Biomedical subjects

A Ishimoto

Publications and source records attributed to A Ishimoto.

At least 19 recordsLinked to original sources

PubMed2003

Genistein, a protein tyrosine kinase inhibitor, suppresses the fusogenicity of Moloney murine leukemia virus envelope protein in XC cells.

XC cells are highly susceptible to syncytium formation by infection of ecotropic murine leukemia viruses (MLVs) and by expression of their envelope protein (Env). By this property, XC cells are widely used to determine titers of ecotropic MLVs. Number of plaques resulted from the syncytium formation in XC cells by ecotropic MLV infection is corresponding to number of the viral particles. XC cells had been established from a v-src-induced rat tumor. It has been reported that transformed cells are more sensitive to Mo-MLV-induced syncytium formation than non-transformed cells. To assess whether the transformation by v-src oncogene in XC cells is involved in the high sensitivity to ecotropic MLV-induced syncytium formation, XC cells were treated with genistein, a protein tyrosine kinase inhibitor. Genistein suppressed the syncytium formation between XC cells and ecotropic Env-expressing 293T cells. This result indicates that protein tyrosine kinase activity is associated with the high sensitivity of XC cells to ecotropic Env-induced syncytium formation.

Animals↗

PubMed2001

Analysis of dominant-negative effects of mutant Env proteins of human immunodeficiency virus type 1.

The Env protein of human immunodeficiency virus type 1 is assembled into a stable trimer, and oligomerization is required for maintenance of viral infectivity. This property of Env suggests that Env mutants may have a dominant-negative effect on virus infectivity. To investigate this possibility, we established a packaging cell line in which both wild-type and mutant Env proteins could be expressed simultaneously in a single cell. We analyzed the effects of two types of Env mutants: cytoplasmic tail-truncated TM mutants and a mutant defective in gp120/gp41 cleavage. The cytoplasmic tail-truncated proteins were found to be incorporated into virions by forming an oligomer with wild-type TM, but could not inhibit the wild-type function. In contrast, phenotypic mixing of cleavage-defective Env with the wild-type protein caused dramatic inhibition of infectivity, indicating that this mutant has a strong dominant-negative phenotype.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Modification of virus infectivity by cytoplasmic tail of HIV-1 TM protein.

Envelope glycoprotein incorporation is an essential process in formation of infectious particles of human immunodeficiency virus. Accumulated data have indicated that the cytoplasmic tail of Env gp41 is required for efficient incorporation. By analyzing mutant viruses with truncated cytoplasmic tails, we found that the domain was required in a cell-type-dependent manner for maintaining virus infectivity. Although the viruses with truncated cytoplasmic tails produced from HeLa, A3.01 and SupT1 cells showed a greatly reduced infectivity, those from SW480 and MT-4 cells retained a significant infectivity. To clarify the different effect of the cytoplasmic tail mutation on virus infectivity, we performed biochemical studies on the virions produced from HeLa and SW480 cells. Although the truncation of cytoplasmic tail appeared to reduce the Env incorporation in both cell lines, it caused a significant incorporation of Env precursor with HeLa cells. The results suggested that the cytoplasmic tail regulated selective incorporation of processed Env into virions in a cell-type-dependent manner.

Cell Line↗

PubMed2000

Biochemical characterization of the Drosophila axin protein.

In the Wnt/Wingless pathway, accumulation of beta-catenin/Armadillo protein is a key regulatory step. Vertebrate Axin is a negative regulator of Wnt signaling, promoting glycogen synthase kinase-3beta-mediated phosphorylation of beta-catenin and thereby destabilizing beta-catenin. Using Drosophila cell culture systems, we demonstrated that a Drosophila homolog of Axin (Daxin) inhibits Wingless-induced Armadillo accumulation and Drosophila T-cell factor-dependent transcription induced by Wingless, Dishevelled, and Armadillo. The carboxy-terminal portion of Daxin is not essential for the inhibitory activity, but a mutant only consisting of this portion behaves as a dominant-negative protein. Moreover, interactions between Daxin and Zeste-white 3, Armadillo, Dishevelled, protein phosphatase 2A and Daxin itself were shown, providing direct evidence that Daxin is a scaffold protein in the Wingless pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Functional association between the nef gene product and gag-pol region of HIV-1.

Nef gene function is diverse among virus isolates of primate immunodeficiency viruses. We found differential effects of nef mutation on the virus replication between two HIV-1 clones, NL432 and LAI. The nef mutation in NL432 affected the infectivity more severely compared with that in LAI, although the Nef functions of both clones were comparable. Analysis with a series of chimeric viruses between NL432 and LAI revealed that the gag-pol region was responsible for the differential effect of nef mutation. The functional association between Nef and gag-pol suggested that one of the potential targets of Nef was located within the gag-pol region.

Cell Line↗

PubMed2000

Resistance to Friend murine leukemia virus infection conferred by the Fv-4 gene is recessive but appears dominant from the effect of the immune system.

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). However, the resistance caused by Fv-4 is recessive in nude mice, which suggests that immunological effects play important roles in this resistance in vivo (K. Higo, Y. Kubo, Y. Iwatani, T. Ono, M. Maeda, H. Hiai, T. Masuda, K. Kuribayashi, F. Zhang, T. Lamin, A. Adachi, and A. Ishimoto, J. Virol. 71:750-754, 1997). To determine the immunological effect on the resistance in vivo, we infected immunologically immature newborn mice homozygous (Fv-4(r/r)) and heterozygous (Fv-4(r/-)) for Fv-4. Although the Fv-4(r/r) mice showed complete resistance to F-MuLV whether infected neonatally or as adolescents, the Fv-4(r/-) mice showed high sensitivity to viral proliferation and disease induction when infected as newborns but complete resistance when infected as adolescent mice. To confirm the immunological effect on the resistance in adolescent mice with the Fv-4(r/r) and Fv-4(r/-) genotypes, we examined the effect of an immunosuppressant drug, FK506, on the resistance. The mice with the Fv-4(r/r) genotype treated with FK506 still showed resistance, but the mice with the Fv-4(r/-) genotype became highly sensitive to F-MuLV infection. Flow cytometric analysis to detect the Fv-4 gene product showed that the Fv-4 gene product was expressed on the cells from newborn and adolescent mice. The Fv-4 gene product was also detected on the cells from the FK506-treated mice as well as on those from untreated mice. However, a quantitative difference in the gene product between the cells with the Fv-4(r/r) and Fv-4(r/-) genotypes was detected by indirect staining for flow cytometry. These results show that the resistance to F-MuLV infection conferred by the Fv-4 gene is originally recessive, but it looks dominant in adolescent mice mainly because of the effect of the immune system.

Animals↗

PubMed2000

Identification of Notch1 as a frequent target for provirus insertional mutagenesis in T-cell lymphomas induced by leukemogenic mutants of mouse mammary tumor virus.

In contrast to wild-type mouse mammary tumor virus (MMTV), the MMTV mutants with specific deletions in the U3 region of their long terminal repeats cause T-cell lymphomas. In 30% of T-cell lymphomas arising in BALB/c mice infected with MLA-MMTV, a leukemogenic MMTV mutant, we have found that MMTV proviruses were integrated into a short region of the Notch1 genome, so that truncated Notch1 transcripts encoding the transmembrane and the cytoplasmic domains of Notch1 protein could be expressed. Thus, Notch1 is a major target of provirus insertional mutagenesis in these T-cell lymphomas.

Animals↗

PubMed2000

Mechanisms of human papillomavirus E2-mediated repression of viral oncogene expression and cervical cancer cell growth inhibition.

The papillomavirus E2 gene product plays a pivotal role in viral replication. E2 has multiple functions, including (i) transcriptional activation and repression of viral promoters and (ii) the enhancement of viral DNA replication. It was previously reported that E2 suppressed the growth of papillomavirus-positive cervical carcinoma cell lines. In the present study, we investigated the mechanisms of E2 growth inhibition. We found that the transcriptional activation function of E2 is required for inhibition of the growth of HeLa cells as well as for transcriptional repression of the viral E6/E7 promoter. It had been previously postulated that transcriptional repression of the E6/E7 promoter results from E2 binding its cognate sites proximal to the E6/E7 promoter and displacing other cellular transcriptional factors. In this study, we report a requirement for the transcription activation function for the binding of E2 to transcriptionally active templates.

Cell Division↗

PubMed1999

Murine leukemia provirus-mediated activation of the Notch1 gene leads to induction of HES-1 in a mouse T lymphoma cell line, DL-3.

Constitutive activation of Notch signaling is known to be associated with tumorigenesis. In a mouse T lymphoma cell line, DL-3, we found that a murine leukemia provirus was inserted in the Notch1 locus, which led to marked expression of a virus-Notch1 fusion mRNA encoding an intracellular portion of the Notch1 protein. Furthermore, expression and nuclear localization of this constitutively active form of Notch1 protein were confirmed. Corresponding to this finding, the transcription of the hairy/enhancer of split (HES-1) gene, a known target of Notch1 signaling, was elevated in this cell line. A potential role for overexpressed HES-1 in the development of the lymphoma was discussed.

Amino Acid Sequence↗

PubMed1999

Characterization of mouse dishevelled (Dvl) proteins in Wnt/Wingless signaling pathway.

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, were biochemically characterized in mouse and Drosophila cell culture systems. We found that treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic beta-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with our finding in a Drosophila wing disc cell line, clone-8, that Wg treatment induced hyperphosphorylation of Dsh (Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J., and Nusse, R. (1995) Genes Dev. 9, 1087-1097). Furthermore, we showed that mouse Dvl proteins affect downstream components of Drosophila Wg signaling as Dsh does; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of beta-catenin) and Drosophila E-cadherin levels, hyperphosphorylation of Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II was shown to coimmunoprecipitate with Dvl proteins, and Dvl proteins were phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Induction of B-cell lymphoma in BALB/c nude mice with an ecotropic, B-tropic helper virus present in the murine AIDS virus stock.

The pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.

Animals↗

PubMed1999

Intracisternal type A particle-mediated activation of the Notch4/int3 gene in a mouse mammary tumor: generation of truncated Notch4/int3 mRNAs by retroviral splicing events.

The int3 oncogene was discovered as a frequent target in mouse mammary tumor virus-induced mammary tumors and encodes the intracellular domain of a Notch4/int3 protein. In one spontaneous mammary tumor, no. 9, that developed in a BALB/c mouse, we have found an insertion of a 1.2-kb sequence, consisting of a 5' long terminal repeat and gag sequences of an intracisternal type A particle (IAP) as well as an extra copy of the Notch4/int3 genomic sequences containing exons 23 and 24, into the intron between exons 24 and 25 of the Notch4/int3 gene. In this tumor, unique splicing events between the IAP and the Notch4/int3 sequences generated two types of IAP-Notch4/int3 fusion transcripts encoding two different portions of the intracellular domain of Notch4/int3 proteins: one with a RAM domain and the other without. Interestingly, these two proteins showed different subcellular localizations in a mouse mammary epithelial cell line, HC-11.

Alleles↗

PubMed1998

Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling.

Wingless (Wg) treatment of Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Dfrizzled-2. However, we found that a line of S2 cells obtained from another source express Dfrizzled-2 and Dfrizzled-1. Thus, we designated this cell line as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and DE-cadherin protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induced the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells was phosphorylated as a consequence of expression of Dfrizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells but can grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling.

Animals↗

PubMed1997

Accumulation of Armadillo induced by Wingless, Dishevelled, and dominant-negative Zeste-White 3 leads to elevated DE-cadherin in Drosophila clone 8 wing disc cells.

Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (ZW-3), and Armadillo (Arm), work sequentially; wg and dsh negatively regulate zw-3, which in turn down-regulates arm. To biochemically analyze interactions between the Wg pathway and Drosophila E-cadherin (DE-cadherin) which bind to Arm, we overexpressed Dsh, ZW-3, and Arm, in the Drosophila wing disc cell line, clone 8, which responds to Wg signal. Dsh overexpression led to accumulation of Arm primarily in the cytosol and elevation of DE-cadherin at cell junctions. Overexpression of wild-type and dominant-negative forms of ZW-3 decreased and increased Arm levels, respectively, indicating that modulation in zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevated DE-cadherin levels, suggesting that Dsh-induced DE-cadherin elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative ZW-3-, and truncated Arm-induced accumulation of DE-cadherin protein was accompanied by a marked increase in the steady-state levels of DE-cadherin mRNA, suggesting that transcription of DE-cadherin is activated by Wg signaling. In addition, overexpression of DE-cadherin elevated Arm levels by stabilizing Arm at cell-cell junctions.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Growth ability of auxiliary gene mutants of human immunodeficiency virus types 1 and 2 in unstimulated peripheral blood mononuclear cells.

Mutational studies on the Vif, Vpr, Vpu, Vpx, and Nef genes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses derived from HIV-1 NL strain and HIV-2 GH strain in unstimulated peripheral blood mononuclear cells were determined. Vif- viruses of both HIV-1 and HIV-2 did not grow at all in these cells. Similarly, no replication of HIV-2 Vpx- mutant was detected. In contrast, both of Vpr- and Nef- viruses of HIV-1 and HIV-2, and Vpu- virus of HIV-1 grew quite well in the cells. These results show, together with the data previously reported, that only Vif and Vpx are essential for HIV replication in primary blood cell cultures.

Cells, Cultured↗

PubMed1997

Human immunodeficiency virus type 1 Vpu modifies viral cytopathic effect through augmented virus release.

We characterized two human immunodeficiency virus type 1 (HIV-1) strains, HIVMCK and HIV213, which have different cytopathic effects in infected cells. HIV213 was highly cytopathic, whereas HIVMCK was not. Biological analyses of chimeric viruses from the cloned infectious DNAs of HIVMCK and HIV213 showed that the Vpu region was responsible for the differing cytopathicity of these viruses. Although HIVMCK expressed Vpu protein, HIV213 did not because of a mutation at the start codon for Vpu. The amounts of envelope glycoprotein and virus particles associated with the cell surface were significantly increased on cells infected with Vpu-deficient viruses compared with Vpu-positive viruses. These data suggest that the highly cytopathic effects of HIV213 (Vpu-deficient) are due to an accumulation of envelope glycoprotein at the infected-cell surface, which would be caused by the retention of progeny virions in the absence of Vpu-facilitated virion release.

Base Sequence↗

PubMed1997

Susceptibility of nude mice carrying the Fv-4 gene to Friend murine leukemia virus infection.

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). Despite complete resistance to ecotropic MuLV infection in mice carrying the Fv-4 gene, it is known that cells carrying the resistance gene in tissue culture do not always show resistance as extensive as that in vivo (H. Yoshikura and T. Odaka, JNCI 61:461-463, 1978). To investigate the immunological effect on resistance in vivo, we introduced the Fv-4 gene into BALB/c nude mice (Fv-4-/- nude[nu/nu]) by mating them with Fv-4 congenic BALB/c mice (Fv-4r/r nude+/+) and examined the susceptibility of the F2 progeny to F-MuLV. All BALB/c nude mice without the Fv-4 gene (Fv-4-/- nude[nu/nu]) were permissive to F-MuLV and developed erythroleukemia within 2 weeks after virus inoculation. The BALB/c nude mice with the Fv-4 gene (Fv-4r/r nude[nu/nu]) did not develop leukemia, and no or little virus was detected in the spleen 7 weeks after virus inoculation. The resistance to F-MuLV was dominant in (Fv-4 congenic BALB/c x BALB/c nude) F1 mice with the Fv-4r/- nude(nu/+) genotype as strictly as in (Fv-4 congenic BALB/c x BALB/c) F1 mice with the Fv-4r/- nude+/+ genotype. However, almost all BALB/c nude mice with the Fv-4r/- nude(nu/nu) genotype developed the disease within 7 weeks, and the virus was detected in all of their spleens even in the mice without leukemia. These results show that the resistance caused by the Fv-4 gene is recessive in nude mice and dominant in BALB/c mice. Some immunological effects, perhaps cell-mediated immunity, may play important roles in the resistance to F-MuLV infection in vivo in addition to the dosage effect of the Fv-4 product.

Animals↗

PubMed1997

Possible origin of murine AIDS-inducing sequence.

The murine AIDS (MAIDS) virus has a unique sequence in its gag p12 region. A transcript which hybridizes with this sequence is expressed in normal C57BL/6 mice. This transcript has been proposed to be the origin of the MAIDS virus, since the virus was originally isolated from radiation-induced leukemic C57BL/6 mice. The transcript, designated Edv, was molecularly cloned and sequenced. Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic defective MAIDS virus has 16-bp deletions and a 1-bp insertion in the 5' and 3' regions of the gag p12 sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the gag p12 region of the MAIDS virus is less homologous to that of the helper virus and Edv transcript due to the frameshift mutations. This suggested that the MAIDS virus was generated by such frameshift mutations in the gag p12 region during recombination between the helper virus and the Edv or a related sequence.

Animals↗