Biomedical subjects

F Tashiro

Publications and source records attributed to F Tashiro.

At least 37 records · Page 2Linked to original sources

PubMed1999

Molecular cloning and chromosomal mapping of mouse intronless myc gene acting as a potent apoptosis inducer.

Our previous findings suggest that the activation of the rat intronless myc gene provides a selective advantage in tumor suppression through apoptosis induction. In the present study, to examine whether intronless myc gene acting as an apoptosis inducer is evolutionarily conserved in mammalian cells, we isolated the mouse intronless myc gene and characterized it. A sequence analysis demonstrated that mouse intronless myc gene, ms-myc, has a linearly opened translatable frame consisting of 1293bp with 90% homology with that of rat s-myc. The chromosomal locus of ms-myc was identified on chromosome 19B by a fluorescent in situ hybridization (FISH) analysis. Gene transfection experiments showed that the transient overexpression of ms-Myc with transactivation activity effectively induces cell death in a wild-type p53-independent manner. In addition, cells stably expressing transfected ms-myc became more susceptible to apoptosis induced by genotoxic stress such as UV-irradiation and hydrogen peroxide compared with untransfected control cells. These observations suggest that the rodents commonly contain an s-myc-type of intronless myc gene with apoptosis-inducing activity.

Amino Acid Sequence↗

PubMed1999

Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker.

Gene targeting in embryonic stem (ES) cells via homologous recombination can occur at very low frequency. In order to enrich homologous recombinants before screening, a negative selection marker, such as thymidine kinase (TK) and diphtheria toxin A fragment (DT-A), has been commonly used. In this study, we developed a negative selection marker using DT-A gene with polyadenylation signal and it was designated DT-ApA. To determine the difference in targeting efficiency of the negative selections, we constructed three different targeting vectors for each negative selection (first, TK at the 3' end, second, TK at both the 5' and 3' ends < 2 X TK >, and third, DT-ApA at the 5' end of the homologous sequences). Gene targeting experiments using these constructs clearly showed that negative selection using DT-ApA was more efficient than that using TK for homologous recombination and that negative selection using DT-ApA was as efficient as that using 2 X TK. Considering the fact that the use of DT-ApA is more convenient for construction of targeting vectors than that of 2 X TK, DT-ApA is an efficient negative selection marker. In addition, we examined long and accurate PCR (LA-PCR) for screening gene targeted clones. The use of LA-PCR with genomic DNAs from ES cell clones facilitated simple detection of homologous recombinants, suggesting that the screening with LA-PCR is compatible with the use of longer homologous sequences of both arms in vector design. Our results indicate that the use of DT-ApA for negative selection together with the application of LA-PCR for screening ensures efficient and time-saving screening for homologous recombinants.

Animals↗

PubMed1999

Enhancement of glutathione S-transferase placental-form positive liver cell foci development by microcystin-LR in aflatoxin B1-initiated rats.

The objective of this study was to elucidate whether microcystin-LR (MC-LR), a hepatotoxic blue-green algal toxin in drinking water, is carcinogenic or possesses the ability to modulate aflatoxin B1 (AFB1)-induced hepatocarcinogenicity. In a medium-term liver bioassay, male Fischer 344 rats were given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) followed by an i.p. injection of MC-LR for 6 weeks after 2 weeks of DEN treatment. To study the synergism between AFB1 and MC-LR, DEN-treated rats were given an i.p. injection of AFB1 (0.5 mg/kg) dissolved in dimethyl sulfoxide (DMSO) followed by MC-LR at 2 weeks after the treatment. In a separate experiment, the rats were first given AFB1 (0.5 mg/kg) and 2 weeks later an i.p. injection of 1 or 10 microg/kg of MC-LR twice a week for 6 weeks. Most rats were subjected to a two-thirds partial hepatectomy (PH) at week 3 and were killed under anesthesia at week 8. Liver sections were analyzed for glutathione S-transferase placental form (GST-P) expression, and subjected to histopathological examination for phenotypic alteration of hepatocellular foci. In rats that did not receive DEN, MC-LR did not cause a significant increase in the numbers of GST-P-positive foci, whereas AFB1 induced a slight increase in GST-P-positive foci development. In rats given DEN, MC-LR enhanced the expression of GST-P-positive foci, as did AFB1 but no synergism was observed. Histopathological analysis revealed that the area of eosinophilic foci, a biomarker for preneoplastic liver lesion, markedly increased because of MC-LR. In rats given AFB1 as an initiator, treatment with MC-LR resulted in a synergistic increase in the development of GST-P-positive foci. These results suggest that the hepatocarcinogenicities of MC-LR and AFB1 can be predicted in experimental animals with a medium-term bioassay. Furthermore, tumor promoting activity of MC-LR was demonstrated in rats treated with AFB1.

Aflatoxin B1↗

PubMed1998

Pancreatic beta cell-specific expression of thioredoxin, an antioxidative and antiapoptotic protein, prevents autoimmune and streptozotocin-induced diabetes.

The cytotoxicity of reactive oxygen intermediates (ROIs) has been implicated in the destruction of pancreatic beta cells in insulin-dependent diabetes mellitus (IDDM). Thioredoxin (TRX), a redox (reduction/oxidation)-active protein, has recently been shown to protect cells from oxidative stress and apoptosis. To elucidate the roles of oxidative stress in the development of autoimmune diabetes in vivo, we produced nonobese diabetic transgenic mice that overexpress TRX in their pancreatic beta cells. In these transgenic mice, the incidence of diabetes was markedly reduced, whereas the development of insulitis was not prevented. Moreover, induction of diabetes by streptozotocin, an ROI-generating agent, was also attenuated by TRX overexpression in beta cells. This is the first direct demonstration that an antioxidative and antiapoptotic protein protects beta cells in vivo against both autoimmune and drug-induced diabetes. Our results strongly suggest that oxidative stress plays an essential role in the destruction of beta cells by infiltrating inflammatory cells in IDDM.

Animals↗

PubMed1998

Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice.

ATP-sensitive K+ (KATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated KATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K+ ion-selective pore of the channel. The homozygous mice (Kir6.2(-/-)) lack KATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca2+]i) of pancreatic beta cells in Kir6.2(-/-) are significantly higher than those in control mice (Kir6.2(+/+)), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca2+]i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2(-/-), as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2(-/-) show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2(-/-), which could protect Kir6.2(-/-) from developing hyperglycemia. Our data indicate that the KATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the KATP channel in skeletal muscle might be involved in insulin action.

Animals↗

PubMed1998

Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice.

We previously demonstrated that intramuscular plasmid injection serves as a useful method of long-term systemic delivery of cytokines. In the present study, we assess intramuscular DNA injection as a means of systemically delivering interleukin 10 (IL-10), a cytokine with immunosuppressive properties, and preventing the progression of autoimmune diabetes in the nonobese diabetic (NOD) mouse, an excellent model for human insulin-dependent diabetes mellitus (IDDM). We injected IL-10 expression plasmid (pCAGGS-IL10) or a control pCAGGS plasmid into the muscles of NOD mice twice at 3 and 5 weeks of age. IL-10 was detectable by ELISA in the sera of mice injected with pCAGGS-IL10 for more than 2 weeks after the injection. Although the severity of insulitis at 13 weeks of age was not improved by the intramuscular injection of pCAGGS-IL10, the incidence of diabetes was markedly reduced in NOD mice injected with pCAGGS-IL10 as compared with those injected with pCAGGS or as compared with nontreated NOD mice. These results show that the progression of autoimmune diseases in mice can effectively be suppressed by intramuscular DNA injection, and suggest that this method is potentially applicable to the treatment of human autoimmune diseases.

Animals↗

PubMed1998

Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse biological activities in addition to its well-known ability to stimulate platelet aggregation. Pharmacologic studies had suggested a role for PAF in pregnancy, neuronal cell migration, anaphylaxis, and endotoxic shock. Here we show that disruption of the PAF receptor gene in mice caused a marked reduction in systemic anaphylactic symptoms. Unexpectedly, however, the PAF receptor-deficient mice developed normally, were fertile, and remained sensitive to bacterial endotoxin. These mutant mice clearly show that PAF plays a dominant role in eliciting anaphylaxis, but that it is not essential for reproduction, brain development, or endotoxic shock.

Anaphylaxis↗

PubMed1998

Suppression of arachidonic acid cascade-mediated apoptosis in aflatoxin B1-induced rat hepatoma cells by glucocorticoids.

It has been shown that hypophysectomy protects aflatoxin B1 (AFB1) hepatocarcinogenesis and the prevention of apoptosis is a critical process for tumorigenesis. In this paper, we analyzed the cell death of AFB1-induced rat hepatoma Kagura-2 (K2) cells elicited by an estrogen antagonist, tamoxifen (TAM), and transforming growth factor-beta1 (TGF-beta1) to elucidate the function of endocrine factors in AFB1 hepatocarcinogenesis. TAM and TGF-beta1 induced a typical apoptosis in K2 cells. The apoptotic cell death was efficiently suppressed by glucocorticoids (GCs), but not by other steroid compounds including 17beta-estradiol (E2). Cyclo-oxygenase (COX) inhibitors such as aspirin (ASP) and indomethacin (IND) also inhibited the apoptosis, while inhibitory effects of general lipoxygenase (LOX) inhibitors such as nordihydroguaiaretic acid (NDGA) and 5,8,11-eicosatrienoic acid (ETI) were not observed. TAM and TGF-beta1 enhanced the release of [3H]arachidonic acid (AA) from pre-labeled K2 cells, which was inhibited by dexamethasone (DEX). Furthermore, cytosolic phospholipase A2 (cPLA2) activity in K2 cells treated with TAM for 2 h was higher than that in the control. Prostaglandin J2 (PGJ2) and delta12-PGJ2, AA metabolites formed in the COX pathway, induced K2 cell death. These results suggest that AA metabolites are involved in apoptotic K2 cell death elicited by TAM and TGF-beta1, and GCs could act as a tumor promoter in AFB1 hepatocarcinogenesis through the prevention of apoptosis induced by AA metabolites formed in vivo.

Aflatoxin B1↗

PubMed1997

Role of cytosolic phospholipase A2 in allergic response and parturition.

Phospholipase A2 (PLA2) comprises a superfamily of enzymes that hydrolyse the ester bond of phospholipids at the sn-2 position. Among the members of this superfamily, cytosolic PLA2 has attracted attention because it preferentially hydrolyses arachidonoyl phospholipids and is activated by submicromolar concentrations of Ca2+ ions and by phosphorylation by mitogen-activated protein kinases (MAP kinases). Here we investigate the function of cytosolic PLA2 in vivo by using homologous recombination to generate mice deficient in this enzyme. These mice showed a marked decrease in their production of eicosanoids and platelet-activating factor in peritoneal macrophages. Their ovalbumin-induced anaphylactic responses were significantly reduced, as was their bronchial reactivity to methacholine. Female mutant mice failed to deliver offspring, but these could be rescued by administration of a progesterone-receptor antagonist to the mother at term. Considered together with previous findings, our results indicate that cytosolic PLA2 plays a non-redundant role in allergic responses and reproductive physiology.

Anaphylaxis↗

PubMed1997

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel.

ATP-sensitive K+ (KATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of KATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the KATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. KATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the KATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.

Adenosine Triphosphate↗

PubMed1997

Intramuscular injection of expression plasmid DNA is an effective means of long-term systemic delivery of interleukin-5.

It has been demonstrated that intramuscularly injected expression plasmid DNA is taken up by myofibers and subsequently expresses exogenous genes. In the present study, we assessed intramuscular DNA injection as a means of systemically delivering interleukin-5 (IL-5). We constructed an IL-5 expression plasmid, pCAGGS-IL-5, containing murine IL-5 cDNA under the control of the CAG promoter. The soleus muscle of mice was pretreated with bupivacaine. Two days later, mice were injected with pCAGGS-IL-5 or a control pCAGGS plasmid DNA at the same site. At 2 weeks after DNA injection, eosinophils had increased from 2-3% to 8-29% of peripheral white blood cells in 9 of 10 mice injected with pCAGGS-IL-5, while eosinophils never exceeded 3% in control mice injected with pCAGGS. IL-5 mRNA was present in the muscle area injected with pCAGGS-IL-5. IL-5 was also detectable by ELISA in the sera of most mice injected with pCAGGS-IL-5, but in none of the control mice. These results demonstrate that intramuscular plasmid injection serves as a useful method of systemically delivering cytokines by combining the strong CAG promoter and bupivacaine pretreatment.

Animals↗

PubMed1997

Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor.

Although platelet-activating factor (PAF) has been shown to exert pleiotropic effects on isolated cells or tissues, controversy still exists as to whether it plays significant pathophysiological roles in vivo. To answer this question, we established transgenic mice over-expressing a guinea-pig PAF receptor (PAFR). The transgenic mice showed a bronchial hyperreactivity to methacholine and an increased mortality when exposed to bacterial endotoxin. An aberrant melanogenesis and proliferative abnormalities in the skin were also observed in the transgenic mice, some of which spontaneously bore melanocytic tumors in the dermis after aging. Thus, PAFR transgenic mice proved to be a useful model for studying the basic pathophysiology of bronchial asthma and endotoxin-induced death, and screening of therapeutics for these disorders. Furthermore, our findings provide new insights regarding the role of PAF in the morphogenesis of dermal tissues as well as the mitogenic activity of PAF and PAFR in vivo.

Animals↗

PubMed1997

Comparison of amyloid deposition in two lines of transgenic mouse that model familial amyloidotic polyneuropathy, type I.

We previously produced a transgenic mouse line designated MT-hMet30 by introducing the human mutant transthyretin (TTR) gene carrying the mouse metallothionein promoter, and showed that the presence of human variant TTR is sufficient for amyloid deposition in various tissues of these transgenic mice. However, the expression pattern of human mutant transthyretin gene in the mouse was different from that in man. To analyse pathologic processes, it is essential to establish a transgenic mouse line in which the development and tissue-specific expression of the human mutant TTR gene is the same as in man. Thus, we produced two additional transgenic mouse lines carrying the human mutant TTR gene containing either 0.6 kb (0.6-hMet30) or 6.0 kb (6.0- hMet30) of the upstream region. The expression levels of 6.0-hMet 30 gene in the liver and serum were the same as in man and about 10 times higher than those of 0.6-hMet30 gene in the liver and serum were the same as similar tissues to human patients except for the peripheral and autonomic nervous tissues. The amyloid deposition started earlier and was more extensive in 6.0-hMet30 than 0.6-hMet30 mice, suggesting that the serum levels of human mutant TTR are correlated with the occurrence and degree of amyloid deposition, to some extent. Neither amyloid deposition nor degenerative changes were observed in the peripheral and autonomic nervous systems despite the transgene expression in the choroid plexus of the 6.0-hMet30 mice. In the 6.0-hMet30 mice, amyloid deposition started at 9 months of age, although the serum level of human mutant TTR reached the adult level at 1 month. These results suggest that intrinsic environmental factors other than the mutant gene are involved in the late-onset deposition of amyloid fibrils. Transgenic mice described here should be useful for analysing such factors.

Age Factors↗

PubMed1996

Transient elevation of intracellular calcium ion levels as an early event in T-2 toxin-induced apoptosis in human promyelotic cell line HL-60.

Recently we have reported that T-2 toxin, a trichothecene mycotoxin produced by Fusarium species, is a potent inducer of apoptosis in the human promyelotic cell line HL-60. To clarify the signal transduction pathway of apoptosis primed by T-2 toxin, T-2 toxin-induced apoptosis was investigated in detail using confocal laser microscopy and flow cytometry. Apoptosis in HL-60 cells induced by T-2 toxin was dose dependent when the cells were treated with concentrations of 5-100 ng/ml for more than 2 hr. The apoptosis proceeds through various cell cycle stages of HL-60 cells. Prior to apoptosis, the intracellular calcium ion (Ca+2i) level was markedly elevated within 3-5 min after exposure to T-2 toxin and returned to normal level thereafter. A well-known chelator for Ca+2i, ethylene-N,N,N', N'-tetraacetic acid 4K acetoxymethyl ester (BAPTA-AM), a Ca+2-dependent endonuclease inhibitor ZnCl2, and calpain inhibitor 1 sharply blocked T-2 toxin-induced apoptosis. These results strongly suggest that the Ca+2 signal triggered by T-2 toxin is transduced by the activation of endonuclease and protease, and ultimately evokes apoptosis.

Apoptosis↗

PubMed1996

Clonal prevalence of T cells infiltrating into the pancreas of prediabetic non-obese diabetic mice.

The non-obese diabetic (NOD) mouse spontaneously develops T-cell-mediated autoimmune insulitis. We analyzed the clonotypes of T cell infiltrates of the NOD mouse islets using a new method we have developed recently, which consists of RT-PCR amplification of the CDR3 region of the TCR beta chain mRNA and subsequent single-strand conformation polymorphism (SSCP) analysis. NOD mice of 10-32 weeks of age were shown to accumulate oligoclonal T cells in the pancreas. To examine whether each T cell clone stays in a small area of the pancreas or spreads over the whole pancreas, a pancreas was divided into two pieces, which were then subsequently analyzed in a pair by the above PCR-SSCP method. When a pair produces common bands with the same mobility in SSCP gel, they are likely to represent the presence of the same T cell clones between these two parts of the pancreas. Aged mice (24-32 weeks old) with severe insulitis obviously produced more common bands for most of the Vbeta subfamilies than younger mice (10 weeks old) with only periinsulitis. DNA sequencing verified that these common bands have the same TCR junctional sequences, suggesting that they were derived from the same T cell clones. These results suggest that clonal prevalence of T cells infiltrating into the pancreas occurs in the late stage of insulitis development and that a limited number of T cell clones finally predominate over the whole pancreas.

Animals↗

PubMed1996

Immune response to heat-shock protein correlates with induction of insulitis in I-E alpha d transgenic NOD mice.

To evaluate the correlation between heat-shock protein (HSP) and insulitis, we compared lymphocyte proliferative response to Mycobacterium leprae HSP65 of NOD mice with that of I-E alpha d transgenic NOD (I-E+NOD) mice, which show no insulitis. We found that splenocytes from 15-week-old NOD mice showed a more marked proliferative response to HSP than did those from age-matched I-E+NOD mice (P < 0.05). We then transferred splenocytes from 12-week-old NOD mice into I-E+NOD mice to induce insulitis in the recipients and examined antibody levels against HSP. By 6 weeks posttransfer, insulitis was successfully transferred to four out of five recipients of NOD splenocytes and antibody levels against HSP were significantly higher in the NOD splenocyte-transferred group than in controls, which showed no insulitis (P < 0.01). These results suggest that immune response to HSP correlates with insulitis in NOD mice. Our results support the assertion that HSP is a useful antigen for investigating the etiology of IDDM.

Animals↗

PubMed1995

Inhibition of pancreatic beta-cell glucokinase by antisense RNA expression in transgenic mice: mouse strain-dependent alteration of glucose tolerance.

We have generated transgenic mice, in either C57BL/6 or C3H background, expressing antisense glucokinase mRNA in beta-cells. The glucose phosphorylating activity at 60 mM glucose in transgenic islets was significantly lower than that in controls, and the insulin secretory response to glucose was lower in transgenic islets than in those of controls in both strains. Following i.p. glucose challenge, higher blood glucose levels were observed in transgenic mice than in controls in the C57BL/6 but not the C3H background. These data suggest that a beta-cell secretory defect, in combination with other undefined genetic factors, causes impaired glucose homeostasis in mice.

Animals↗

PubMed1995

An inhibitory role for phosphatidylinositol 3-kinase in insulin secretion from pancreatic B cell line MIN6.

Phosphatidylinositol 3-kinase (PI3-kinase) has been implicated in the regulation of vesicular transport. We examined the roles of PI3-kinase in the glucose-induced insulin secretion from the pancreatic beta cell line MIN6 by using wortmannin, a potent inhibitor of PI3-kinase. Low concentrations of wortmannin markedly potentiated the glucose-induced insulin secretion. This effect was probably mediated by PI3-kinase inhibition. Furthermore, wortmannin completely canceled the suppressive effect of insulin-like growth factor-I on insulin secretion from MIN6 cells. On the basis of these results, we discuss a possible role of PI3-kinase in the negative feed-back regulation of insulin secretion.

Androstadienes↗