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Biomedical subjects

M Okuda

Publications and source records attributed to M Okuda.

At least 73 records · Page 4Linked to original sources
PubMed2000

Reactive oxygen species activate p90 ribosomal S6 kinase via Fyn and Ras.

Reactive oxygen species and growth factors stimulate similar intracellular signal transduction events including activation of Src kinase family members and extracellular signal-regulated kinases (ERK1/2). A potentially important downstream effector of Src and ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth by activating several transcription factors as well as the Na(+)/H(+) exchanger. In the present study, we determined whether H(2)O(2) activates p90RSK to gain insight into signal transduction mechanisms activated by reactive oxygen species. H(2)O(2) (200 microM) stimulated ERK1/2 and p90RSK activity in lymphocytes, endothelial cells, and fibroblasts. The MEK-1 inhibitor, PD98059 (30 microM), inhibited H(2)O(2)-mediated activation of ERK1/2 but not of p90RSK. An essential role for Fyn and Ras in p90RSK activation was suggested by five findings. 1) The tyrosine kinase inhibitor, herbimycin A, and the specific Src kinase family inhibitor, PP1, blocked p90RSK activation by H(2)O(2) in a concentration-dependent manner. 2) p90RSK activation by H(2)O(2) was significantly reduced in fibroblasts derived from transgenic mice deficient in Fyn, but not c-Src. 3) H(2)O(2) rapidly activated Ras (peak at 2-5 min), which preceded p90RSK activation (peak at 20 min). 4) Dominant negative Ras completely blocked H(2)O(2)-induced activation of p90RSK. 5) In Fyn-/- fibroblasts, activation of Ras by H(2)O(2) was significantly attenuated. These results show essential roles for Fyn and Ras in H(2)O(2)-mediated activation of p90RSK and establish redox-sensitive regulation of Ras and p90RSK as a new function for Fyn.

Animals↗
PubMed2000

Role of apoptosis in cisplatin-induced toxicity in the renal epithelial cell line LLC-PK1. Implication of the functions of apical membranes.

The role of apoptosis and the implications of the functions of apical membranes in cisplatin-induced nephrotoxicity were investigated using the kidney epithelial cell line LLC-PK1. When LLC-PK1 cells were treated with 30 microM cisplatin, the number of floating cells was increased markedly. However, the number was not increased by treatment with 1 mM cisplatin, suggesting that different mechanisms were involved in the toxicities of these two treatments. DNA fragmentation, condensation of nuclear chromatin, and the absence of trypan blue staining suggested that cellular toxicity following treatment with 30 microM cisplatin for 24 hr was mediated predominantly by apoptosis. Specific activities of apical enzymes (gamma-glutamyltransferase, EC 2.3.2.2; and alkaline phosphatase, EC 3.1.3.1) in LLC-PK1 cells were decreased markedly by treatment with 30 microM cisplatin for 24 hr, whereas neither lactate dehydrogenase (LDH; EC 1.1.1.27) release nor a decrease in cellular protein content was observed following the same treatment. In addition, concomitant treatment with reduced glutathione completely attenuated both the apoptosis and the decrease of apical enzyme activities induced by 30 microM cisplatin. Neither DNA fragmentation nor condensation of chromatin was induced by treatment with 1 mM cisplatin for 12 hr. However, LDH release and a decrease in cellular protein level were induced by 1 mM cisplatin, suggesting that the toxic effect was due to necrosis. Under these conditions, specific activities of apical enzymes were not decreased. These results suggested that apoptosis was more responsible than necrosis for the loss of apical functions in cisplatin-induced toxicity in LLC-PK1 cells.

Animals↗
PubMed2000

Cloning of the beta(2a) subunit of the voltage-dependent calcium channel from human heart: cooperative effect of alpha(2)/delta and beta(2a) on the membrane expression of the alpha(1C) subunit.

Expression and membrane localization of an epitope-tagged human Ca(2+) channel alpha(1C) subunit were monitored in Xenopus oocytes by confocal microscopy and electrophysiological recording. When alpha(2)/delta and beta(2a) were separately coexpressed with the alpha(1C) subunit, assessment by confocal microscopy showed an 86 and 225% increase of the channel density, respectively. Simultaneous coexpression of alpha(2)/delta and beta(2a) subunits resulted in a cooperative (470%) increase. Electrophysiological measurements performed in parallel revealed that the current augmentation by the alpha(2)/delta subunit is totally attributable to an increase in channel density, whereas the beta(2a) subunit, in addition to increasing channel density, also facilitates channel opening.

Amino Acid Sequence↗
PubMed2000

Cancer prevention by natural carotenoids.

Various natural carotenoids were proven to have anticarcinogenic activity. Epidemiological investigations have shown that cancer risk is inversely related to the consumption of green and yellow vegetables and fruits. Since beta-carotene is present in abundance in these vegetables and fruits, it has been investigated extensively as possible cancer preventive agent. However, various carotenoids which co-exist with beta-carotene in vegetables and fruits also have anti-carcinogenic activity. And some of them, such as alpha-carotene, showed higher potency than beta-carotene to suppress experimental carcinogenesis. Thus, we have carried out more extensive studies on cancer preventive activities of natural carotenoids in foods; i.e., lutein, lycopene, zeaxanthin and beta-cryptoxanthin. Analysis of the action mechanism of these natural carotenoids is now in progress, and some interesting results have already obtained; for example, beta-cryptoxanthin was suggested to stimulate the expression of RB gene, an anti-oncogene, and p73 gene, which is known as one of the p53-related genes. Based on these results, multi-carotenoids (mixture of natural carotenoids) seems to be of interest to evaluate its usefulness for practice in human cancer prevention.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2000

Monkey pulpal response and microtensile bond strength beneath a one-application resin bonding system in vivo.

OBJECTIVES: The aim of this in vivo study was to investigate the biocompatibility and microtensile bond strength of a one-application resin bonding system. METHODS: Class V cavities were prepared on the facial surfaces of 36 intact monkey teeth, and the cavities were restored with an experimental one-application resin bonding system (TOF-1; Tokuyama Corp., Tokuyama, Japan) and a hybrid resin composite (PALFIQUE ESTELITE; Tokuyama Corp., Tokuyama, Japan). Histopathological changes of the restored teeth were evaluated at 3, 30 and 90 days after operation (N = 10). Microtensile bond tests were performed at 3 and 90 days after operation (N = 10). RESULTS: Only two of 30 pulps showed a slight inflammatory cell infiltration. There were no statistically significant differences in the incidence of slight inflammatory cell infiltration among time periods. Bacterial penetration along the cavity walls could not be detected in any specimen. The mean microtensile bond strength at 3 days after operation was 20.6 MPa, and that at 90 days was 14.9 MPa. Differences in bond strengths between the 3 day specimen and the 90 day specimen were statistically significant (p < 0.05). CONCLUSIONS: The one-application resin bonding system exhibited acceptable biologic compatibility to the monkey pulp. Although there were statistically significant differences in bond strengths between the 3 day specimen and the 90 day specimen, this material provided a hermetic seal, eliminating bacterial microleakage.

Acid Etching, Dental↗
PubMed2000

Hepatitis C virus quasispecies and response to interferon therapy in patients with chronic hepatitis C: a prospective study.

We prospectively examined whether the complexity of hepatitis C virus (HCV) quasispecies is related to the response to interferon (IFN) therapy. Among 64 patients who had histologically proven chronic hepatitis and were treated with natural IFN-alpha, 53 patients were analysed. The other 11 patients discontinued therapy because of adverse effects of IFN. The complexity of the hypervariable region 1 (HVR 1) in quasispecies was determined using both clone number determined by fluorescence single-strand conformation polymorphism (SSCP) and nucleotide diversity determined by direct sequencing. These parameters were measured not only before treatment but also at completion and 6 months after therapy, if serum HCV RNA was detectable. This population of patients was different from the general Japanese population with regard to the high prevalence of patients infected with genotype 2a or 2b (49%), who had a higher viral load than those with genotype 1b (P = 0.021). Twenty-two patients (41.5%) were sustained responders. Genotype non-1b (P = 0.0009) and a smaller clone number (P = 0.008) were significantly associated with a sustained response. In multivariate analysis, these variables were independently associated with a sustained response (i.e. genotype: odds ratio 6.84, 95% CI 1.84-30. 12; and clone number: odds ratio 1.26, 95% CI 0.99-1.68). The clone number and nucleotide diversity did not change significantly between pretreatment and at completion or 6 months after therapy. These results suggest that lower complexity of HVR 1 quasispecies predicts a preferable response to IFN therapy that is independent of viral load, especially in the population of the relatively high prevalence of patients infected with genotype 2.

Adolescent↗
PubMed2000

GB virus C/hepatitis G viremia and antibody response to the E2 protein of hepatitis G virus in hemodialysis patients.

The aim of this study was to assess the relationship between the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) RNA and that of antibody to the putative E2 protein (anti-E2) in hemodialysis patients. GBV-C/HGV RNA in serum was detected by a reverse transcription polymerase chain reaction (RT-PCR) assay, and anti-E2 was measured in 244 hemodialysis patients by enzyme-linked immunosorbent assay using recombinant E2 protein. The GBV-C/HGV RNA level was determined by competitive RT-PCR with an interval of 1 year. GBV-C/HGV RNA, anti-E2. and both together were detected in 11 (4.5%), in 19 (7.8%), and in 3 patients (1.2%), respectively. Comparison of clinical characteristics between GBV-C/HGV RNA-positive patients and negative patients revealed the longer duration of hemodialysis (9.8 years vs. 6.0 years; p < 0.05), and the greater frequency of anti-hepatitis C virus (HCV) (63.6% vs. 20.3%; p < 0.05) and HCV RNA (36.4% vs. 12.9%; p < 0.05) in GBV-C/HGV RNA-positive patients. The GBV-C/HGV RNA levels of patients who were positive for anti-E2 remained under detection limit (< 10(2) copies/mL), whereas only one of eight patients who were negative for anti-E2 showed a GBV-C/HGV RNA level under detection limit (p < 0.05). The presence of anti-E2 in serum was associated with loss of detectable GBV-C/HGV RNA or with a very small amount of HCV RNA in hemodialysis patients.

Antibodies, Viral↗
PubMed2000

Molecular cloning and application of a gene complementing pantothenic acid auxotrophy of sake yeast Kyokai no. 7.

Kyokai no. 7 is the most widely used yeast in sake brewing. This yeast is a pantothenic acid auxotroph at 35 degrees C, and this phenotype has been used to distinguish Kyokai no. 7 from other sake yeasts. We cloned a DNA fragment complementing the pantothenic acid auxotrophy from a genomic library of a Saccharomyces cerevisiae laboratory strain. DNA sequence analysis revealed that the DNA fragment encodes ECM31, the deletion of which had previously been identified as a calcofluor white-sensitive mutation. The ECM31 product is similar to the Escherichia coli ketopantoate hydroxymethyltransferase. Disruption of ECM31 in a laboratory S. cerevisiae strain resulted in pantothenic acid auxotrophy, indicating that ECM31 is also involved in pantothenic acid synthesis in yeast. A hybrid of a Kyokai no. 7 haploid and the ecm31 disruptant required pantothenic acid at 35 degrees C for its growth, suggesting that Kyokai no. 7 possesses a temperature-sensitive allele of ECM31. Thus, the ECM31 gene can be used as a selective marker in the transformation of Kyokai no. 7.

Journal Article↗
PubMed2000

Molecular analysis of multidrug resistance in feline lymphoma cells.

OBJECTIVE: To evaluate the mechanism of multidrug resistance in feline lymphoma cell lines. SAMPLE POPULATION: A feline lymphoma cell line (FT-1) and its adriamycin (ADM)-resistant subline (FT-1/ADM). PROCEDURES: The FT-1 cell line was cultivated in the presence of a gradually increasing concentration of ADM to generate its ADM-resistant subline (FT-1/ADM). Susceptibility of cells from the parental FT-1 cell line and the FT-1/ADM subline to antineoplastic drugs was determined. From the complementary DNA (cDNA) template of FT-1/ADM cells, feline MDR1 cDNA was amplified by use of polymerase chain reaction (PCR) and sequenced. Reverse transcription (RT)-PCR and Western blot analyses were performed to assess expression of the MDR1 gene and P-glycoprotein (P-gp) in FT-1/ADM cells, compared with that in FT-1 cells. RESULTS: A drug sensitivity assay revealed that FT-1/ADM cells were much more resistant to ADM and vincristine than the parental FT-1 cells. The feline MDR7 cDNA amplified by use of PCR was 3,489 base pairs long, corresponding to approximately 90% of the whole open reading frame of human MDR1 cDNA; its amino acid sequence was 91.5, 87.0, and 79.4% identical to that of human MDR1, mouse mdr1a, and mdr1b cDNA, respectively. By RT-PCR analysis, expression of MDR1 messenger RNA was clearly detected in FT-1/ADM cells but not in the parental FT-1 cells. Western blot analysis also revealed the expression of P-gp encoded by the MDR1 gene in FT-1/ADM cells but not in FT-1 cells. CONCLUSIONS: The basic structure of the feline MDR1 gene was essentially the same as that of multidrug-resistance genes of other species. Expression of P-gp appeared to be one of the mechanisms responsible for the development of multidrug resistance in feline lymphoma cell lines in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Estimation of the lethal toluene concentration from the accidental death of painting workers.

To determine the potentially lethal level of thinner concentration in the air, we measured the concentration of toluene in the blood and others of three patients who suffered severe acute thinner intoxication between 4 January 1996 and 21 April 1997 in Ube city. The concentration of toluene in blood were 30.2 mg/L in died patient, and 13.7 mg/L and 17.5 mg/L in recovered patients, respectively. By extrapolation from the results of our previous toxicokinetic research on toluene poisoning in anesthetized dogs, the fatal concentration of toluene was estimated to be approximately 1800 to 2000 ppm for 1-hour exposure.

Accidents, Occupational↗
PubMed2000

[The effects of intravenous milrinone for the patient undergoing CABG].

The hemodynamic effects of intravenous infusion of milrinone were evaluated in 25 patients undergoing CABG using an internal mammary artery graft. Milrinone was administered to 9 patients at the time of weaning from cardiopulmonary bypass, at a dosage of 3 to 5 micrograms/kg/min. Postoperative cardiac function was compared in this group versus the other 17 patients who were treated without milrinone. We determined such parameters as cardiac index, wedge pressure and mean pulmonary pressure. Our findings did not show any significant difference between the 2 groups. We also studied a subject of low-output patients (EF < 0.5). In the patients with low-cardiac output, the use of milrinone in addition to standard postoperative administration of low-dose dopamine reduced mean pulmonary pressure and wedge pressure. Thus, milrinone not only improved the left ventricular function, but also expanded the pulmonary vascular bed.

Aged↗
PubMed1999

Gender differences in expression of organic cation transporter OCT2 in rat kidney.

The organic cation transporter (OCT) mediates translocation of various cationic molecules including drugs, toxins and endogenous substances. We examined gender differences in the expression of rat (r) OCT2 in the kidney. Slices and basolateral membrane vesicles of male rat kidney showed a higher transport activity for tetraethylammonium than those of female rat kidney. The expression levels of rOCT2 mRNA and protein in the kidney of males were much higher than those in females. There was no gender difference in mRNA expression of rOCT1 and rOCT3. These findings suggest that rOCT2 is responsible for the gender differences in renal basolateral membrane organic cation transport activity.

Animals↗
PubMed1999

Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation.

Fluid shear stress (flow) modulates endothelial cell function via specific intracellular signaling events. Previously we showed that flow activated ERK1/2 in an integrin-dependent manner (Takahashi, M., and Berk, B. C. (1996) J. Clin. Invest. 98, 2623-2631). p130 Crk-associated substrate (Cas), a putative c-Src substrate, was originally identified as a highly phosphorylated protein that is localized to focal adhesions and acts as an adapter protein. Recent reports have shown that Cas is important in cardiovascular development and actin filament assembly. Flow (shear stress = 12 dynes/cm(2)) stimulated Cas tyrosine phosphorylation within 1 min in human umbilical vein endothelial cells. Phosphorylation peaked at 5 min (3.5 +/- 0.7-fold) and was sustained to 20 min. Tyrosine phosphorylation of Cas was functionally important because flow stimulated association of Cas with Crk in a time- and force-dependent manner. Flow-mediated activation of c-Src, phosphorylation of Cas, and association of Cas with Crk were all inhibited by calcium chelation and pretreatment with the Src family-specific tyrosine kinase inhibitor PP1. To determine the role of c-Src in flow-stimulated phosphorylation of Cas, we transduced cells with adenovirus encoding kinase-inactive Src. Expression of kinase-inactive Src prevented flow-induced Cas tyrosine phosphorylation but not ERK1/2 activation. Calcium-dependent activation of c-Src and tyrosine phosphorylation of Cas defines a new flow-stimulated signal pathway, different from ERK1/2 activation. This pathway may be involved in focal adhesion remodeling and actin filament assembly.

Benzoquinones↗
PubMed1999

Anti-tumor-promoting effects of phenylpropanoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis.

In our joint project in the search for anti-tumor promoters from natural plant sources, we carried out a primary screening of 12 phenylpropanoids isolated from Boronia pinnata Sm. (Rutaceae) by examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All tested compounds in this study showed inhibitory activity against the EBV activation even at 1 x 10 mol ratio without any cytotoxicity. Among 12 phenylpropanoids tested, boropinal-C (1), boropinol-A (5), boropinol-C (9) and 3-(3'-methoxy-4'-prenyloxy)phenyl-1-propene (10), all having a 4'-(3-methylbut-2-enyloxy) group, a so-called prenyloxy group, showed more potent activities. Furthermore, 3-(3'-methoxy-4'-prenyloxy)phenyl-1-propene (10) also exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. This investigation indicated that certain phenylpropanoids might be valuable anti-tumor promoters.

Animals↗