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Y Amuro

Publications and source records attributed to Y Amuro.

At least 37 records · Page 2Linked to original sources
PubMed1991

Boronate affinity chromatography of gamma-glutamyltransferase in patients with hepatocellular carcinoma.

We analyzed the serum gamma-glutamyltransferase (gamma-GT) by boronate affinity chromatography to ascertain the presence or absence of any changes in the binding properties of gamma-GT toward boronate gels in patients with hepatocellular carcinoma and liver cirrhosis, and in normal controls. The mean gamma-GT activity ratio of the bound (peak 2) and nonbound (peak 1) fraction in patients with hepatocellular carcinoma was significantly higher than that in patients with liver cirrhosis or in normal controls. Thus, the gamma-GT, which has adjacent cis-hydroxyl groups in its carbohydrate moieties, was found to increase in the serum of patients with hepatocellular carcinoma. The positivity rate was examined in patients with hepatocellular carcinoma and liver cirrhosis, using a cut-off level for the peak 2:peak 1 ratio of 1.05 (mean + 2 SD of liver cirrhosis). Nineteen (42.2%) patients with hepatocellular carcinoma had a ratio of peak 2:peak 1 higher than 1.05. Nine of the 19 patients who had serum alpha-fetoprotein levels below 100 ng/ml had an elevated peak 2:peak 1 ratio. In total, 77.8% of the occurrence of hepatocellular carcinoma could be detected by a combination of these two markers. Three patients who had developed hepatocellular carcinoma during the course of cirrhosis but remained negative for alpha-fetoprotein throughout the course developed higher levels of peak 2:peak 1 ratio when hepatocellular carcinoma occurred. These results indicate that the two markers, the peak 2:peak 1 ratio of serum gamma-GT activity and serum alpha-fetoprotein level, may be considered to serve as complementary markers for the diagnosis of hepatocellular carcinoma.

Adult↗
PubMed1990

Purification and properties of 3 alpha-hydroxysteroid dehydrogenase as a 3-keto bile acid reductase from human liver cytosol.

The NADPH-dependent 3 alpha-hydroxysteroid dehydrogenases (peaks 1, 2 and 3) acting on 3-keto-5 beta-cholanoic acid separated by carboxymethyl-cellulose chromatography from human liver cytosol were purified to homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using Affi-Gel blue, phenyl-Sepharose CL-4B, TSKgel G3000 SW chromatography and chromatofocusing. The overall purifications of the enzymes from cytosol were 316-fold (peak 1), 232-fold (peak 2) and 345-fold (peak 3) and the recoveries of the enzymes were 0.4% (peak 1), 7.1% (peak 2) and 3.7% (peak 3). The isoelectric points of the enzymes were found to be 7.34, 7.46 and 7.88 by chromatofocusing. The molecular weights of the enzymes were similar and estimated to be about 32,000 by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymological properties were nearly identical among the three forms. The reaction was reversible and the optimum pH of the enzymes for oxidation was about 8.4 and that for reduction was about 7.4. The enzymes could not reduce 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid, 7 alpha-hydroxy-5 beta-cholestan-3-one and 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one to the corresponding 3 alpha-hydroxysteroids, whereas the enzymes could reduce 3,7-disubstituted 3-keto bile acids. Thus, the enzymes purified in this study were found to have a stereospecific character for some 3-ketosteroids. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of dithiothreitol to the reaction mixture, indicating that the enzyme required a sulfhydryl group for activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Purification and some properties of the fast migrating alkaline phosphatase in FL-amnion cells (the Kasahara isoenzyme) and its cDNA cloning.

One of two main FL-amnion cell alkaline phosphatase (AP), the fast migrating one (FL-APF) has been reported to be identical to Kasahara isoenzyme (K.I.), which occurs preferentially in sera of patients with primary hepatoma. We purified FL-APF of which the apparent molecular weight was 135,000 by gel filtration, and that of the subunit was 62,000 on SDS/PAGE, indicating homodimeric structure of FL-AL-APF. FL-APF was found to react with monoclonal antibody against adult intestinal AP, but not with monoclonal antibody to placental AP. We isolated FL-APF cDNA clone from FL-amnion cells, of which cDNA was 2525 base pairs in length. Nucleotide sequence of the coding region and the 3' untranslated region was identical to the sequence of human adult intestinal AP cDNA. But the untranslated region of the 5' end of the isolated clone was slightly longer than that of intestinal AP. Hence, FL-APF (K.I.) may occur by altered glycosylation of intestinal AP.

Alkaline Phosphatase↗
PubMed1990

Discrimination of liver cirrhosis from chronic hepatitis by analysis of serum cholinesterase isozymes using affinity electrophoresis with concanavalin A or wheat germ agglutinin.

Isozymic alteration of serum cholinesterase (ChE) was investigated in patients with chronic liver diseases using affinity electrophoresis with concanavalin A (Con A) or wheat germ agglutinin (WGA). On Con A-containing agarose gel electrophoresis, three bands with enzyme activity (named bands I to III, from the anodic side to the cathodic) were observed in sera of normal controls. Disappearance of band II was observed in 50% (15/30) of cirrhotic patients, but only one of 20 patients with chronic hepatitis lacked band II of the serum ChE isozymes. Meanwhile, WGA-containing agarose gel electrophoresis revealed that normal controls had four ChE isozymes (named bands I to IV from the anodic side to the cathodic). These four isozymes were also observed in patients with chronic hepatitis. However approximately 67% (20/30) of cirrhotic patients lacked band II of ChE isozymes. When these two affinity electrophoreses were used in combination, 22 (73%) of 30 cirrhotic patients had isozymic alteration of their serum ChE on either Con A-containing or WGA-containing agarose gel electrophoresis, or both. Thus, affinity electrophoreses with Con A and WGA seemed to be useful methods in differentiating liver cirrhosis from chronic hepatitis.

Cholinesterases↗
PubMed1990

Monoclonal antibody to pseudouridine used to develop a radioimmunoassay.

Pseudouridine, a component of tRNA, was modified to yield the derivatives: succinyl, palmitoyl pseudouridine, and protein conjugates. These derivatives were used in preparation of monoclonal antibodies specifically directed to pseudouridine. MAbs from three hybridomas (OAL 881, 812 and 814) were established and shown to be directed to pseudouridine, uridine and uracil. OAL 881 was equally reactive with the three substrates, OAL 812 showed the same reactivity for pseudouridine and uracil, while OAL 814 showed a reactivity mainly for pseudouridine. MAb OAL 814 was used in a radioimmunoassay (RIA) system unique for pseudouridine. A good dose-response curve was observed in the range between 31.3 and 2000 nmol/ml. Intra- and inter-assay CV values were below 4.1% and 7.4% respectively, and good results were obtained from recovery of added material and dilution tests. The ratio of pseudouridine/creatinin in urine was significantly higher in patients with cancer than in normal subjects.

Antibodies, Monoclonal↗
PubMed1989

Reduction of 7-ketolithocholic acid by human liver enzyme preparations in vitro.

The formation of chenodeoxycholic and ursodeoxycholic acids from 7-ketolithocholic acid by human liver preparations was examined in vitro. Liver preparations were incubated with 7-ketolithocholic acid at pH 5.5 in a sodium-potassium-phosphate buffer containing NADPH or NADH. The products formed were analyzed by gas chromatography and gas chromatography-mass spectrometry. Results showed that chenodeoxycholic and ursodeoxycholic acids could be formed from 7-ketolithocholic acid by human liver enzyme(s). The enzyme(s) required NADPH but not NADH as coenzyme and was localized largely in the microsomes. The conjugated 7-ketolithocholic acid, especially the taurine conjugated, was predominantly reduced to chenodeoxycholic acid, whereas the unconjugated 7-ketolithocholic acid was not reduced well to either chenodeoxycholic acid or ursodeoxycholic acid. Thus the reduction of 7-ketolithocholic acid by human liver enzyme(s) was found to be dependent on whether the substrate was conjugated or not.

Chenodeoxycholic Acid↗
PubMed1988

Serum pseudouridine as a biochemical marker in patients with hepatocellular carcinoma.

The concentration of serum pseudouridine, a degradation product of transfer ribonucleic acid, was determined by high-performance liquid chromatography in patients with hepatocellular carcinoma, liver cirrhosis, other benign hepatobiliary diseases, and healthy controls. The serum pseudouridine concentration in patients with hepatocellular carcinoma was significantly higher than that in patients with cirrhosis or the controls. Twenty-seven (51.9%) of 52 patients with hepatocellular carcinoma had serum pseudouridine concentrations higher than the mean value for healthy controls plus 2 SD. Fourteen of the 36 patients who had serum alpha-fetoprotein levels below 400 ng/ml, had elevated serum pseudouridine concentration. In total, 36 of the 52 patients (69.2%) could be detected by combination of these two markers. Two patients who had developed hepatocellular carcinoma during the course of cirrhosis and were continuously negative for alpha-fetoprotein, had higher levels of the pseudouridine concentration when hepatocellular carcinoma occurred. Furthermore, 4 of the 7 patients who had a very small cancer and were negative for alpha-fetoprotein, had elevated serum pseudouridine concentration. These results indicate that serum pseudouridine is a useful biochemical marker and that serum pseudouridine and alpha-fetoprotein in combination are considered to serve as complementary markers, for the diagnosis of hepatocellular carcinoma.

Aged↗
PubMed1988

Direct determination of urinary pseudouridine by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method was developed for the determination of pseudouridine in urine. This method does not need pretreatment by boronate affinity gel. Therefore, it can be used in screening patients with malignant disease and for monitoring clinical response to chemotherapy with other tumor markers.

Biomarkers, Tumor↗
PubMed1988

Urinary pseudouridine as a biochemical marker in the diagnosis and monitoring of primary hepatocellular carcinoma.

The urinary level of pseudouridine, primarily a degradation product of transfer ribonucleic acid (tRNA), was determined in 38 patients with primary hepatocellular carcinoma, 18 with liver cirrhosis, 12 with chronic hepatitis, nine with acute hepatitis, and 28 healthy subjects. The mean urinary pseudouridine concentration was significantly higher in the patients with hepatoma [38.2 +/- 12.8 (SD) nmol/mumol creatinine] than in those with liver cirrhosis (20.3 +/- 6.8), chronic hepatitis (24.4 +/- 8.2), and acute hepatitis (21.7 +/- 8.2), and in healthy subjects (23.8 +/- 4.9). Urinary pseudouridine level was elevated above the mean value plus 2 SD for the healthy subjects (33.6 nmol/mumol creatinine) in 71% of our hepatoma cases. Serum alpha-fetoprotein levels correlated poorly with urinary pseudouridine levels, thus, the combination assay for urinary pseudouridine and serum alpha-fetoprotein could detect 79% of the patients with hepatoma. Moreover, urinary pseudouridine level was reduced after effective transcatheter arterial embolization therapy.

Adult↗
PubMed1987

Partial purification and characterization of 7 alpha-hydroxysteroid dehydrogenase from rat liver microsomes.

An NADPH-dependent 7 alpha-hydroxysteroid dehydrogenase acting on 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was partially purified 160-fold with a yield of 13% from rat liver microsomes using DEAE-cellulose, hydroxyapatite and Affi-Gel Blue column chromatography. The specific activity of the purified enzyme was 91.3 nmol chenodeoxycholic acid formed/min per mg of protein. The reaction was reversible, and the optimum pH of the enzyme for the oxidation was about 8.5, whereas that for the reduction was about 5.0 A molecular weight of the enzyme was estimated to be about 130,000 by Superose 6TM gel filtration chromatography. The apparent Km value for 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was 35.7 microM and that for NADPH was 90.9 microM. The preferred substrate for the enzyme was 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid rather than 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, a 7-keto-bile acid analogue. The enzyme also preferred the unconjugated form to the conjugated forms. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of reduced form of glutathione to the reaction mixture, indicating that the enzyme requires a sulfhydryl group for activity.

Animals↗
PubMed1987

Autoimmune hepatitis or hepatic involvement in SLE?--A case report.

We report a patient with autoimmune hepatitis, who simultaneously satisfied the 1982 revised ARA criteria for systemic lupus erythematous and emphasize the difficulty in differentiating these two diseases. In addition, current concepts of a possible immunological distinction between autoimmune hepatitis and hepatic involvement in SLE are reviewed.

Autoimmune Diseases↗
PubMed1987

Novel cholinesterase expression in the HuH-7 cell line.

On polyacrylamide gradient gel electrophoresis, normal serum cholinesterase was separated into seven isozymes (I-VII from the anodic to the cathodic side). The enzyme of the conditioned medium of the HuH-7 cell line, established from a human hepatoma, had two main isozymes. The one migrating faster was located slightly to the cathodic side of band II of the normal serum enzyme, and the other, a slower one, electrophoresed at the same position as that of band VI of the normal serum enzyme. Aside from these two isozymes, a faint band with enzyme activity sometimes appeared at a position just cathodic or very close to the position of band I of the normal serum isozymes. The effect of some inhibitors and activators on both the normal serum enzyme and the enzyme of the conditioned medium was similar, but lectin-binding properties of the two enzymes were different with Ricinus communis agglutinin I, concanavalin A and wheat germ agglutinin. These results suggest that the difference in sugar moieties of both enzymes is expressed in D-galactose, D-mannose and N-acetylglucosamine.

Carcinoma, Hepatocellular↗
PubMed1987

Erythrocyte 2,3-diphosphoglycerate in liver diseases.

The erythrocyte 2,3-diphosphoglycerate (2,3-DPG) was studied in patients with liver diseases, chronic obstructive pulmonary disease, and in normal subjects. The level of 2,3-DPG in liver diseases occurred in the following increasing order: chronic persistent hepatitis, chronic active hepatitis, liver cirrhosis, and cirrhosis with hepatocellular carcinoma. A significant negative correlation between the 2,3-DPG concentration and serum albumin concentration was found in the liver diseases. The 2,3-DPG level was correlated to the serum concentration of total bile acids and to the arterial blood pH. A negative correlation was found between the arterial blood pH and the serum albumin concentration. The level of 2,3-DPG in hepatocellular carcinoma and/or liver cirrhosis was higher than that in more hypoxic chronic obstructive pulmonary disease. And an increased level of 2,3-DPG was also shown in nonhypoxic patients with liver diseases. These results suggest that the level of erythrocyte 2,3-DPG increases according to the severity of the liver disease, and compared to the level in hypoxic chronic obstructive pulmonary disease, the level of erythrocyte 2,3-DPG is higher in both hepatocellular carcinoma and liver cirrhosis.

2,3-Diphosphoglycerate↗
PubMed1986

Isocholic acid formation from 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid with human liver enzyme.

The formation of isocholic acid from 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid by human liver preparations was examined in vitro. Liver preparations were incubated with 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid at pH 7.4 in a phosphate buffer containing NADPH or NADH. The products formed were analyzed by gas chromatography and gas chromatography/mass spectrometry. Results showed that 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid was reduced mainly to isocholic acid and to cholic acid in a smaller amount in the presence of NADPH, while it was reduced only to cholic acid in the presence of NADH. The reducing enzyme participating in the formation of isocholic acid was localized largely in the cytosol and had more specificity to the unconjugated form as substrate than to the conjugated forms. 3-Keto bile acid analogues, 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids were not reduced to the corresponding iso-bile acids by the cytosol in the same conditions used in the isocholic acid formation and the activity of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was not inhibited by the addition of 3-keto-5 beta-cholanoic acid or 7 alpha-hydroxy-3-keto-5 beta-cholanoic acid to the reaction mixture. Furthermore, on column chromatography of Affi-Gel Blue, the peak of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was clearly distinguished from that of the enzyme catalyzing the reduction of 3-keto-5 beta-cholanoic acid to isolithocholic acid and that of alcohol dehydrogenase. These results indicate that this enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid is different from the enzyme(s) catalyzing the reduction 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids to the corresponding iso-bile acids and from alcohol dehydrogenase, and has a stereospecific character for 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid.

Bile Acids and Salts↗
PubMed1986

Microheterogeneity of Kasahara isozyme.

The microheterogeneity of Kasahara isozyme was investigated by affinity electrophoresis with Con A as the affinity ligand in combination with polyacrylamide gradient gel electrophoresis. On two-dimensional Con A-containing agarose gel electrophoresis, the Kasahara isozyme was separated into three molecular species. Kasahara isozyme electrophoresed as two distinct bands with enzyme activity on polyacrylamide gradient gel, but liver, intestinal or placental alkaline phosphatase showed only one distinct spot or band on both electrophoreses. One of the three molecular species of Kasahara isozyme separated by Con A-containing agarose gel electrophoresis was extracted from the gel and applied to the polyacrylamide gradient gel electrophoresis again, resulting in the same electrophoretic pattern as that of the original Kasahara isozyme. These findings indicated that the Kasahara isozyme consists of at least four molecular species. The same analysis was conducted with alkaline phosphatase of the HuH-6 cl-5 cell line, which has been reported to release an alkaline phosphatase closely resembling the Kasahara isozyme, and the results were compared with those obtained with the Kasahara isozyme.

Alkaline Phosphatase↗