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Biomedical subjects

S Hase

Publications and source records attributed to S Hase.

At least 55 records · Page 3Linked to original sources
PubMed1997

Purification and characterization of neutral alpha-mannosidase from hen oviduct: studies on the activation mechanism of Co2+.

Neutral alpha-mannosidase was purified to homogeneity from hen oviduct. The molecular mass of the enzyme was 480 kDa on gel filtration, and the 110-kDa band on SDS-PAGE in the presence of 2-mercaptoethanol indicated that it is composed of four subunits. The activated enzyme hydrolyzed both p-nitrophenyl alpha-D-mannoside and high mannose-type sugar chains. This substrate specificity is almost the same as that reported for the neutral a-mannosidase from Japanese quail oviduct [Oku and Hase (1991) J. Biochem. 110, 982-989]. Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GlcNAc (Km =0.44 mM) was hydrolyzed four times faster than Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GIcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)++ +Manbeta1-4GlcNAc was obtained as the end product from Man9GlcNAc on digestion with the activated alpha-mannosidase. The enzyme was activated 24-fold on preincubation with Co2+. The activation with other metal ions, like Mn2+, Ca2+, Fe2+, Fe3+, and Sr2+, was less than 5-fold, and Zn2+, Cu2+, and Hg2+ inhibited the enzyme activity. The optimum pHs for both the enzyme activity and activation with Co2+ were around 7. The cobalt ion contents of the purified, EDTA-treated, and Co2+-activated enzymes were 1.5, 0.0, and 3.9, respectively, per molecule. Since the Co2+-activated enzyme gradually lost its activity on incubation with EDTA and the activity was restored promptly on the addition of Co2+, the binding of Co2+ to the enzyme seems to be essential for its activation. The results obtained with protease inhibitors together with those of the SDS-PAGE before and after activation, showed that the proteolytic cleavage reported for the activation of monkey brain alpha-mannosidase seems not to be involved.

Animals↗
PubMed1997

Relationship between local immune response to Helicobacter pylori and the diversity of disease: investigation of H. pylori-specific IgA in gastric juice.

In order to evaluate the relationship between local immune response to Helicobacter pylori and the diversity of disease, 77 asymptomatic subjects who underwent a health examination were studied. Helicobacter pylori-specific IgG in serum and H. pylori-specific IgA in gastric juice were measured by ELISA, and the measured IgA titre was classified into two grades, low or high. Histological classification of gastritis was performed according to the Sydney system. Cytokines in gastric juice were also measured, and the cytotoxin-associated gene A (cagA) status of H. pylori was tested by PCR. Of the 65 subjects who were positive for H. pylori-specific IgG in serum, 38 (58.5%) were classified as H. pylori-specific IgA low titre in gastric juice and 27 (41.5%) had high titres. In the IgG-positive, IgA-low group, the rate of peptic ulcers (especially duodenal ulcers) in endoscopic findings was higher (P < 0.05); the score of activity and the density of H. pylori were higher (P < 0.001 and P < 0.05, respectively); the score of metaplasia was lower (P < 0.05); and the level of interleukin-1 beta was lower (P < 0.05) than in the IgG-positive, IgA-high group. The positive rate of the cagA gene was 84.4% and there was no significant difference between the two groups. There were differences in endoscopic and histological findings between the IgG-positive, IgA-low and the IgG-positive, IgA-high groups. It is suggested that persons infected with H. pylori can be divided into two different states of disease according to local immune response.

Antibodies, Bacterial↗
PubMed1997

Kinetics of fibroblasts in ulcer healing in rats: interference with indomethacin.

OBJECTIVE: The authors reported that the repair of submucosal tissue is important in ulcer healing. The kinetics of fibroblasts have not been well known in gastric ulcer healing. The effect of subcutaneous administration of indomethacin (1 mg/kg) to submucosal tissue was also examined. METHOD: Immunohistological staining was done using the antibodies for the proliferating cell nuclear antigen prolyl 4-hydroxylase and alpha-smooth muscle actin in acetic acid induced gastric ulcers in rats. The terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end labeling method was also employed. RESULTS: The granulation tissue consisted predominantly of lymphocytes in indomethacin-treated rats. In the control group, proliferative cell nuclear antigen positive cells were at their peak 5 days after ulcer formation (day 5). Prolyl 4-hydroxylase positive fibroblasts were maximal on day 10. Alpha-smooth muscle actin positive myofibroblasts were few on day 5 and increased on day 15. In terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end labeling, positive cells were sporadically seen on days 20 and 30. In indomethacin-administered rats, the fibroblasts proliferated weakly. The numbers of prolyl 4-hydroxylase and alpha-smooth muscle actin positive spindle-shaped cells were fewer and their appearance delayed. Nick end labeling positive cells were few before day 30 and sporadically observed on days 50 and 80. CONCLUSION: The results indicate that fibroblasts differentiated into several phenotypes and finally underwent apoptosis.

Acetic Acid↗
PubMed1997

Three-dimensional endoscopic ultrasonography of gastrointestinal lesions using an ultrasound probe.

BACKGROUND: This study was carried out to ascertain the clinical usefulness and problems of three-dimensional (3D) images obtained with the ultrasound probe. METHODS: Eighteen resected specimens and 21 patients were examined. After the images were recorded on video, 3D displays were produced using a workstation. RESULTS: In the in vitro study the surface images were quite consistent with the macroscopic findings in 17 cases. In 2 esophageal cancers, 7 of 10 gastric cancers, and 2 colonic cancers the depth of tumor invasion was assessed accurately from the reconstructed images. In the in vivo study although 3 D display had some limitations, it was useful in esophageal and rectal lesions. CONCLUSIONS: This new diagnostic method could visualize the 3D extent of gastrointestinal lesions and appears to have a useful clinical application.

Adult↗
PubMed1997

Effect of histamine on human fibroblast in vitro.

The effects of histamine (CAS 51-45-6) on cell growth, collagen synthesis of fibroblasts derived from human foreskin, and on fibroblast-mediated collagen remodelling were studied. The cellmat DNA content was measured 2 days after human fibroblasts were plated at a split ratio of 1:10. Effect of histamine (10(-9)-10(-4) mol/l) on the increase of DNA content was not observed. Fibroblasts at confluence were cultured with histamine only, and with pyrilamine or cimetidine in addition to histamine for 2 h. Type I procollagen C-peptide in the medium was measured by enzyme immunoassay and was corrected by DNA content. Type I collagen synthesis was stimulated by histamine (10(-6)-10(-4) mol/l) and its stimulation was inhibited by cimetidine, but not by pyrilamine. Collagen solution containing fibroblast was incubated until gelation. It was incubated with histamine only, and with pyrilamine or cimetidine in addition to histamine. The gel contraction was stimulated by histamine (10(-4) mol/l) and its stimulation was inhibited by pyrilamine, but not by cimetidine. These facts suggests that histamine stimulates type I collagen synthesis of fibroblast and collagen remodeling via H2 and H1 receptors, respectively.

Cell Division↗
PubMed1996

Preparation of neoglycolipids with a definite sugar chain moiety from glycoproteins.

A method for preparing neoglycolipids possessing a sugar chain with a definite structure using two N-linked sugar chains as model compounds was developed. Pyridylaminated sugar chains obtained from glycoproteins were purified by reversed-phase HPLC and a pyridylaminated sugar chain thus obtained was converted to a 1-amino-1-deoxy derivative. The product was conjugated by reductive amination with phosphatidylglycolaldehyde prepared by periodate oxidation of dipalmitoyl phosphatidylglycerol. Neoglycolipids formed were purified by gel filtration from the reaction mixture. The structures of the purified neoglycolipids were confirmed by composition analysis for sugar, fatty acid, phosphorus, and 2-acetamido-1,2-dideoxy-1-[(hydroxyethyl)amino]-D-glucitol, the linkage region of the sugar chain, and lipid moieties. The method provides a convenient means of preparing neoglycolipids having a definite structure.

Carbohydrates↗
PubMed1996

Classification of sugar chains of glycoproteins by analyzing reducing end oligosaccharides obtained by partial acid hydrolysis.

Sugar chain types were classified on the basis of reducing end di- and trisaccharide structures. Sugar chains liberated from glycoproteins by the hydrazinolysis-N-acetylation method were pyridylaminated, and pyridylamino (PA-) sugar chains were purified by HPLC. The PA-sugar chains thus purified were partially hydrolyzed with 1 M trifluoroacetic acid. The acid hydrolysis conditions were investigated with the object of obtaining PA-di- and trisaccharides with high yields for different types of PA-sugar chains. The acid hydrolysates were separated by size-fractionation HPLC into PA-mono-, PA-di-, and PA-trisaccharides, and each fraction was analyzed by reversed-phase HPLC. The structures were then identified by comparing the HPLC elution positions with those of authentic PA-oligosaccharides derived from N-linked sugar chains and 12 types of O-linked sugar chains.

Acetylation↗
PubMed1996

Analysis of pyridylaminated O-linked sugar chains by two-dimensional sugar mapping.

Two-dimensional HPLC mapping of O-linked sugar chains was studied. Twenty-seven pyridylamino (PA-) derivatives of O-linked sugar chains liberated from glycoproteins by hydrazinolysis-N-acetylation were separated by reversed-phase and size-fractionation HPLC. Combining the elution positions obtained by these two HPLC techniques allows the construction of a two-dimensional sugar map which can be used to analyze the structure of PA-O-linked sugar chains. As in the case of PA-N-linked sugar chains, the additivity rule which correlates chemical structures with elution positions was also found in the case of PA-O-linked sugar chains. This separation system was effectively applied to the elucidation of minute amounts of PA-O-linked oligosaccharides.

Aminopyridines↗
PubMed1996

Preoperative staging of superficial esophageal carcinoma: comparison of an ultrasound probe and standard endoscopic ultrasonography.

BACKGROUND: In diagnosing superficial esophageal carcinoma, it is necessary to differentiate mucosal carcinoma from submucosal carcinoma because mucosal carcinoma has a good prognosis and local treatment is likely to be successful. We evaluated an ultrasound probe and endoscopic ultrasonography (EUS) in the staging of superficial esophageal carcinoma. METHODS: From October 1992 to September 1994, 22 patients with 25 lesions (7 mucosal carcinomas, 18 submucosal carcinomas) were examined preoperatively with both the probe and EUS. The ultrasound findings were compared with histologic findings in all cases. RESULTS: The accuracy rates of the depth of invasion by the ultrasound probe were 86% (6 to 7) for mucosal carcinoma and 94% (17 to 18) for submucosal carcinoma, total 92% (23 to 25); by EUS 71% (5 to 7) for mucosal carcinoma and 78% (14 to 18) for submucosal carcinoma, total 76% (19 to 25). In the evaluation of lymph node metastasis, the overall accuracy was 56% by the ultrasound probe (sensitivity 25% and specificity 80%) and 67% by EUS (sensitivity 50% and specificity 80%). CONCLUSIONS: The ultrasound probe was more convenient to use and more accurate than EUS in the evaluation of the depth of invasion of superficial esophageal carcinoma.

Adult↗
PubMed1996

Substrate specificity of bovine liver cytosolic neutral alpha-mannosidase activated by Co2+.

A cytosolic neutral alpha-mannosidase was purified from bovine liver. Its molecular weight was found to be 500,000 on gel filtration. The activity of the enzyme toward Man alpha 1-6-(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc-PA was increased 26-fold by preincubation with 1 mM Co2+. Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc was hydrolyzed by the enzyme to Man alpha 1-3Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc, which was further hydrolyzed to Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc. The rate of hydrolysis was 15-fold greater than that of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. This substrate specificity suggested that the enzyme could be involved in the degradation of oligomannose-type sugar chains with one GlcNAc residue released from glycoproteins by endo-beta-N-acetylglucosaminidase, and supported a pathway for glycoprotein catabolism via oligomannosyl glycans with one GlcNAc residue proposed on the basis of an earlier study on a cytosolic neutral alpha-mannosidase from Japanese quail oviduct [Oku, H. and Hase, S. (1991) J. Biochem. 110, 982-989].

Acetylglucosamine↗
PubMed1996

Purification and characterization of hen oviduct alpha 1,2-mannosidase.

An alpha-mannosidase capable of hydrolyzing three Man alpha 1,2-residues from pyridylamine-(PA-) labeled Man9GlcNAc2 was purified from hen oviduct. The purity of the preparation was analyzed by PAGE; its molecular weight was 42,000 by SDS-PAGE or 50,000 by gel filtration. The pH optimum was 6.5. The enzyme was inactivated with EDTA; enzyme activity was restored by the addition of Ca2+. The enzyme activity was inhibited by 1-deoxymannojirimycin, but not by swainsonine. The substrate specificity of the purified enzyme was analyzed using PA-oligomannose-type sugar chains. When Man9GlcNAc2-PA was digested, Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4Glc-NAc-PA was obtained as an end product, and the enzyme was incapable of hydrolyzing p-nitrophenyl alpha-D-mannoside and Man alpha 1,3- or Man alpha 1,6-residues. Judging from these characteristics, the enzyme was classified as a Man9-mannosidase or Golgi mannosidase I and speculated to participate in the processing or catabolism of glycoproteins.

1-Deoxynojirimycin↗
PubMed1996

Detection of UDP-D-xylose: alpha-D-xyloside alpha 1-->3xylosyltransferase activity in human hepatoma cell line HepG2.

We previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xyl alpha 1-3Glc) and a trisaccharide (Xyl alpha 1-3Xyl alpha 1-3 Glc) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors. The structures of these sugar chains suggested the presence of an alpha 1-->3xylosyltransferase for their biosynthesis. We report here on the detection of alpha 1-->3xylosyltransferase activity which catalyzes the transfer of xylose to Xyl alpha 1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xyl alpha 1-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor. The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xyl alpha 1-3Xyl alpha 1-3Glc by Smith degradation, mass spectrometry, and alpha- and beta-xylosidase digestions. The apparent K(m) value for pyridylaminated Xyl alpha 1-3Glc was 52 mM and for UDP-D-Xyl 0.28 mM. Optimum pH was 7.2. The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xyl alpha 1-3Glc, forming Xyl alpha 1-3Xyl alpha 1-3Glc in human cells.

Animals↗
PubMed1995

Structures of the N-linked oligosaccharides on human plasma vitronectin.

The structures of N-linked oligosaccharides present on human plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N-glycosidase F digestion and tagged with 2-aminopyridine; the pyridylamino-oligosaccharides were then fractionated by anion-exchange and reverse-phase HPLC. Ten major pyridylamino-oligosaccharides were isolated. The linkages and locations of sialic acid residues were determined by desialylation with Salmonella sialidase in combination with acid. The asialo forms were then analyzed by two-dimensional sugar mapping, component sugar analysis and 400-MHz 1H-NMR spectroscopy. The major oligosaccharides of human vitronectin were of the diantennary N-acetyllactosamine type, with a lesser amount of the tri- and a small amount of the mono-antennary type, to which 1-3 mol sialic acid residues were linked, mostly through alpha 2-6 linkages, although alpha 2-3 linkages were also present. The possibility that several binding activities of vitronectin can be ascribed to its glycan moiety was discussed, based on the specific features of the N-linked oligosaccharides on human vitronectin revealed here.

Carbohydrate Sequence↗
PubMed1995

Flow cytometric analysis of DNA heterogeneity in superficial carcinoma of the esophagus.

BACKGROUND: There are few studies of flow cytometric analysis for DNA heterogeneity of patients with superficial carcinoma of the esophagus limited to the epithelium or superficially invading the lamina propria or submucosa. METHODS: Flow cytometric analysis of cellular DNA content was performed on superficial carcinomas of the esophagus using paraffin embedded blocks of the surgically resected specimens from 56 patients. To evaluate the intratumoral DNA heterogeneity, a total of 141 samples of the 56 tumors were analyzed, depending upon the tumor size. RESULTS: One or two of the samples was available from 18 of 19 patients with tumors 2 cm or less in greatest dimension, whereas more than three of the samples were available from 22 of 37 patients with tumors 2.1 cm or greater in dimension (P < 0.003). Of 56 tumors, 40 (71.4%) exhibited DNA aneuploidy; DNA heterogeneity was found in 26 tumors (46.4%). The remaining 16 tumors exhibited DNA diploidy. Two of the five tumors that were limited to the epithelium had DNA heterogeneity. The mean dimension of the tumors with DNA heterogeneity was significantly greater (5.8 +/- 2.8 cm) than those exhibiting DNA diploidy (2.3 +/- 1.1 cm) and DNA aneuploidy without heterogeneity (2.9 +/- 2.4 cm). Recurrences after esophagectomy were detected in 6 of the 56 patients; the DNA ploidy pattern of these six patients exhibited DNA heterogeneity. CONCLUSION: The incidence of DNA heterogeneity increases as tumor size increases and is associated with an increased risk of tumor recurrence after esophagectomy in patients with superficial carcinoma of the esophagus.

Adult↗