchange of sequence motifs and the accumulation of point mutations.

Alleles↗
PubMed2001

Differentiation of mouse hepatitis viruses in animal facilities in Japan by use of nucleotide analysis of the nucleocapsid gene.

The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains,although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.

Animals↗
PubMed2000

Replication of enterotropic and polytropic murine coronaviruses in cultured cell lines of mouse origin.

To understand the virus-cell interactions that occur during murine coronavirus infection, six murine cell lines (A3-1M, B16, CMT-93, DBT, IC-21 and J774A.1) were inoculated with eight murine coronaviruses, including prototype strains of both polytropic and enterotropic biotypes, and new isolates. All virus strains produced a cytopathic effect (CPE) with cell-to-cell fusion in B16, DBT, IC-21 and J774A.1 cells. The CPE was induced most rapidly in IC-21 cells and was visible microscopically in all cell lines tested. In contrast, the coronaviruses produced little CPE in A3-1M and CMT-93 cells. Although most virus-infected cells, except KQ3E-infected A3-1M, CMT-93 and J774A.1 cells, produced progeny viruses in the supernatants when assayed by plaque formation on DBT cells, the kinetics of viral replication were dependent on both the cell line and virus strain; replication of prototype strains was higher than that of new isolates. There was no significant difference in replication of enterotropic and polytropic strains. B16 cells supported the highest level of viral replication. To determine the sensitivity of the cell lines to murine coronaviruses, the 50% tissue culture infectious dose of the coronaviruses was determined with B16, DBT, IC-21 and J774A.1 cells, and compared to that with DBT cells. The results indicate that IC-21 cells were the most sensitive to murine coronaviruses. These data suggest that B16 and IC-21 cells are suitable for large-scale preparation and isolation of murine coronaviruses, respectively.

Animals↗
PubMed1999

Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.

Secondary lymphoid organ chemokine (SLC) is expressed in high endothelial venules and in T cell zones of spleen and lymph nodes (LNs) and strongly attracts naive T cells. In mice homozygous for the paucity of lymph node T cell (plt) mutation, naive T cells fail to home to LNs or the lymphoid regions of spleen. Here we demonstrate that expression of SLC is undetectable in plt mice. In addition to the defect in T cell homing, we demonstrate that dendritic cells (DCs) fail to accumulate in spleen and LN T cell zones of plt mice. DC migration to LNs after contact sensitization is also substantially reduced. The physiologic significance of these abnormalities in plt mice is indicated by a markedly increased sensitivity to infection with murine hepatitis virus. The plt mutation maps to the SLC locus; however, the sequence of SLC introns and exons in plt mice is normal. These findings suggest that the abnormalities in plt mice are due to a genetic defect in the expression of SLC and that SLC mediates the entry of naive T cells and antigen-stimulated DCs into the T cell zones of secondary lymphoid organs.

Animals↗
PubMed1998

MHV-induced fatal peritonitis in mice lacking IFN-gamma.

IFN-gamma gene was disrupted by homologous recombination in A3-1 embryonic stem cells. Germinally transmitted chimeric mice were successfully obtained and backcrossed with C57BL/6 (B6) mice 5 or 6 times. Deficiency of IFN-gamma in homozygous mice was confirmed by northern blot analysis of spleen cells stimulated with phorbor esther and calcium ionophore and also by IFN-gamma production in the culture supernatant of spleen cells stimulated with the same reagents. B6 mice lacking IFN-gamma were infected intraperitoneally (ip) with 10(6) PFU of JHMV and monitored for their survival. Approximately 90% of the mice died at 50 days post-infection (pi) and the mean survival time was 28 days. Mice sacrificed at 3 weeks pi showed severe peritonitis accompanying the accumulation of a viscous fluid in the abdominal and thoracic cavities. Microscopically, the disease was characterized by disseminated granulomatous inflammation and exudative fibrinous serositis in the abdominal cavity. Infectious virus was isolated in most tissues including the liver, spleen, kidney, pancreas and lung during the experimental periods. The disease was not observed in wild-type or heterozygous littermates infected i.p. with JHMV. These results suggest that IFN-gamma plays a critical role in MHV infection in mice. This experimental model may provide a unique opportunity to address the pathogenesis of virus-induced peritonitis such as feline infectious peritonitis in cats.

Acute Disease↗
PubMed1998

Murine coronavirus-induced subacute fatal peritonitis in C57BL/6 mice deficient in gamma interferon.

Gamma interferon-deficient (IFN-gamma-/-) mice with a C57BL/6 background were infected intraperitoneally with mouse hepatitis virus strain JHM (JHMV). In contrast to IFN-gamma-+/- and IFN-gamma+/+ mice, JHMV persisted in IFN-gamma-/- mice and induced death during the subacute phase of the infection. Unexpectedly, infected IFN-gamma-/- mice showed severe peritonitis accompanying the accumulation of a viscous fluid in the abdominal and thoracic cavities in the subacute phase. Destructive changes of hepatocytes were not observed. Administration of recombinant IFN-gamma protracted the survival time of IFN-gamma-/- mice after JHMV infection. These results demonstrate that IFN-gamma plays a critical role in viral clearance in JHMV infection. They also show that a resultant persistent JHMV infection induces another form of disease in IFN-gamma-/- mice, which bears a resemblance to feline infectious peritonitis in cats.

Alanine Transaminase↗
PubMed1998

Asparagine-linked glycosylation of the rat leukemia inhibitory factor expressed by simian COS7 cells.

The leukemia inhibitory factor (LIF) is a secretory glycoprotein and a pluripotent growth factor which acts in diverse cell systems. LIF has been reported to be heavily glycosylated. In this paper, we examine the transient expression of rat LIF (rLIF) in COS7 cells and its glycosylation by a PNGaseF treatment and lectin blot. rLIF expression in COS7 cells resulted in seven molecular species being produced with zero to six N-glycosyl moieties. Mutated rLIF proteins with substitutions at the seven possible N-glycosylation sites were also expressed. An analysis of the molecular weight of the mutated rLIF confirmed the six N-glycosylation sites. Bioassays of mouse leukemia cell lines were performed to analyze the contribution of the glycosyl moieties to their functions. We found that the glycosyl moieties at each of the N-glycosylation sites were not essential to their function of the protein, but the reduced functions to promote the proliferation of DA-1a cells that had been observed for some mutants suggests a biochemical role for the in vitro function.

Amidohydrolases↗
PubMed1997

Characterization of embryonic stem-like cell lines derived from embryoid bodies.

Embryoid bodies (EB) were formed by TT2 embryonic stem (ES) cells in vitro. ES-like cell lines (ESLC) were established by culturing cells obtained by disaggregation of EB at 4, 8 and 20 days after culture, and designated ESLC4, ESLC8 and ESLC20, respectively. Flow cytometric analysis indicated that the cell surface expression of Le(a) on ESLC was less than that of original TT2 ES cells, but the expression of L-CAM was comparable. After suspension culture, all of the ESLC cells formed cystic EB in vitro. In addition, some ESLC4- and ESLC8-derived EB showed signs of beating. Although coat color chimeras were able to be produced with ESCL4 at a lower rate than parental ES cells, the cells did not contribute to germ line cells in chimeras. These results suggest that the ESLC had less pluripotent than parental ES cells and also that EB formation is not useful in obtaining pluripotent cells.

Animals↗
PubMed1997

Replication of murine coronaviruses in mouse embryonic stem cell lines in vitro.

Replication of murine coronaviruses in eight mouse embryonic stem (ES) cell lines of several genetic backgrounds was examined. Both mouse hepatitis virus (MHV) type 2 and MHV, strain A59 replicated well with no or minimal cytopathic effect in all the ES cell lines tested. The results suggest the possibility that MHV-infected ES cells may disseminate MHV in mouse colonies due to embryo manipulation.

Animals↗
PubMed1996

Maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus.

A persistently coronavirus-infected embryonic stem (ES) cell line A3/MHV was established by infecting an ES cell line, A3-1, with mouse hepatitis virus type-2. Although almost all A3/MHV cells were found infected, both A3/MHV and A3-1 cells expressed comparable levels of cell surface differentiation markers. In addition, A3/MHV cells retained the ability to form embryoid bodies. These results suggest that persistent coronavirus infection does not affect the differentiation of ES cells.

Animals↗
PubMed1996

Characterization of T cells expanded in vivo during primary mouse hepatitis virus infection in mice.

After intraperitoneal infection with mouse hepatitis virus, strain JHM (JHMV), JHMV replicated in the spleen of C57BL/6 mice for a few days but cleared within a week. The acute viral clearance coincided with moderate expansion of CD8+T cells and modest expansion of CD4+T cells, and was impaired moderately in mice depleted of CD8+T cells and completely in mice depleted of both CD4+ and CD8+T cell subsets. Flow cytometric analysis showed that expression of cell surface markers on the spleen T cells changed during JHMV infection. CD8+T cells expressing increased amounts of CD11a, CD43, CD44 and CD49d, and those expressing decreased levels of T cell receptor alpha beta, CD8, CD45RB and L-selectin were expanded in the spleen after JHMV infection. However, they did not express CD11b, CD25 or NK1.1. They used highly heterogenous V beta chains for their T cell receptors. In addition to CD11ahighCD8+T cells, CD11ahighCD4+T cells were detected transiently after JHMV infection. The virus-specific cytotoxic T lymphocyte (CTL) activity was observed in both CD4+ and CD8+ spleen T cells from mice 7 days post-infection. The present study shows the dynamics of CD8+ and CD4+T cells in the spleen during JHMV infection in mice and suggests that CD11ahighT cells may be involved in JHMV clearance in vivo because their appearance was temporally correlated with T cell-mediated viral clearance in vivo and antiviral CTL activity in vitro.

Animals↗
PubMed1996

Generation of antiviral CD11ahigh T cells in CD4+ T cell-depleted mice and adult thymectomized mice after mouse hepatitis virus infection.

The mechanism of CD11ahighCD8+ T cell induction after mouse hepatitis virus infection, which has been suggested to play a key role in the elimination of infectious virus from the spleen in C57BL/6 mice, was studied. In CD4+ T cell-depleted mice, CD11ahighCD8+ T cells were induced in the spleen and spleen cells showed virus-specific cytotoxic T lymphocyte activity after mouse hepatitis virus infection. The same results were obtained in adult thymectomized mice. These results indicate that CD11ahighCD8+ T cells can be generated after mouse hepatitis virus infection in the absence of either CD4+ T cells or the thymus.

Aging↗
PubMed1996

Role of CD4+ and CD8+ T cells in mouse hepatitis virus infection in mice.

Viral growth and histopathological changes in the liver after intraperitoneal infection with mouse hepatitis virus, strain JHM were compared among normal C57BL/6 mice, those depleted of CD4+ T cells, CD8+ T cells and both T cell subsets. Viral growth in mice depleted of CD4+ T cells increased slightly, but pathological changes resembled those in normal mice. In contrast, the hepatitis was exacerbated in mice depleted of CD8+ T cells and those depleted of both T cell subsets. These results suggest that CD8+ T cells play a key role although both T cell subsets are involved in protection against mouse hepatitis virus infection in mice.

Animals↗