Search PubMed⌕ Search

Biomedical subjects

T Asano

Publications and source records attributed to T Asano.

At least 289 records · Page 16Linked to original sources
PubMed1997

Massive actin polymerization induced by phosphatidylinositol-4-phosphate 5-kinase in vivo.

The Rho family GTP-binding proteins have been known to mediate extracellular signals to the actin cytoskeleton. Although several Rho interacting proteins have been found, downstream signals have yet to be determined. Many actin-binding proteins are known to be regulated by phosphatidylinositol 4,5-bisphosphate in vitro. Rho has been shown to enhance the activity of phosphatidylinositol-4-phosphate 5-kinase (PI4P5K), the phosphatidylinositol 4,5-bisphosphate synthesizing enzyme. Recently we isolated several isoforms of type I PI4P5K. Here we report that PI4P5K Ialpha induces massive actin polymerization resembling "pine needles" in COS-7 cells in vivo. When truncated from the C terminus to amino acid 308 of PI4P5K Ialpha, both kinase activity and actin polymerizing activity were lost. Although the dominant negative form of Rho, RhoN19, alone decreased actin fibers, those induced by PI4P5K were not affected by the coexpression of RhoN19. These results suggest that PI4P5K is located downstream from Rho and mediates signals for actin polymerization through its phosphatidylinositol-4-phosphate 5-kinase activity.

Actins↗
PubMed1997

p85alpha gene generates three isoforms of regulatory subunit for phosphatidylinositol 3-kinase (PI 3-Kinase), p50alpha, p55alpha, and p85alpha, with different PI 3-kinase activity elevating responses to insulin.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85alpha, but the SH3 and bcr homology domains of p85alpha were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85alpha gene product. We suggest that this protein be called p50alpha. Northern blotting using a specific DNA probe corresponding to p50alpha revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50alpha protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50alpha. Taken together, these observations indicate that the p85alpha gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85alpha, p55alpha, and p50alpha), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50alpha forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85alpha and p55alpha associate only with p110 that can be wortmannin-labeled. Furthermore, p50alpha exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50alpha expression in the liver and its marked responsiveness to insulin, p50alpha appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three alpha isoforms has a different function and may have specific roles in various tissues.

Amino Acid Sequence↗
PubMed1997

Tacrine inhibits nicotinic secretory and current responses in adrenal chromaffin cells.

Tacrine enhanced acetylcholine-induced catecholamine secretion with a concentration of up to 10 microM, but inhibited it at over 10 microM in perfused adrenal glands. Qualitatively the same result was obtained with physostigmine. Both tacrine and physostigmine only inhibited the secretory responses to carbachol and/or nicotine in perfused glands and dispersed chromaffin cells. Acetylcholinesterase activity of adrenal homogenates was inhibited by tacrine and physostigmine in a concentration-dependent manner. In whole-cell patch-clamp experiments, tacrine and physostigmine caused reversible inhibition of nicotine-evoked inward currents with a dose range similar to that for the inhibitory action on the secretory response. These results suggest that the enhancing effect of tacrine and physostigmine on acetylcholine-induced catecholamine secretion results from the prevention of enzymatic hydrolysis of acetylcholine in adrenal glands and that the inhibitory effect is due to the inhibition of nicotinic receptor-mediated membrane currents in adrenal chromaffin cells.

Acetylcholine↗
PubMed1997

Screening of patients with maternally transmitted diabetes for mitochondrial gene mutations in the tRNA[Leu(UUR)] region.

To investigate the prevalence and clinical characteristics of diabetes mellitus caused by mitochondrial gene mutations in the tRNA[Leu(UUR)] region, a known 'hot spot' for pathogenic mutations, we screened 440 diabetic patients with diabetic mothers for 11 mitochondrial gene mutations reported in mitochondrial neuromuscular disorders; nucleotide pairs (np) 3250, 3251, 3252, 3254, 3256, 3260, 3271, 3291, 3302 and 3303 in addition to an A to G transition at np 3243. The dot-blot hybridization method using 32P-labelled sequence-specific oligonucleotides as probes was used. One subject carrying a T to C transition at np 3271 and seven carrying the A to G transition at np 3243 were identified, while none of the other diabetic patients screened had these mutations in the tRNA[Leu(UUR)] region. The patient with the 3271 mutation, a 39-year-old male, had excellent glycaemic control with diet alone and had neither hearing impairment nor symptoms suggesting mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Thus, among patients with maternally transmitted diabetes, the prevalence of the 3271 mutation was approximately one-seventh that of the 3243 mutation, and other mutations are even more rare in the mitochondrial tRNA[Leu(UUR)] region.

Adult↗
PubMed1997

Detection of hepatitis B virus x transcripts in human hepatocellular carcinoma tissues.

BACKGROUND: The hepatitis B virus (HBV) or hepatitis C virus (HCV) sequences were frequently detected in hepatocellular carcinoma (HCC) tissues by the method of polymerase chain reaction (PCR). However, the expression levels of HBV and HCV in HCC tissues remain to be documented. This study evaluates the mRNA expression levels of those virus sequences in the human HCC tissues through Northern blot hybridization. METHODS: We performed Northern blot hybridization to identify the HBV x mRNA and HCV RNA in HCC tissues of 14 cases excised at Chiba University Hospital. RESULTS: The HBV x transcripts could be identified in the tumor tissues from all three serum HBsAg-positive cases and two cases among 11 HBsAg-negative patients. One HBsAg-negative case showed the most intensive signal in those five cases. However, HCV RNA could not be identified by this method. The HBV x gene transcript is predicted to have a size of about 0.7 kb; however, the molecular weight of HBV x specific mRNA is not consistent and is distributed between approximately 2.0 and 9.0 kb. The hybridization signal intensity is also variable according to the cases. CONCLUSION: These observations suggest that HBV x gene may be transcribed with flanking cellular sequences and the carcinogenic activity may be different according to the expressed levels. The identification of HBV x transcripts in HCC tissues on the Northern blot level also suggests that HBV x mRNA expression might be significant and concerned with the development of human HCC even in HBsAg-negative cases.

Adult↗
PubMed1997

Immunostimulatory therapy with anti-CD3 monoclonal antibodies and recombinant interleukin-2: heightened in vivo expression of mRNA encoding cytotoxic attack molecules and immunoregulatory cytokines and regression of murine renal cell carcinoma.

The response rate to IL-2 immunotherapy, currently used in the treatment of metastatic renal cell cancer, is limited. Based on our earlier demonstration that a combined regimen of monoclonal antibodies directed at the T cell surface protein CD3 (anti-CD3 mAbs) and IL-2 is synergistic in constraining tumor progression in a murine fibrosarcoma hepatic metastasis model, we have explored the efficacy of an anti-CD3 mAbs plus IL-2 regimen in a murine renal cell cancer model. Our studies demonstrate that a regimen of anti-CD3 mAbs plus IL-2 is superior to treatment with anti-CD3 mAbs alone or IL-2 alone in reducing the number of pulmonary metastases and in prolonging survival. Moreover, the efficacious regimen is associated with heightened intrapulmonary expression of mRNA encoding cytotoxic attack molecules (perforin, granzyme B) and immunoregulatory cytokines (IL-4, IL-10 and IFN- gamma).

Animals↗
PubMed1997

Thromboxane A2 synthetase inhibitor failed to ameliorate the arterial narrowing during the chronic phase of cerebral vasospasm.

To determine the pathogenetic mechanism underlying the maintenance of arterial narrowing during the chronic phase of cerebral vasospasm caused by subarachnoid hemorrhage (SAH), we examined the effect of ozagrel, a thromboxane A2 (TXA2) synthetase inhibitor, on chronic vasospasm in a canine two-hemorrhage model in comparison with that of fasudil, an inhibitor of protein kinases. The magnitude of the vasospasm was determined angiographically. On SAH day 7, a vasospasm was observed in every dog. Intraarterial or intravenous administration of ozagrel (3 mg/kg/30 min) did not reverse the vasospasm but tended to increase bleeding. In contrast, intraarterial administration of fasudil (3 mg/kg/30 min) significantly reversed the vasospasm. These findings suggest that: 1) TXA2 does not participate in the maintenance of chronic vasospasm after SAH; and 2) the protein kinases, particularly myosin-light chain kinase and protein kinase C, are involved in the pathogenesis of arterial narrowing during the chronic phase of cerebral vasospasm.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Prevalence of the Trp64Arg missense mutation of the beta3-adrenergic receptor gene in Japanese subjects.

Prompted by the recent findings that a tryptophan to arginine (Trp64Arg) mutation in the beta3-adrenergic receptor gene was associated with an earlier onset of non-insulin-dependent diabetes mellitus (NIDDM) in Pima Indians, with abdominal obesity and insulin resistance in Finns, and with an increased capacity to gain weight in French whites, we studied the prevalence of this mutation in 231 diabetic and 95 nondiabetic Japanese subjects and assessed its contribution to the development of obesity and NIDDM. The allelic frequencies of the mutation were 0.18 in diabetic and 0.23 in nondiabetic subjects, showing no significant difference between the two groups (P = .067). In nondiabetic subjects, body mass index (BMI) did not differ between those with and without the mutation (22.2 +/- 3.5 v 21.4 +/- 3.2 kg/m2, P = .252). In NIDDM subjects, BMI at the time of study and maximal BMI before the start of treatment did not differ between those with and without the mutation (22.8 +/- 2.6 v 23.2 +/- 3.7 kg/m2, P = .678, and 24.7 +/- 2.6 v 24.9 +/- 3.1 kg/m2, P = .277). Homozygotes for the mutation did not have trends to have increased BMI in either diabetic or nondiabetic subjects. The age at diagnosis of NIDDM also did not differ between the two groups (48.8 +/- 9.9 v 47.8 +/- 12.5 years, P = .796). Fasting serum cholesterol and triglyceride levels and systolic and diastolic blood pressure before the start of treatment did not differ between NIDDM subjects with and without the mutation. In conclusion, although the Trp64Arg mutation is not uncommon in Japanese, it does not appear to be associated with obesity, NIDDM, age at diagnosis of NIDDM, or dyslipidemia. Our results suggest that the mutation has minor effects, if any, on the development of obesity and NIDDM in Japanese.

Adult↗
PubMed1997

Identification of hepatitis B virus integration in hepatitis C virus-infected hepatocellular carcinoma tissues.

BACKGROUND/AIMS: The integration of HBV DNA is thought to be involved in the initial stage of hepatocarcinogenesis, and it has been reported that transactivating factors encoded by the X and preS2/S genes stimulate transcription of multiple viral and cellular genes. We assessed the possible contributions of hepatitis B virus integration to the occurrence of hepatocellular carcinoma in hepatitis C virus-infected as well as in hepatitis B virus-infected patients by identifying the integrated HBV DNA sequence, and the X and preS2/S regions were further investigated in HBV DNA-integrated cases. METHODS: Southern blot hybridization for detecting HBV DNA in tumor tissues from 28 hepatocellular carcinoma patients was carried out with full-length HBV DNA, and then with X and preS2/S regions as probes. We also carried out reverse transcription-polymerase chain reaction for detecting HCV RNA to confirm hepatitis C virus-infection in liver tissues. RESULTS: Clonally integrated HBV DNA sequences were demonstrated in 16 of 28 patients (57.1%), including five HBsAg seropositive and 11 HBsAg seronegative patients. Of these 11 HBsAg seronegative patients, 10 were also positive for anti-HCV in their sera, and all nine examined cases had HCV RNA in liver. Furthermore, the X region was identified in 14 of 16 HBV DNA integrated cases (87.5%), and the preS2/S region in 6/16 (37.5%). CONCLUSIONS: The present Southern blot analysis demonstrates that clonally integrated HBV DNA sequences were identified even in hepatitis C virus-infected hepatocellular carcinoma patients at a high rate (10/18, 55.6%), and suggests that integrated hepatitis B virus, whose major component is the X gene, may play an important role in hepatocarcinogenesis in hepatitis B virus-integrated cases with and without hepatitis C virus infection.

Adult↗
PubMed1997

Hepatic protein synthesis rate of liver specimens as a predictor of viability in rat cold ischemia liver transplantation model.

BACKGROUND/AIMS: We have previously reported that the hepatic protein synthesis rate, calculated as the uptake rate of L-[4.5 3H] leucine by the protein fraction during a 10-min incubation of a 16-G needle biopsy specimen of liver tissue, represents a high level of liver function and is therefore useful for evaluating liver function. We investigated the hepatic protein synthesis rate level in a pre-transplant liver to learn if it might predict the outcome in a rat orthotopic liver transplantation model. METHODS: Grafts were stored, liver specimens were obtained using a 21-G Chiba type II skinny needle, and the hepatic protein synthesis rate was calculated. Subsequently, liver transplantation was performed, and the hepatic protein synthesis rate level of revascularized liver, tissue blood flow rate, serum alanine aminotransferase, lactate dehydrogenase, hyaluronic acid, ketone body rate, and 2-week survival were examined. RESULTS: The hepatic protein synthesis rate of pretransplant liver was correlated with parameters of post-transplant liver function: hepatic protein synthesis rate of the revascularized liver (r=0.92, p<0.0001), tissue blood flow rate (r=0.77, p<0.004), serum alanine aminotransferase (r=-0.69, p<0.003), lactate dehydrogenase (r=-0.54, p<0.03), hyaluronic acid (r=-0.86, p<0.0002), and ketone body rate (r=0.57, p<0.02). Pretransplant hepatic protein synthesis rate in survivors was 263.6+/-54.2 nmol/mg protein/10 min, while that in nonsurvivors was significantly lower at 162.0+/-39.0 (p<0.0001). When evaluation was made using a logistic regression model, the accuracy predicted using the value of hepatic protein synthesis rate was 95% (19/20). CONCLUSIONS: These results suggest that measuring the hepatic protein synthesis rate of the grafts with a 21-G Chiba type II skinny needle may be a predictive criterion in the assessment of graft viability.

Alanine Transaminase↗
PubMed1997

Beta-galactosidase-deficient mouse as an animal model for GM1-gangliosidosis.

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid beta-galactosidase, which hydrolyses the terminal beta-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted beta-galactosidase gene showed beta-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7-10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis.

Animals↗
PubMed1997

TCR beta-independent development of CD4+ CD8+ thymocytes observed in a strain of scid mice.

Mice homozygous for severe combined immunodeficiency (scid) mutation usually halt their thymocyte development at the CD4-CD8- double negative (DN) stage due to their inability of TCR gene rearrangement. In this study, we report that SCID-bg mice, which were originally generated by mating CB-17-scid mice with KSN-bg mice, spontaneously develop dominant CD4+ CD8+ double positive (DP) thymocytes. Their thymi were mainly composed of DN, CD4-CD8+ and DP cells, and the majority of them did not present CD3. Similarly, they lacked TCR beta expression both on cell surface and in cytoplasm, which suggests that the thymocyte development to the DP stage observed in SCID-bg mice, was independent of CD3 and TCR beta expression. In spite of significant DP thymocytes in SCID-bg mice, the histology of their thymi was not so different from those of CB-17-scid mice. Analysis of bone marrow cells in SCID-bg mice showed that the development of B lineage cells was not altered when compared with CB-17-scid mice. These findings point out the fact that thymocytes in SCID-bg mice have a peculiar characteristic compared to CB-17-scid mice, and provide evidence of TCR beta-independent development of thymocytes to the DP stage.

Animals↗
PubMed1997

Characterization of CD4+CD8+ thymocytes observed in SCID-bg mice.

Previously, we reported that a strain of severe combined immunodeficient (SCID) mice, namely SCID-bg, spontaneously develop CD4+CD8+ double positive (DP) thymocytes despite their scid mutation. In the present study, we intend to further characterize the DP thymocyte population in SCID-bg mice with Southern hybridization and flow cytometry. Southern hybridization analyses of sorted DP thymocytes in SCID-bg mice showed rearrangement of T cell receptor (TCR) beta and TCR gamma gene segments, although the expression of TCR beta and gamma delta TCR molecules was absent. The phenotype of the DP thymocytes in SCID-bg mice was CD44- heat-stable antigen (HSA)+CD25-, and they did not express CD69. Interestingly, the expression of thymus leukaemia (TL) antigen was observed in the DP thymocytes of SCID-bg mice but not in their CD4-CD8- double negative (DN) fraction. Fas expression was higher and bcl-2 expression was down-regulated in the DP thymocytes as compared to the DN thymocytes in SCID-bg mice, suggesting that they are not immortalized cells having escaped from apoptosis. Taken together, these results show that the phenotype of the DP thymocytes in SCID-bg mice is similar to that of the earliest phase of the DP thymocytes in normal mice, although the expression of the molecules in the pre-TCR complex is absent.

Animals↗
PubMed1997

Differential localization of the gamma 3 and gamma 12 subunits of G proteins in the mammalian brain.

The localization of two forms of the gamma subunit of G proteins, gamma 3 and gamma 12, was examined in the mammalian brain. Concentrations of these two gamma subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of gamma 3 tended to decrease with age, whereas that of gamma 12 in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that gamma 3 was abundant in the neuropil, whereas gamma 12 was localized in glial cells. In the hippocampal formation of aged human brains, levels of gamma 12-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of gamma 12-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that gamma 12 is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that gamma 12 was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of gamma 3 in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that gamma 3 and gamma 12 are selectively expressed in neuronal and glial cells, respectively, and that concentrations of gamma 3 and gamma 12 in the brain are related to the numbers and/or extent of maturation of these cells.

Age Factors↗
PubMed1997

N omega-nitro-L-arginine attenuates early ischemic neuronal damage of prolonged focal cerebral ischemia and recirculation in rats.

The present study aimed to examine the effects of N omega-nitro-L-arginine (LNA) on the early ischemic neuronal damage (EIND). All the experiments were carried out under general anesthesia, maintaining the blood gases and the body temperature within the physiological ranges. The local CBF, the topographically corresponding cortical specific gravity, and the volume of EIND were determined in each rat, which was subjected to prolonged or temporary occlusion of middle cerebral artery (MCA) using our original miniclip. Significant cortical edema developed only in the brain area where the local CBF value was below 200 ml 100 g-1 min-1. The prolonged MCA occlusion for 1, 2, and 4 h induced a time-dependent increase in the severity of cortical edema and the volume of EIND. Removal of the clip invariably induced recirculation. Compared to that induced by 4 h prolonged ischemia, the brain damage was improved by 1 h MCA occlusion followed by 3 h recirculation, whereas it was significantly worsened by 2 h ischemia followed by 2 h recirculation. While LNA [1 mg, i.p., given two times during the experiment] only partially inhibited the activity of brain nitric oxide synthase, it remarkably ameliorated EIND of both prolonged ischemia and recirculation in this model. The above findings indicate the pathogenic role of nitric oxide in prolonged ischemia as well as recirculation.

Animals↗
PubMed1997

Ebselen (DR3305) ameliorates delayed cerebral vasospasm in a canine two-hemorrhage model.

Ebselen, a seleno-organic compound which inhibits arachidonic acid lipoxygenase activity and exerts glutathione peroxidase-like activity, ameliorated delayed cerebral vasospasm in a canine two-hemorrhage model. Twenty-five dogs were exposed to subarachnoid hemorrhage and divided into two groups. In the Ebselen-treated group (6 dogs), 50 mg kg-1 of Ebselen was administered twice a day, for 7 days. The other 19 dogs without administration of Ebselen were used as a control group. In the Ebselen-treated group, the basilar artery on Day 7 after subarachnoid hemorrhage was constricted to 68.1 +/- 6.4% (mean +/- SD, n = 6) of the angiographic diameter on Day 0, before subarachnoid hemorrhage. This percentage was significantly larger than the 41.3 +/- 4.6% (n = 19) in the control group. The basilar artery segment obtained on Day 7 in the Ebselen-treated group produced 3.32 +/- 1.82 nmol of 5-hydroxyeicosatetraenoic acid/mg protein/5 minutes (n = 5), significantly less than the 6.73 +/- 0.95 (n = 3) produced by the artery in the control group. Thus, administration of Ebselen suppressed arachidonate 5-lipoxygenase activation and had a beneficial effect on angiographically detected delayed vasospasm.

Animals↗