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E Ohashi

Publications and source records attributed to E Ohashi.

At least 19 recordsLinked to original sources
PubMed2005

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours.

We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3sigma, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3sigma, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours.

14-3-3 Proteins↗
PubMed2001

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.

2-Acetylaminofluorene↗
PubMed2001

Translesion DNA synthesis catalyzed by human pol eta and pol kappa across 1,N6-ethenodeoxyadenosine.

1,N(6)-Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) eta and kappa, which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol eta than with pol kappa and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol eta, may contribute to the translesion DNA synthesis events observed for 1,N(6)-ethenodeoxyadenosine in human cells.

Animals↗
PubMed2001

Effect of retinoids on growth inhibition of two canine melanoma cell lines.

Two new canine melanoma cell lines (CMM1 and CMM2) were established from the patients with oral malignant melanomas. Histopathological type of both CMM1 and CMM2 was a mixed cell type consisted of spindle-shaped cells, polygonal cells, and oval cells. Doubling time of CMMI and CMM2 were 18.4 +/- 1.96 hr and 21.0 +/- 0.73 hr, respectively. The effect of two kinds of retinoids (all-trans retinoic acid and 9-cis retinoic acid) on the proliferation of these cells were examined by morphological changes, proliferation assay and apoptosis assay. However, the retinoids did not suppress growth rate of these cells. This result suggests that retinoids used in this study did not induce differentiation, apoptosis, and growth inhibition of the canine melanoma cell lines.

Alitretinoin↗
PubMed2001

Investigation of gene expressions related to cholesterol metabolism in rats fed diets enriched in n-6 or n-3 fatty acid with a cholesterol after long-term feeding using quantitative-competitive RT-PCR analysis.

We have developed a method to quantitate hepatic apolipoprotein (apo) B, LDL receptor, 3-hydroxy-3-methylglutary coenzyme A reductase (HMG-CoA reductase) and cholesterol 7alpha-hydroxylase mRNA expression in rats fed a cholesterol-enriched diet after long-term feeding using competitive RT-RCR. Rats (8 wk of age) fed a conventional diet were shifted to diets containing 10% perilla oil (PEO, oleic acid+linoleic acid+alpha-linolenic acid), borage oil (BRO, oleic acid+linoleic acid+gamma-linolenic acid), evening primrose oil (EPO, linoleic acid+gamma-linolenic acid), mixed oil (MIO, oleic acid+linoleic acid+gamma-linolenic acid+alpha-linolenic acid), or palm oil (PLO, palmitic acid+oleic acid+linoleic acid) with 0.5% cholesterol for 15 wk. There were no significant differences in the food intake and body weight gain among the groups. The liver weight in the PEO and PLO groups was significantly higher than other groups. The serum total cholesterol and very low density lipoprotein (VLDL)+intermediate density lipoprotein (IDL)+low density lipoprotein (LDL)-cholesterol concentrations were consistently higher in PLO group than in the other groups. The serum high density lipoprotein cholesterol concentration was significantly lower in the PEO group than in the other groups. The liver cholesterol concentration group was significantly higher in the PEO than in the other groups. There were no significant differences in the hepatic LDL receptor mRNA level among the groups. Hepatic apo B, HMG-CoA reductase and cholesterol 7alpha-hydroxylase mRNA levels were not affected by the experimental conditions. However, hepatic cholesterol 7alpha-hydroxylase mRNA level in the PEO and MIO groups tended to be higher than in the other groups. The fecal cholesterol extraction was significantly higher in the MIO and PLO groups than in the PEO and EPO groups and the total bile acid extraction was significantly higher in the PEO and MIO groups than in the PLO group. The results of this study demonstrated that both n-6 fatty acid and n-3 fatty acids such as gamma-linolenic acid and alpha-linolenic acid lowered serum total cholesterol and VLDL+IDL+LDL-cholesterol concentrations of rats in the presence of excess cholesterol in the diet compared with dietary saturated fatty acid.

Animals↗
PubMed2000

Fidelity and processivity of DNA synthesis by DNA polymerase kappa, the product of the human DINB1 gene.

Mammalian DNA polymerase kappa (pol kappa), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol kappa copies undamaged DNA with average single-base substitution and deletion error rates of 7 x 10(-3) and 2 x 10(-3), respectively. These error rates are high when compared to those of most other DNA polymerases. pol kappa also has unusual error specificity, producing a high proportion of T.CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/DinB family, pol kappa can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol kappa is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.

Base Pair Mismatch↗
PubMed2000

Error-prone bypass of certain DNA lesions by the human DNA polymerase kappa.

The Escherichia coli protein DinB is a newly identified error-prone DNA polymerase. Recently, a human homolog of DinB was identified and named DINB1. We report that the DINB1 gene encodes a DNA polymerase (designated polkappa), which incorporates mismatched bases on a nondamaged template with a high frequency. Moreover, polkappa bypasses an abasic site and N-2-acetylaminofluorene (AAF)-adduct in an error-prone manner but does not bypass a cis-syn or (6-4) thymine-thymine dimer or a cisplatin-adduct. Therefore, our results implicate an important role for polkappa in the mutagenic bypass of certain types of DNA lesions.

Bacterial Proteins↗
PubMed2000

Retinoid receptors and the induction of apoptosis in canine osteosarcoma cells.

Retinoids, all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA), induced morphological changes and apoptosis-like cell death characterized by cell shrinkage, chromatin condensation and nuclear disintegration in three canine osteosarcoma cells, OOS, HOS and POS, at a concentration of 10(-5) M. Both retinoid receptors, RARs and RXRs, were identified in these cells. 9-cis-RA bound to both the RXRs and the RARs, whereas ATRA bound to only the RARs in these cells. Those results indicate that the induction of apoptosis in canine osteosarcoma cells may be mediated by the specific control of RARs and RXRs.

Alitretinoin↗
PubMed1999

Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and their application to the detection of B. cereus in rice.

As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B. cereus JCM 2152(T), Bacillus thuringiensis IAM 12077(T), Bacillus mycoides ATCC 6462(T), and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.

Amino Acid Sequence↗
PubMed1998

Inhibition in a microgravity environment of the recovery of Escherichia coli cells damaged by heavy ion beams during the NASDA ISS phase I program of NASA Shuttle/Mir mission no. 6.

We participated in a space experiment, part of the National Space Development Agency of Japan (NASDA) Phase I Space Radiation Environment Measurement Program, conducted during the National Aeronautics and Space Administration (NASA) Shuttle/Mir Mission No. 6 (S/MM-6) project. The aim of our study was to investigate the effects of microgravity on the DNA repair processes of living organisms in the in orbit. Heavy ion beam radiation- or ç-irradiation-damaged biological samples of Escherichia coli and the radioresistant bacterium Deinococcus radiodurans were prepared and placed in a biospecimen box, which was loaded into the RRMD III sensor unit of the Space Shuttle. Two identical sets of samples were left in the Spacehab's Payload Processing Facility (SPPF) in Florida, USA, as a control. (flight No. STS-84) was launched from NASA John F. Kennedy Space Center (KSC) in Florida, USA, on May 15, 1997. The mission duration was 9.22 days. An astronaut activated the biological samples in the biospecimen box in the Spacehab during orbit in order to start repair of the DNA damaged by heavy ion beams or ç-irradiation and the samples were incubated for 19 h 35 min at about 22ûC, the cabin temperature. The control specimens in the SPPF were subjected to the same treatment under terrestrial gravity. After returned to earth, we investigated cell recovery by comparing the repair of the radiation-damaged DNA of E. coli and D. radiodurans in the microgravity environment in space with that on Earth. The results indicated that the DNA repair process of E. coli, but not of D. radiodurans, cells was inhibited in a microgravity environment.

Cell Survival↗
PubMed1997

A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR.

Primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, were coupled with oligonucleotides for the shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. A minimum of 10(2) CFU per PCR (10 microliters) was necessary to amplify E. coli O157:H7-specific bands by multiplex PCR. Food particles as well as various unknown metabolic by-products of bacteria inhibited the PCR, but a simple two-step filtration procedure eliminated this inhibition. To reliably generate PCR products, an E. coli inoculum of 10(3) CFU g of food slurry-1 in a nonspecific medium was required with 6 h of enrichment at 37 degrees C. However, when the food homogenate was incubated overnight, E. coli O157:H7 at an initial inoculum of even 1 CFU g-1 was detected.

Animals↗
PubMed1995

Development of a thin membrane glucose sensor using beta-type crystalline chitin for implantable biosensor.

A biocompatible thin membrane glucose sensor has been developed using beta-type crystalline chitin as a supporting material for the purpose of implantable biosensor. Gold electrode was formed by vapor deposition of the surface of glucose oxidase-immobilized beta-chitin membrane. The sensor showed rapid responses to glucose and H2O2 (< 45 s in batch operation), and addition of 2,6-dichlorophenolindophenol as a mediator caused expansion of the measuring range. To investigate the possible uses for the implantable, the sensor sterilization and toxicity were studied. Sterilization with ethylene oxide was achieved by exposing samples, and a decrease in activity was permissible for measurements of body fluid. From the results of the toxicity assay using mouse fibroblast cells, beta-chitin sensor, constructed with chitin, glucose oxidase and gold electrode, showed no particular toxicity.

Animals↗
PubMed1992

Mesenchymal hamartoma of the liver: report of an adult case and review of the literature.

A case of mesenchymal hamartoma of the liver incidentally found to be a hyperechoic mass by abdominal ultrasonography in a 62-year-old male is described. Computed tomography and magnetic resonance imaging of the liver confirmed the presence of a well-demarcated inhomogeneous mass in the left medial segment. Hepatic arteriography revealed the mass to be generally hypovascular. Histopathologic diagnosis of the resected specimen was a solid form of benign mesenchymal hamartoma, a rather rare lesion for the patient's age. Fifteen adult cases of hepatic mesenchymal hamartoma in the literature including the present case are reviewed.

Diagnosis, Differential↗