PubMed Health⌕ Search

Biomedical subjects

S Yonehara

Publications and source records attributed to S Yonehara.

At least 19 recordsLinked to original sources
PubMed1999

Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production.

Tumor necrosis factor-alpha (TNF-alpha) exerts two separate effects on neutrophils, stimulating effector functions while simultaneously inducing apoptosis. We examined here the involvement of caspases in neutrophil apoptosis and the effect of TNF-alpha-induced apoptosis on reactive oxygen production. Immunoblotting and affinity labeling showed activation of caspase-8, caspase-3, and a caspase with a large subunit of 18 kD (T18) in TNF-alpha-treated neutrophils. Active caspase-6 and -7 were not detectable in this cell type. Caspase-8 activated caspase-3 and T18 in neutrophil cytoplasmic extracts. zVAD-fmk blocked neutrophil apoptosis, in parallel with the inhibition of caspase activation. TNF-alpha-induced caspase activation was accompanied by a decrease in the ability of neutrophils to release superoxide anion. Conversely, TNF-alpha treatment in the presence of zVAD-fmk caused a prolonged augmentation of superoxide release. Granulocyte-macrophage colony-stimulating factor inhibited TNF-alpha-induced caspase activation and apoptosis, while reversing the diminution in superoxide release. These observations not only suggest that a caspase cascade mediates apoptotic events and downregulates oxygen radical production in TNF-alpha-treated neutrophils, but also raise the possibility that suppression of caspase activation with enhanced proinflammatory actions of TNF-alpha may underlie the pathogenesis of inflammatory diseases.

Adult↗
PubMed1998

The development of autoimmune inflammatory arthropathy in mice transgenic for the human T cell leukemia virus type-1 env-pX region is not dependent on H-2 haplotypes and modified by the expression levels of Fas antigen.

Previously, we reported that human T cell leukemia virus type-1 env-pX region-introduced transgenic (pX-Tg) mice develop an inflammatory polyarthropathy. Although autoimmune pathogenesis was suggested, the detailed mechanisms remain to be elucidated. In this report, we examined effects of the MHC and fas genes on the development of the disease. When pX-Tg mice were backcrossed with different inbred strains, the incidence of arthritis differed among strains; 64% and 72% in BALB/cAn (H-2d), 25% and 46% in C3H/HeN (H-2k), and 0% and 2% in C57BL/6J (H-2b) background at 3 and 6 months of age, respectively. Rheumatoid factor levels in the serum correlated with the susceptibility to the disease, whereas IL-1beta and MHC gene expression were similarly elevated in all of these strains, suggesting involvement of immune regulatory genes in this strain difference. However, introduction of the H-2d locus into C57BL/6J pX-Tg mice did not increase the incidence of arthritis, and substitution of the BALB/cAn H-2 locus with the H-2b did not decrease it. The results indicate that the H-2 locus is not the major determinant of the disease. Then, since previous study indicated a defect in Fas-mediated apoptosis of transgenic T cells, the effects of fas gene modification on the disease were examined. The incidence increased when these pX-Tg mice were crossed with lpr/lpr mice, while it decreased when crossed with fas-transgenic mice. These observations suggest that aberration of Fas-mediated apoptosis of peripheral lymphocytes, rather than negative selection in the thymus, is involved in the development of autoimmune arthropathy in pX-Tg mice.

Animals↗
PubMed1998

Redox regulation of caspase-3(-like) protease activity: regulatory roles of thioredoxin and cytochrome c.

Oxidative stress induces a variety of cellular responses, including apoptosis, and caspase family proteases are known to be involved in apoptosis. Caspase-3(-like) protease activity was examined in Jurkat T cells to investigate the mechanism of apoptosis induced by a thioloxidant, diamide. Caspase-3 was activated when cells were cultured with 200 microM diamide that induced apoptosis, whereas no caspase-3 activation was detected with 500 microM diamide that induced necrosis. When apoptosis was induced in cells with exposure to 200 microM diamide, the intracellular thioredoxin (TRX) levels were maintained and the intracellular generation of reactive oxygen intermediates was marginal. The cytosolic fractions of cytochrome c were increased earlier than the activation of caspase-3. In contrast, when cells were exposed to 500 microM diamide, intracellular reactive oxygen intermediate generation was increased and processing of caspase-3 was not detected despite cytochrome c release, resulting in necrosis. Caspase-3 activity in cell lysate precultured with anti-Fas Ab was suppressed dose dependently by diamide and restored by thiol-reducing agents, DTT or TRX. When cells were precultured with 5 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, intracellular TRX levels were maintained, and as low as 20 microM diamide could induce apoptosis associated with the increase of cytosolic cytochrome c and the activation of caspase-3. These results indicate that the activation of caspase-3 in diamide-induced apoptosis is mediated, at least partly, by cytochrome c release from mitochondria, and the cellular reducing environment maintained by TRX, as well as glutathione, is required for caspase-3 activity to induce apoptosis.

Antibodies, Monoclonal↗
PubMed1998

Expression of fas antigen in the normal mouse brain.

The Fas antigen (Fas/Apo-1/CD95) is a cell surface receptor protein that mediates apoptosis-inducing signals and plays an important role in the immune system. In the central nervous system, during the period of naturally occurring cell death many neurons appear to die by apoptosis. We investigated the involvement of Fas in these events. The expression of Fas transcripts and protein was examined in the juvenile mouse brain. By RT-PCR analysis, Fas mRNA was detected in the cerebrum, cerebellum, and hippocampus of the brain. By immunohistochemistry, we found Fas in neurons localized in the CA2 and CA3 sectors of the hippocampus and in the cortical III layer of the cerebrum, but not neurons in the cerebellum. Furthermore, Fas was expressed on the primary cultured hippocampal and cerebral cells using immunofluorescence and flow cytometry. These results suggest that Fas is specifically expressed on neurons in the mouse brain during postnatal development.

Animals↗
PubMed1998

Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin- mediated cell-cell adhesion.

The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target of Cdc42 and Rac1, was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.

Animals↗
PubMed1998

Purification, molecular cloning, and characterization of TRP32, a novel thioredoxin-related mammalian protein of 32 kDa.

We purified a protein of 32 kDa from human thymoma HPB-ALL cells that was co-purified with a catalytic fragment of MST (mammalian STE-20-like), a kinase of the STE20 family, which is proteolytically activated by caspase in apoptosis (Lee, K.-K., Murakawa, M., Nishida, E., Tsubuki, S., Kawashima, S., Sakamaki, K., and Yonehara, S. (1998) Oncogene 16, in press). Molecular cloning of the gene encoding this 32-kDa protein (TRP32) reveals that it is a novel protein of 289 amino acid residues and contains an NH2-terminal thioredoxin domain with a conserved thioredoxin active site. The human and mouse TRP32 proteins have 99% homology, and the thioredoxin domains are completely identical. The thioredoxin domain of TRP32 has thioredoxin-like reducing activity, which can reduce the interchain disulfide bridges of insulin in vitro. However, the thioredoxin domain of TRP32 is more sensitive to oxidation than human thioredoxin. Northern blot analysis showed that TRP32 is expressed in all human tissues. Expression of TRP32 was also confirmed in all mammalian cell lines tested by Western blot analysis using anti-TRP32 monoclonal antibody. Subcellular fractionation and immunostaining analysis showed TRP32 is cytoplasmic protein. These findings suggest that TRP32 is a novel cytoplasmic regulator of the redox state in higher eukaryotes.

Amino Acid Sequence↗
PubMed1998

Proteolytic activation of MST/Krs, STE20-related protein kinase, by caspase during apoptosis.

The Fas system has been extensively investigated as a model of apoptosis and the caspase cascade has been shown to be a characteristic mechanism of signaling of apoptosis. We have identified and purified a kinase that was activated after the stimulation of Fas on human thymoma-derived HPB-ALL cells. Partial amino acid sequencing of the purified kinase revealed it to be MST/Krs, member of the yeast STE20 family of protein kinases. MST/Krs was activated by proteolytic cleavage and proteolytic activation was blocked by the caspase inhibitor, Z-VAD-FK. A mutant MST with Asp-->Asn replacement at a putative caspase cleavage site was resistant to either the proteolytic cleavage or the activation of the kinase activity. These findings suggest that proteolytic activation is one activation mechanism of MST and plays a role in apoptosis.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of mouse caspase-8.

Fas (APO-1/CD95) is a transmembrane receptor protein which induces apoptosis upon activation. In apoptosis triggered by Fas, a subset of cysteine proteases designated caspases is activated, playing a central role as effector molecules. Among these caspases, human caspase-8 (FLICE/MACH/Mch5) has been isolated and shown to be indispensable for Fas-mediated apoptotic signaling. In this study, we isolated the mouse homologue to human caspase-8 from a BaF3 cell cDNA library. This molecule conserved the death effector domain (DED) and protease domain as detected in human caspase-8, and was capable of inducing apoptosis in KB and Rat-1 cells when overexpressed. Expression of caspase-8 was detected in the various tissues of adult mouse and in embryos at 9.5 days and 17.5 days of development by Northern-blot analysis. Further, we isolated a chromosomal gene for caspase-8 from a mouse genomic library and analyzed the genomic structure of the isolated gene. This gene consisted of eight exons and seven introns spanning about 26 kb in the coding region.

Amino Acid Sequence↗
PubMed1998

Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis.

Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.

Antigens, Nuclear↗
PubMed1998

Potential withdrawal of rheumatoid synovium by the induction of apoptosis using a novel in vivo model of rheumatoid arthritis.

OBJECTIVE: To investigate whether Fas-mediated apoptosis has potential as a new therapeutic strategy in rheumatoid arthritis (RA) by use of a novel model of RA in which human RA tissue is grafted into SCID mice. METHODS: Fresh rheumatoid synovial tissue including joint cartilage was grafted subcutaneously into the backs of SCID mice. Six weeks after engraftment, anti-Fas monoclonal antibody was injected intraperitoneally. Time-related apoptotic changes caused by anti-Fas monoclonal antibody in grafted synovium were evaluated by nick end-labeling histochemistry. RESULTS: Thirty-six hours after the injection, diffuse apoptotic changes were observed in the grafted synovia. Four weeks after the injection, rheumatoid synovial tissue diminished. CONCLUSION: This is the first report concerning the present effectiveness of anti-Fas monoclonal antibody in diminishing rheumatoid synovium in vivo, and suggests the possibility of a new strategy for treating rheumatoid arthritis by inducing Fas-mediated apoptosis.

Animals↗
PubMed1998

Fas antigen expression and apoptosis of lymphocytes in macaques infected with simian immunodeficiency virus strain mac.

To investigate the role of apoptosis in the pathogenesis of HIV infection we used macaques infected with simian immunodeficiency virus (SIV) as a primate model and examined the characteristics of the apoptosis of lymphocytes in SIV mac-infected macaques. In vitro apoptosis was more strongly induced in peripheral blood mononuclear cells (PBMC) from SIV mac239-infected macaques than those from uninfected controls. We found that the frequency of Fas antigen-positive cells was higher in PBMC from SIV mac-infected macaques than from uninfected controls, and in vitro apoptosis of PBMC was suppressed by an inhibitor of the interleukin-1 beta converting enzyme (ICE) family proteases. In biopsied lymph nodes, the number of apoptotic nuclei in T cell-dependent areas was higher in SIV mac-infected macaques than in uninfected controls. A higher number of apoptotic nuclei in lymph nodes of SIV mac-infected macaques was observed in the stage of persistent general lymphadenopathy than in those with AIDS-related complex, while there was no significant difference in the extent of apoptosis of cultured PBMC among the SIV mac-infected macaques. These results suggest that in vitro apoptosis is mediated by the Fas/Fas ligand and ICE system and that apoptosis in lymph nodes may be more closely related to the stage of SIV mac infection than is that of cultured PBMC.

Animals↗
PubMed1998

CD95 predicts responsiveness to tretinoin in acute promyelocytic leukemia.

We describe a predictive marker (CD95) for the responsiveness to tretinoin (RA) in acute promyelocytic leukemia (APL). Functional CD95 expression during RA treatment have been observed only in those patients who responded to RA. Expression of CD95 (Fas antigen), which plays a major role in apoptosis, was determined by fluorescence activated cell sorter (FACS) analysis. APL cases in which no enhancement of CD95 expression was observed showed no response to RA and did not obtain complete remission. We propose that CD95 can predict the clinical response to RA probably due to differentiation.

Adult↗
PubMed1997

Resistance to fas-mediated apoptosis of peripheral T cells in human T lymphocyte virus type I (HTLV-I) transgenic mice with autoimmune arthropathy.

Transgenic mice carrying the env-pX region of human T lymphocyte virus type I (HTLV-I) develop autoimmune arthropathy in high incidence. Adopting the approach that Fas-mediated apoptosis has a critical function in the elimination of self-reactive T cells, we examined the involvement of this apoptosis in the induction of autoimmunity in HTLV-I transgenic mice. Splenic T cells derived from the transgenic mice were more resistant to apoptosis induced by anti-Fas mAb than those of the nontransgenic mice, whereas no appreciable difference in apoptosis was detected for thymocytes from either mouse's type. The resistance of transgenic T cells may be due to Tax coded in the pX region, since Tax mediates the inhibition of anti-Fas- induced apoptosis in mature T cell line, Jurkat. Among the transgenic mice, the extent of the resistance to Fas-mediated apoptosis was further enhanced in transgenic T cells with disease. These results suggest that the escape of self-reactive T cells from Fas-mediated apoptosis in the periphery, is critical for the development of autoimmune arthropathy in HTLV-I transgenic mice.

Animals↗
PubMed1997

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.

The activation of multiple interleukin-1beta converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

Affinity Labels↗
PubMed1997

Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control.

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4 coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-XL or adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-XL or E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.

Adenovirus E1B Proteins↗
PubMed1997

Involvement of Fas antigen in ovarian follicular atresia and luteolysis.

The Fas antigen (Fas) is a cell-surface receptor protein that mediates apoptosis-inducing signals and plays an important role in the immune system. Significant amounts of Fas mRNA can be detected not only in lymphoid organs but also in the liver, heart, and ovary. In the ovary, apoptosis is thought to cause follicular atresia and luteolysis. We have investigated the involvement of Fas in these events. Here we report that Fas protein is expressed on granulosa and luteal cells but not on oocytes in the ovary. An injection of anti-Fas monoclonal antibody with apoptosis-inducing activity into adult mice enhanced follicular atresia and luteolysis. After the injection, the corpora lutea disappeared and the number of follicles containing pyknotic granulosa cells increased. There were also fewer ovulated ova and lower levels of luteal cell-produced progesterone. Furthermore, as the result of a non-functional Fas/Fas ligand system, mature ovaries from the mouse mutant/pr (lymphoproliferation) were histologically abnormal in terms of follicular development, in that the number of secondary follicles significantly increased. These results suggested that Fas plays an important role in follicular atresia and luteolysis in the ovarian physiology of adult mice.

Animals↗
PubMed1997

Rheumatic diseases in an MRL strain of mice with a deficit in the functional Fas ligand.

OBJECTIVE: To characterize Fas antigen expression on the cell surface, and to determine the effect of this expression in rheumatic diseases using a newly established gld-congenic MRL strain of mice (MRL/gld), which is defective in its functional Fas ligand (Fas-L). METHODS: Flow cytometric analyses of lymphoid cells and macrophages were performed using anti-Fas and other cell surface markers. Histopathologic manifestations were examined using immunochemistry and light and electron microscopy. Serum levels of IgG and anti-DNA antibodies were measured by single radial immunodiffusion and enzyme-linked immunosorbent assay, respectively. RESULTS: MRL/gld mice developed systemic lymphadenopathy with an accumulation of Thy1.2+, B220+ and CD4-, CD8- T cells, which both express the Fas antigen. Splenic B cells positive for surface IgM and/or surface IgD, and resident peritoneal macrophages exhibited up-regulated expression of the Fas antigen, at much higher levels than those observed in MRL/MpJ-+/+ (MRL/+) mice. Forms of rheumatic disease were observed in these mice, although not in C3H/HeJ-gld/gld mice. These forms included diffuse glomerulonephritis, granulomatous arteritis, and arthritis, and were associated with the infiltration of mononuclear cells expressing the Fas antigen. Serum levels of IgG and anti-DNA antibodies were significantly increased in MRL/gld mice compared with MRL/+ mice. CONCLUSION: Rheumatic disease was generated by the gld gene in mice with an MRL background, as it is by the lpr gene, which is a Fas deletion mutant, associated with autoimmune traits. Rheumatic disease in this MRL strain was initiated by an incapacity for Fas/Fas-L-induced apoptosis, resulting in the development of autoimmunity and allowing for a persistent immune response in the affected lesions.

Animals↗