Biomedical subjects

Shinwa Shibata

Publications and source records attributed to Shinwa Shibata.

5 recordsLinked to original sources

PubMed2004

Tsix transcription- versus RNA-based mechanisms in Xist repression and epigenetic choice.

Recent inquiries have revealed a surprisingly large number (>2500) of naturally occurring antisense transcripts, but their function remains largely undiscovered. A better understanding of antisense mechanisms is clearly needed because of their potentially diverse roles in gene regulation and disease. A well-documented case occurs in X inactivation, the mechanism by which X-linked gene expression is equalized between XX females and XY males. The antisense gene Tsix determines X chromosome choice and represses the noncoding silencer, Xist. In principle, Tsix action may involve RNA, the act of transcription, or local chromatin. Here, we create novel Tsix alleles to distinguish transcription- versus RNA-based mechanisms. When Tsix transcription is terminated before Xist (TsixTRAP), Tsix cannot block Xist upregulation, suggesting the importance of overlapping antisense transcription. To separate the act of transcription from RNA, we knocked in Tsix cDNA in the reverse orientation (Tsix(cDNA)) to restore RNA levels in cis without concurrent transcription across Xist. However, Tsix(cDNA) cannot complement TsixTRAP. Surprisingly, both mutations disrupt choice, indicating that this epigenetic step requires transcription. We conclude that the processed antisense RNA does not act alone and that Tsix function specifically requires antiparallel transcription through Xist. A mechanism of transcription-based feedback regulation is proposed.

Cells, Cultured↗

PubMed2003

Characterization and quantitation of differential Tsix transcripts: implications for Tsix function.

In dosage compensation of female mammals, the accumulation of Xist RNA initiates silencing of one X-chromosome. Xist action is repressed by the antisense gene, Tsix, whose full-length RNA product is complementary to Xist RNA in mice. While previous work showed that Tsix transcription blocks the accumulation of Xist RNA, it is still unclear whether this repression requires the antisense RNA product or whether the antisense transcriptional movement is sufficient. A better understanding of potential mechanisms requires elucidation of Tsix RNA structure and determination of Tsix RNA copy number relative to that of Xist RNA. Previous work indicated that at least some of murine Tsix is spliced and that human TSIX truncates within the 3' end of XIST. Here, further characterization and quantitation of murine Tsix RNA reveal three new findings: first, in undifferentiated embryonic stem cells, Tsix RNA is present at 10-100-fold molar excess over Xist RNA. Second, only 30-60% of Tsix RNA is spliced at known exon-intron junctions. The nearly equal abundance of spliced and unspliced species leaves open possible roles for both isoforms. Finally, Tsix is spliced heterogeneously at the 5' end and most detectable splice variants exhibit only a 1.9 kb region of complementarity between sense and antisense RNAs. Implications for Tsix's possible mechanisms of action are discussed.

Animals↗

PubMed2002

Acute hepatic failure in IFN-gamma-deficient BALB/c mice after murine coronavirus infection.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) persists in interferon-gamma (IFN-gamma)-deficient C57BL/6 (B6-GKO) mice and results in subacute fatal peritonitis, which bears a resemblance to feline infectious peritonitis. To examine the role of other host factors in MHV infection in mice, IFN-gamma-deficient mice with a BALB/c background (BALB-GKO) were infected intraperitoneally with MHV and compared with B6-GKO mice. In contrast to B6-GKO mice, BALB-GKO mice died within 1 week due to acute hepatic failure. The viral titer of the liver in BALB-GKO mice was significantly higher than that in B6-GKO mice. All hepatocytes in BALB-GKO mice were necrotic at 5 days post-infection, which was clearly distinct from large but limited lesion in the liver from infected B6-GKO mice. The serum alanine aminotransferase activity of infected BALB-GKO mice were higher than that of B6-GKO mice and was paralleled with the severity of the pathological changes and viral titers in infected mice. Administration of exogenous IFN-gamma to BALB-GKO partially inhibited the acute death. These results indicate that BALB-GKO and B6-GKO mice clearly show different diseases following MHV infection, although wild type counterparts of both mice apparently showed the same clinical course after MHV infection.

Alanine Transaminase↗

PubMed2002

Inhibition of B16 melanoma experimental metastasis by interferon-gamma through direct inhibition of cell proliferation and activation of antitumour host mechanisms.

Interferon-gamma (IFN-gamma) has pleiotropic activities other than its antivirus action, including cell growth inhibition, natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activation, and angiogenesis inhibitory activity, and these activities are supposed to be involved in its antitumour activity. However, it has not been completely elucidated which activity is mainly involved in the tumour suppression in vivo. In this study, we analysed inhibitory mechanisms of endogenous IFN-gamma against B16 melanoma experimental metastasis. After intravenous injection of tumour cells, tumour deposits in the lungs and liver were increased and life span was shorter in IFN-gamma(-/-) mice, indicating important roles for IFN-gamma in antitumour mechanisms. Interestingly, tumour deposits were not increased in IFN-gamma receptor (R)(-/-) mice. Furthermore, only low levels of cell-mediated immunity against the tumour and activation of NK cells were observed, indicating that antimetastatic effects of IFN-gamma is not mediated by host cells. The survival period of B16 melanoma-bearing IFN-gamma R(-/-) mice was, however, shorter than wild-type mice. These observations suggest that IFN-gamma prevents B16 melanoma experimental metastasis by directly inhibiting the cell growth, although antitumour host functions may also be involved in a later phase.

Animals↗

PubMed2002

Genomic structure of the mouse 2',5'-oligoadenylate synthetase gene family.

2',5'-Oligoadenylate synthetase (2-5OAS) is one of the interferon (IFN)-induced proteins and mediates the antiviral action of IFN. In human, three classes of 2-5OAS genes (OAS1, OAS2, and OAS3) and one OAS-like gene (OASL) are reported. In mice, however, OAS genes corresponding to human OAS2 and OAS3 have not been identified. In this report, we identified six novel OAS family genes in mice by screening mouse genomic library and expressed sequence tag (EST) database. These genes include three homologs of the human OAS1 and each homologous gene of the human OAS2, OAS3, and OASL, respectively. Each gene displays 52%-65% amino acid identity to the corresponding human homologs. Nine 2-5OAS genes, except for two OASL genes, locate within the 210-kb genomic region and form a cluster. Each novel 2-5OAS gene showed a characteristic expression pattern among different tissues, and all of them were induced by polyinosinic-polycytidylic acid. Biochemical analyses using recombinant proteins produced in Escherichia coli showed that all the novel mouse 2-5OAS molecules have double-stranded RNA (dsRNA) binding ability, but they do not have 2-5OAS activity except for the OAS2 and OAS3 mouse homologs. These results show that there are at least 11 OAS genes, which are classified into four groups, in the mouse.

2',5'-Oligoadenylate Synthetase↗