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A Ohashi

Publications and source records attributed to A Ohashi.

At least 91 records · Page 5Linked to original sources
PubMed1997

Expression of fibroblast growth factor receptors in naevus-cell naevus and malignant melanoma.

In a previous study, we showed by immunohistochemical analysis that basic fibroblast growth factor (bFGF) is expressed strongly and homogeneously in naevus-cell naevus (NCN), while that in malignant melanoma (MM) is heterogeneous and sometimes non-existent. In order to elucidate the role of bFGF in these pigmented tumours, the expression of its receptors must be determined. In this study, we performed an immunohistochemical analysis of FGF receptors 1, 2 and 3 (FGFR-1, FGFR-2 and FGFR-3, respectively) in NCN and MM and compared their expression and localization with those of bFGF. The expression of bFGF and its three receptors was also examined in melanoma cell lines. None of the 10 NCN that showed strong, homogeneous staining for bFGF expressed FGFR-1 or FGFR-3 proteins; six weakly expressed FGFR-2 protein. Ten primary and 10 metastatic MM showed heterogeneous expression for the three receptors, with larger populations of FGFR-3-negative cells in the primary than in the metastatic tumours. Western blot analysis showed homogeneous expression of bFGF protein in all four melanoma cell lines tested, while FGFR proteins had a heterogeneous distribution in the different cell lines. Cultured NCN and normal melanocytes showed no immunoreactive band for FGFR-1 protein, the only protein tested. Our results suggested that tumour-derived bFGF is involved in melanoma formation through an autocrine mechanism, but is involved mostly through a paracrine or other mechanisms in NCN.

Blotting, Western↗
PubMed1996

Development of CCD-based direct observation equipment for biological samples.

Equipment for the direct observation of biological samples has been developed. The equipment comprises a charge coupled device (CCD) unit and a light-emitting diode (LED), which is placed above the light-sensing face of the CCD. The biological sample is positioned just onto the CCD with no lens system interposed. Because of its small size, the equipment can be operated in an incubator, allowing the continuous observation of biological samples under appropriate temperature, humidity and gas concentration conditions. The equipment is operated periodically at 5-minute periods to reduce the influence of heat generated by the equipment and to maintain a constant sample temperature. E. coli colony formation is observed continuously for 70 hours without any adverse effects occurring during the observation. Hepatocyte morphological change and hepatocyte pattern formation are also observed.

Animals↗
PubMed1996

c-KIT receptor expression in cutaneous malignant melanoma and benign melanotic naevi.

To investigate the role of c-KIT receptor in melanocytic tumour development and progression, we analysed the expression and localization of c-KIT by immunohistochemistry and Western blotting. In contrast to the positive staining shown by melanocytes and naevus cells in the epidermis of common naevi (n=20), all dysplastic naevi (n=13) were negative, as were dermal melanocytic cells of blue naevi (n = 4) and common naevi (n = 26). Three out of four superficial spreading melanomas lost c-KIT expression both in the epidermal and dermal parts, while nodular melanomas showed no expression of c-KIT except in partially positive cells, and six out of seven metastatic melanomas were negative. In acral lentiginous melanomas (n = 8), in contrast to other types of melanoma, all cases with melanoma cells growing basally in the epidermis showed strong c-KIT positivity, but melanoma cells growing at the upper layers of the epidermis and vertically into the dermis lost c-KIT expression. Using the Western blot method on cultured pigment cells, human epidermal melanocytes, junctional naevus cells and one out of three metastatic melanoma cell lines showed 125 and 145 kDa bands corresponding to c-KIT, whereas dermal naevus cells did not. These results suggest that dysplastic naevi are distinct from ordinary naevi in terms of c-KIT expression and that basally growing cells in acral lentigenous melanomas could be at an initial stage of tumour progression, before c-KIT loss occurs.

Blotting, Western↗
PubMed1995

Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.

Cytochrome c oxidase consists of three mitochondrion- and several nucleus-encoded subunits. We previously found that in a mutant of Saccharomyces cerevisiae lacking nucleus-encoded subunit 4 of this enzyme (CoxIV), subunits 2 and 3 (CoxII and CoxIII), both encoded by the mitochondrial DNA, were unstable and rapidly degraded in mitochondria, presumably because the subunits cannot assemble normally. To analyze the molecular machinery involved in this proteolytic pathway, we obtained four mutants defective in the degradation of unassembled CoxII (osd mutants) by screening CoxIV-deficient cells for the accumulation of CoxII. All of the mutants were recessive and were classified into three different complementation groups. Tetrad analyses revealed that the phenotype of each mutant was caused by a single nuclear mutation. These results suggest strongly that at least three nuclear genes (the OSD genes) are required for this degradation system. Interestingly, degradation of CoxIII was not affected in the mutants, implying that the two subunits are degraded by distinct pathways. We also cloned the OSD1 gene by complementation of the temperature sensitivity of osd1-1 mutants with a COXIV+ genetic background on a nonfermentable glycerol medium. We found it to encode a member of a family (the AAA family) of putative ATPases, which proved to be identical to recently described YME1 and YTA11. Immunological analyses revealed that Osd1 protein is localized to the mitochondrial inner membrane. Disruption of the predicted ATP-binding cassette by site-directed mutagenesis eliminated biological activities, thereby underscoring the importance of ATP for function.

ATP-Dependent Proteases↗
PubMed1994

[Development of real-time CT fluoroscopy].

A new CT system that permits real-time monitoring of CT images was developed. Phantom and volunteer studies revealed that the images were displayed at a rate of six per second with a delay time of 0.83 second with clinically sufficient resolution (256 x 256) using the newly developed fast image processor and partial-reconstruction algorithm. The clinical trial of stereotactic aspiration of intracerebral hematoma was successful. The initial trial with CT fluoroscopy revealed potential usefulness of the system in biopsy and other CT-guided interventions.

Biopsy, Needle↗
PubMed1994

Aminopeptidase Y, a new aminopeptidase from Saccharomyces cerevisiae. Purification, properties, localization, and processing by protease B.

A novel aminopeptidase, termed aminopeptidase Y, was purified from yeast, Saccharomyces cerevisiae, as two molecular forms of 70 and 75 kDa, which were identical immunologically, catalytically and in N-terminal sequence up to 14 residues. They contained 0.81 and 0.84 mol of zinc atom/mol of protein, respectively. N-Gly-canase generated a single 53-kDa species from both forms, and endoglycosidase H gave a 54-kDa species. Immunoblotting after subcellular fractionation showed that aminopeptidase Y is localized in the vacuole/lysosome-soluble compartment as the 70-kDa form. Aminopeptidase Y hydrolyzed all amino acid-4-methylcoumaryl-7-amides (MCA) examined, especially Lys- and Arg-MCA. Dipeptidyl-MCAs were far more rapidly hydrolyzed (Lys-Ala-MCA/Lys-MCA = 350-fold, Arg-Arg-MCA/Arg-MCA = 150-fold). Hydrolysis of amino acid-MCAs or dipeptides was markedly enhanced by Co2+, whereas that of dipeptidyl-MCAs, tripeptides, and larger peptides was inhibited. Rabbit anti-aminopeptidase Y antiserum adsorbed over half the aminopeptidase activities in vacuolar extract from wild-type yeast cells. ABYS1 mutant cells from which the genes of four vacuolar proteases had been deleted contained aminopeptidase Y as a 74-kDa proform. This was converted to the mature form (70 kDa) by vacuolar extract of wild-type cells and by purified yeast vacuolar protease B.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of the aminopeptidase Y gene of Saccharomyces cerevisiae. Sequence analysis and gene disruption of a new aminopeptidase.

A yeast genomic DNA encoding a new vacuolar aminopeptidase, aminopeptidase Y, was isolated by using a cDNA fragment obtained by screening the lambda gt11 yeast cDNA library with anti-aminopeptidase Y antibody. The DNA sequence encodes 537 amino acids. The "mature" protein, whose NH2-terminal sequence was determined previously by analysis of the purified enzyme, consists of 481 amino acids, and the calculated molecular weight (52,900) coincides with the value obtained by SDS-polyacrylamide gel electrophoresis of the enzyme after removal of sugar chains, 53 kDa. The 56-residue preprosequence was divided into two parts by putative processing sites for signal peptidase and conversion to the mature form; the 21-residue presequence has a hydrophobic stretch which may function as the signal sequence for transit through the endoplasmic reticulum, and the 35-residue prosequence (4013 Da) accounts for the 4-kDa difference between proaminopeptidase Y in the vacuolar proteases-deleted ABYS1 mutant and wild-type mature enzyme. The aminopeptidase Y gene was localized on chromosome II by genetic mapping. A deletion mutant was constructed by disrupting the aminopeptidase Y gene. Vacuolar aminopeptidase activities toward Ala-4-methylcoumaryl-7-amide (MCA) and Lys-MCA were 13 and 20% of wild-type, and those in the presence of Co2+ were 2.2 and 2.8%, respectively. Mutant cells showed no ability to hydrolyze Lys-Ala-MCA to Lys and Ala-MCA, although vacuolar carboxypeptidase Y activity was similar to that in wild-type cells.

Amino Acid Sequence↗
PubMed1994

ATP-dependent proteolysis in yeast mitochondria.

An ATP-dependent proteolysis system in yeast mitochondria was characterized by examining the hydrolysis of mitochondrial translation products in isolated mitochondria. Degradation of [35S]methionine-labeled polypeptides synthesized in isolated yeast mitochondria was activated by exogenously added ATP. ADP, GTP, and CTP substituted for ATP to some extent, but nonhydrolyzable ATP analogues did not. Adenosine-5'-O-(3'-thio-triphosphate) effectively competed with ATP as activator. Carboxyatractyloside, an inhibitor of adenine nucleotide translocation across the mitochondrial inner membrane, and the metal chelator o-phenanthroline inhibited the ATP-dependent proteolysis. The latter inhibition was abolished by subsequent addition of Mn2+ or Co2+ but not Ca2+ or Zn2+. Hemin inhibited the ATP-dependent proteolysis of mitochondrial translation products with a half-maximum inhibition at 12 microM. Analysis by SDS-polyacrylamide gel electrophoresis showed that [35S]methionine-labeled polypeptides were rapidly degraded into low-molecular-weight species. Submitochondrial particles retained the ATP-dependent proteolytic activity and had the same spectrum of inhibitors as intact mitochondria except for a reduced effect of carboxyatractyloside. These results indicate that yeast mitochondria contain an ATP-dependent and hemin-sensitive proteolysis system which is associated with the inner membrane and can hydrolyze mitochondrial translation products, and that a chelator-sensitive protease is probably involved in this system.

Adenosine Triphosphate↗
PubMed1994

Divalent metal ion-dependent mitochondrial degradation of unassembled subunits 2 and 3 of cytochrome c oxidase.

Intracellular protein degradation plays an important role in maintaining the stoichiometry of the different subunits of an oligomeric enzyme. In a Saccharomyces cerevisiae mutant defective in cytochrome c oxidase subunit 4 encoded in nuclear DNA, mitochondrial-encoded subunits 2 and 3 cannot assemble normally [Dowhan et al. (1985) EMBO J. 4, 179-184]. In this study, we show that those unassembled forms of subunits 2 and 3 in this strain are eliminated rapidly by degradation. Reduction of the intracellular ATP level by inhibiting the glycolytic pathway, or inhibition of the entry of ATP into mitochondria by bongkrekic acid, both of which are expected to reduce the intramitochondrial ATP level in respiratory-deficient cells such as WD1, significantly suppressed the degradation, suggesting that the degradation requires intramitochondrial ATP. The degradation was also inhibited by o-phenanthroline, a membrane-permeable metal chelator, and this inhibitory effect was suppressed by addition of an excess amount of Co2+, Mn2+, or Zn2+, but not by Ca2+ or Mg2+, suggesting a novel metal-dependence of the degradation of unassembled Cox II and Cox III which has not been reported previously for mitochondrial metabolic protein degradation systems. A potential advantage of using this strain for identifying the factor(s) involved by a genetical approach is discussed.

Adenosine Triphosphate↗
PubMed1994

[The usefulness of the bayesian method for the prediction of plasma theophylline levels in asthmatic children during RTC therapy by the sustained preparation of theophylline].

Pharmacokinetic prediction of plasma theophylline levels during RTC therapy by the sustained release preparation of theophylline was studied with a one compartment model in children aged 1-6 years old showing comparatively long sleeping time. Using the parameters measured from the population of 1-17 year olds, the accuracy of the prediction was studied by the Bayesian method. Absolute error (A.E.) and estimation error (E.E.) were about 2.0. The prediction values of E.E. seemed to be rather higher if the measured values of the absorption phase were used, and the values seemed to be rather lower if the values of the elimination phase were used. From these results it might be concluded that the Bayesian method seems to be useful for RTC therapy in children under 6 years old.

Adolescent↗
PubMed1991

Efficacy of endoscopic retrograde cholecystoendoprosthesis (ERCCE) for cholecystitis.

Fourteen patients with cholecystitis were treated by endoscopic placement of an endoprosthesis into the cystic duct. In these patients the administration of antibiotics had failed to change the clinical picture of cholecystitis. Clinical and ultrasonographic findings before and two months after stenting were compared. There was an improvement in abdominal pain in 9 out of 14 cases (64%); fever, present in 5 patients, disappeared in all. No clinical complications were observed. Sonographic findings in terms of gallbladder wall thickening and sludge present within the gallbladder improved in 57% and 88%, respectively. Our results indicate that this endoscopic procedure may be effective in the treatment of cholecystitis.

Aged↗
PubMed1991

Diagnosis of biliary diseases via a naso-biliary catheter.

Nasobiliary double contrast radiography and nasobiliary cytological diagnosis via nasobiliary catheter were performed in 30 patients with biliary diseases to assess the diagnostic usefulness and limitations of the procedures. The results showed that the contour and fine mucosal pattern of the extrahepatic bile duct could be demonstrated adequately (88%) by nasobiliary double contrast radiography. In contrast, the intrahepatic bile duct could not be fully demonstrated (56%) by nasobiliary double contrast radiography because of its anatomical structure. On the other hand the fine reticular pattern of the mucosa was defined in each section of the gallbladder by nasobiliary double contrast radiography. This procedure is thus a valuable method for detection of mucosal lesions of the extrahepatic bile duct and gallbladder, especially carcinoma. The cell collection rate was not as high (59%) using conventional nasobiliary cytological diagnosis. Brush cytology via a nasobiliary catheter was shown to be a useful adjunct to other diagnostic procedures.

Barium Sulfate↗
PubMed1990

Apocarboxypeptidase B-sepharose: a specific adsorbent for peptides.

Apocarboxypeptidase B-Sepharose was prepared by immobilization of porcine carboxypeptidase B, followed by treatment of the column with o-phenanthrolin. This column efficiently adsorbed Met-enkephalin-Arg-Arg (YGGFMRR) in an optimum pH range of 6.5-7.5. The adsorbed Met-enkephalin-Arg-Arg was eluted at pH 4.0 and confirmed to be unaltered. In the apocarboxypeptidase B-Sepharose chromatography, Met-enkephalin-Arg-Arg or dynorphin 1-13 (YGGFLRRIRPKLK), substrates of carboxypeptidase B, was separated from Met-enkephalin (YGGFM), dynorphin B 1-9 (YGGFLRRQF), and beta-neo-endorphin (YGGFLRKYP) which do not react with the immobilized enzyme.

Amino Acid Sequence↗
PubMed1990

[A case of hyperplastic polypoid lesion in the common bile duct hard to differentiate from early bile duct carcinoma].

In a 72-year-old male carcinoma of the bile duct was suspected because of an abnormality on laboratory examination. Ultrasound showed the dilatation of extrabiliary duct. Endoscopic ultrasound revealed localized wall thickening of the common bile duct. ERCP demonstrated the dilatation of the CBD. Within the dilated duct a filling defect was present. Histology of the resected specimen disclosed hyperplasia of the epithelium in the CBD.

Aged↗
PubMed1989

Cloning and expression in Escherichia coli of an extremely thermostable oligo-1,6-glucosidase gene from Bacillus thermoglucosidasius.

The gene for an extremely thermostable oligo-1,6-glucosidase (dextrin-6-alpha-D-glucanohydrolase; EC 3.2.1.10) of obligately thermophilic Bacillus thermoglucosidasius KP1006 was cloned within a 4.2-kilobase HindIII-PvuII fragment of DNA by using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. The gene was transcribed, presumably from its own promoter, in E. coli. E. coli with the hybrid plasmid accumulated oligo-1,6-glucosidase mainly in the cytoplasm. The level of enzyme production was comparable to that observed for B. thermoglucosidasius. The enzyme coincided absolutely with the B. thermoglucosidasius enzyme in its molecular weight (60,000), in its electrophoretic behavior on denaturing and nondenaturing polyacrylamide gels, in the temperature dependency of its stability and activity, and in its antigenic determinants.

Bacillus↗
PubMed1987

New chelator-sensitive proteases in matrix of yeast mitochondria.

Proteases in yeast mitochondria were studied using fluorogenic synthetic substrates containing methylcoumaryl amide (MCA). Among the eleven substrates which are commonly employed for several types of proteases, Leu-MCA, Arg-MCA, Boc-Gln-Arg-Arg-MCA and Boc-Phe-Ser-Arg-MCA were found to be highly susceptible to proteases in mitochondria. All these proteases were localized in the matrix and sensitive to o-phenanthroline but not to phenylmethylsulfonyl fluoride or iodoacetate. The analysis of hydrolyzed products of Boc-Gln-Arg-Arg-MCA indicated that the peptide was cleaved at the site between Gln and Arg. These results demonstrate that there exist chelator-sensitive aminopeptidase(s) and endopeptidases in the matrix of yeast mitochondria.

Aminopeptidases↗
PubMed1987

A simple and rapid assay for heme attachment to apocytochrome c.

A method for assaying the covalent attachment of heme to apoprotein of cytochrome c was developed. 125I-labeled apoprotein was chemically prepared from 125I-labeled yeast cytochrome c (iso-1-cytochrome c). After incubation of 125I-apocytochrome c with yeast mitochondria, the product was extracted with Triton X-100, digested with trypsin in the presence or absence of a reducing agent, and then precipitated in trichloroacetic acid. The resulting precipitates were collected on nitrocellulose membranes and counted for radioactivity. The radioactivity correlated well with the appearance of a heme-containing peptide in the trypsin digested peptide fragments of cytochrome c. This procedure is simpler and faster than the previously reported methods.

Apoproteins↗