Biomedical subjects

K Kobashi

Publications and source records attributed to K Kobashi.

At least 55 records · Page 3Linked to original sources

PubMed1995

Regulation of arylsulfate sulfotransferase from a human intestinal bacterium by nucleotides and magnesium ion.

Arylsulfate sulfotransferase (ASST) from a human intestinal bacterium stoichiometrically catalyzed the transfer of a sulfate group from phenylsulfate esters to phenolic compounds. Pentachlorophenol, one of the selective inhibitors of phenol sulfoconjugation in mammalian tissues, inhibited both phenol and tyramine sulfation by ASST. Nucleotide triphosphates such as ATP, GTP, UTP and CTP, and pyrophosphate inhibited the ASST activity, whereas Mg2+ and Mn2+ activated the enzyme and prevented its inhibition by ATP and pyrophosphate. Equimolar binding of [alpha-] and [gamma-32P]ATP to the enzyme showed that the enzyme protein was not phosphorylated, but bound ATP. These results suggest that nucleotide triphosphates and divalent cations are important modulators in the control of ASST activity.

Adenosine Triphosphate↗

PubMed1994

Enzymic O-sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A-44.

The enzymic O-sulfation of Tyr residues in a recombinant hirudin variant-1 (rHV-1) and its analog in which Glu61 and Glu62 were replaced by Tyr, [E61Y, E62Y]rHV-1, was carried out by use of sulfotransferase isolated from an anaerobic bacterium from the human intestine, Eubacterium A-44. Although rHV-1 was not sulfated by this enzyme, the sulfation of [E61Y, E62Y]rHV-1 was observed, and three kinds of sulfated analog, whose C-terminal six amino acid residues were -PYY(SO3H)YLQ, -PYYY(SO3H)LQ, and -PYY(SO3H)Y(SO3H)LQ, were obtained. Among the sulfated hirudin analogs tested here, the Tyr62 and Tyr63 bisulfated [E61Y, E62Y]rHV-1 showed the strongest thrombin inhibition with the inhibition constant (Ki) of 0.0430 pM, followed by the Tyr63 monosulfated analog (Ki = 0.0593 pM) and the Tyr62 monosulfated one (Ki = 0.158 pM). The Tyr63 monosulfated analog and Tyr62 and Tyr63 bisulfated one were more potent inhibitors of thrombin than unsulfated rHV-1. The increase in affinity caused by sulfation was predominantly due to an increase in the association-rate constant.

Amino Acid Sequence↗

PubMed1994

Improved purification of arylsulfate sulfotransferase from human intestinal bacterium by using polyclonal antibody.

Arylsulfate sulfotransferase (ASST) from a human intestinal bacterium stoichiometrically catalyzed the transfer of the sulfate group of phenylsulfate esters to phenolic compounds. Polyclonal antibodies against ASST were obtained from rabbit sera. These antisera did not inhibit ASST activity. ASST was recognized by the IgG fraction of the antisera, but rat liver phenol sulfotransferase did not show cross-reactivity to ASST on Western blot (immunoblot) analysis. The ASST was purified by an anti-ASST immobilized affinity column chromatography to homogeneity on SDS-PAGE. The NH2-terminal amino acid and partial sequence of the purified enzyme were serine and SVKYSFEDHIINRQYEAEQAMLAKF, respectively. We corrected the previous result that the NH2-terminal of ASST was arginine.

Amino Acid Sequence↗

PubMed1994

Intestinal bacterial hydrolysis is indispensable to absorption of 18 beta-glycyrrhetic acid after oral administration of glycyrrhizin in rats.

Gnotobiote rats were prepared by infecting germ-free rats with Eubacterium sp. strain GLH, a human intestinal bacterium capable of hydrolysing glycyrrhizin to 18 beta-glycyrrhetic acid. Their faeces and caecal contents showed glycyrrhizin-hydrolysing activities (31.7 and 31.3 pmol min-1 (mg protein)-1, respectively) similar to those (81.0 and 39.9 pmol min-1 (mg protein)-1, respectively) of conventional rats, although there was no detectable activity in germ-free rats. When glycyrrhizin (100 mg kg-1) was orally administered to conventional, germ-free and gnotobiote rats, no glycyrrhizin could be detected in plasma 4 or 17 h after the administration, using EIA and HPLC assays. Plasma 18 beta-glycyrrhetic acid was not detected 4 or 17 h after the administration of glycyrrhizin to germ-free rats nor could this compound be detected in caecal contents or in the faeces. However, 18 beta-glycyrrhetic acid (0.6-2.6 nmol mL-1) was detected in plasma of the conventional and the gnotobiote rats 4 and 17 h after the administration, and the caecal contents after 4 h and the cumulative faeces up to 17 h of the conventional and the gnotobiote rats contained considerable amounts of 18 beta-glycyrrhetic acid. These findings indicate that orally administered glycyrrhizin is poorly absorbed from the gut, but is hydrolysed to 18 beta-glycyrrhetic acid by intestinal bacteria such as E. sp. strain GLH, and the resulting 18 beta-glycyrrhetic acid is absorbed.

Administration, Oral↗

PubMed1994

Isolation of a human intestinal anaerobe, Bifidobacterium sp. strain SEN, capable of hydrolyzing sennosides to sennidins.

A strictly anaerobic bacterium capable of metabolizing sennosides was isolated from human feces and identified as Bifidobacterium sp., named strain SEN. The bacterium hydrolyzed sennosides A and B to sennidins A and B via sennidin A and B 8-monoglucosides, respectively. Among nine species of Bifidobacterium having beta-glucosidase activity, only Bifidobacterium dentium and B. adolescentis metabolized sennoside B to sennidin B, suggesting that the sennoside-metabolizing bacteria produce a novel type of beta-glucosidase capable of hydrolyzing sennosides to sennidins.

Anthracenes↗

PubMed1994

Enzymatic sulfation of glycosides and their corresponding aglycones by arylsulfate sulfotransferase from a human intestinal bacterium.

A novel type of arylsulfate sulfotransferase (ASST) from a predominant human intestinal bacterium catalyzes the stoichiometric transfer of a sulfate group from phenolic sulfate esters to phenols. We clarified that polyphenols were better substrates of this enzyme than the corresponding glycosides. Additionally, a coumarin derivative, esculetin, was sulfated by ASST at the 6-position to give 6-monosulfate. Therefore, ASST is more useful for the preparation of sulfated polyphenols at their specific hydroxyl groups and would play an important role in the metabolism of phenolic compounds in vegetable food and traditional medicines.

Arylsulfotransferase↗

PubMed1994

Sulfation of parabens and tyrosylpeptides by bacterial arylsulfate sulfotransferases.

Arylsulfate sulfotransferase purified from Eubacterium A-44 has higher specific activity than the enzymes from Klebsiella K-36 and Haemophilus K-12. Propylparaben and butylparaben were good substrates among several parabens. The antibacterial activity of parabens was reduced by the sulfation of the phenolic hydroxy group. Tyrosine-containing peptides, kyotorphin, enkephalin and cholecystokinin non-sulfate, were effective as acceptor substrates by A-44, K-36 and K-12 sulfotransferases.

Animals↗

PubMed1994

Inhibition of Helicobacter pylori urease activity by hydroxamic acid derivatives.

Helicobacter pylori (HP) produces strong urease [EC 3.5.1.5], which is considered to play a role in the pathogenesis of gastritis and peptic ulcers. Inhibitions against this enzyme have been studied with hydroxamic acid (HXA) derivatives of aliphatic or aromatic carboxylic acids, amino acids and dipeptides. A number of HXAs potently inhibited the urease (I50 values were near the order of 10(-6)M), and H-Ile-Gly-NHOH (I50 = 0.20 x 10(-6)M) was the most potent inhibitor among the derivatives. HP urease was inhibited more potently, in general, than Jack bean (JB) urease by HXAs, and a correlation between the chemical structures of HXA derivatives and their inhibitory effects on HP urease was observed, in comparison with JB urease.

Amino Acids↗

PubMed1994

Enzymic studies on the animal and intestinal bacterial metabolism of geniposide.

Geniposide, a main iridoid glucoside of Gardenia fruit, is transformed to genipin, a genuine choleretic, in vivo in rats (Aburada et al., J. Pharmacobio-Dyn., 1, 81 (1978)). As geniposide was not hydrolyzed to any metabolite by rat liver homogenate, which has beta-D-glucosidase and esterase activities, beta-D-glucosidases in intestinal bacteria seem to be required for an exhibition of its choleretic action. The crude extract of Eubacterium sp. A-44, a human intestinal anaerobe, hydrolyzed geniposide, but that of Ruminococcus sp. PO1-3, another human anaerobe, did not, though both extracts had beta-D-glucosidase activities for p-nitrophenyl beta-D-glucopyranoside. Only one of three beta-D-glucosidases from E. sp. A-44 and none of two from R. sp. PO1-3 hydrolyzed geniposide to genipin. However, carboxylesterases from E. sp. A-44 and pig liver were unable to hydrolyze geniposide to geniposidic acid, but hydrolyzed genipin to an aglycone of geniposidic acid, indicating that geniposide is first hydrolyzed to genipin by beta-D-glucosidases and subsequently to the aglycone of geniposidic acid by esterases. Thus, when geniposide is orally administered, genipin seems to be effectively produced in the intestine and then absorbed to act as a genuine choleretic.

Animals↗

PubMed1994

Characterization of beta-glucosidase and beta-glucuronidase of alkalotolerant intestinal bacteria.

The number of alkalotolerant intestinal bacteria was 1% of the total flora in humans and 0.8% of those in rats. The beta-glucosidase and beta-glucuronidase activity of these intestinal bacteria was induced by elevating the pH of the medium, but the growth was not changed. The enzyme activity in a medium of pH 7 was 5- to 10-fold higher than that in a medium of pH 6. Isolated bacteria from human and rat feces were cultured in a pH 5 general anaerobic medium (GAM) broth to reach a stationary phase, then the pH of the media was changed from 5 to 8. Both beta-glucosidase and beta-glucuronidase were increased 9.2-12.1-fold. The activity of these enzymes was also increased 2-16-fold by adding substrates (p-nitrophenyl-beta-D-glucuropyranoside or p-nitrophenyl-beta-D-glucuronide). beta-Glucuronidase(s) was inhibited by saccharic acid 1,4-lactone or D-glucuronic acid. However, when lactulose was added to the medium, and then intestinal microflora were inoculated in the medium, the productivity of these enzymes dramatically decreased. We thus contend that the induction of the beta-glucosidase and beta-glucuronidase of intestinal bacteria by a high pH can cause colorectal cancer.

Adult↗

PubMed1994

Silymarin and its components are inhibitors of beta-glucuronidase.

Silymarin, a commercial crude drug used as a hepatoprotective, was found to inhibit 53% of beta-glucuronidase activity at a final concentration of 0.8 mg/ml. Of three compounds A, silybin and C, which were isolated from silymarin, A and silybin potently inhibited the enzyme activity, followed by C. beta-Glucuronidases of intestinal bacteria, HGU-1 and HGU-2, and E. coli HB101 were noncompetitively inhibited by silybin. beta-Glucuronidase of the feces of a healthy human and of a human with colon cancer were also inhibited by silybin, silymarin and saccharic acid 1,4-lactone at 0.03-0.15 mg/ml. Silymarin and silybin protected the increase in enzyme activity in the serum of the rats treated with CCl4.

Animals↗

PubMed1994

Kinetic studies on a sulfotransferase from Klebsiella K-36, a rat intestinal bacterium.

Sulfotransferase purified from Klebsiella K-36, a rat intestinal bacterium, stoichiometrically catalyzed the transfer of a sulfate group of phenylsulfate esters to phenolic compounds. One of the reaction products, p-nitrophenol (PNP), non-competitively inhibited the enzyme as to p-nitrophenylsulfate (PNS), a donor substrate, but competitively inhibited the enzyme as to an acceptor substrate, alpha-naphthol. The other reaction product, alpha-naphthol-O-sulfate, non-competitively inhibited the enzyme with regard to both these substrates. These kinetic data suggest that the sulfotransferase reaction proceeds according to an ordered bi bi reaction mechanism. The natural phenolic substances, gallic acid, quercetin, tannic acid, and serotonin were good substrates of K-36 sulfotransferase.

Animals↗

PubMed1994

Purification and characterization of beta-fructofuranosidase from Bifidobacterium infantis.

beta-Fructofuranosidase activities of eight strains of Bifidobacteria, intestinal bacteria, were assayed and Bifidobacterium infantis was selected for purification of the enzyme. beta-Fructofuranosidase activity was recovered in the supernatant fraction after disruption of B. infantis cells with sonication and was purified to homogeneity by ammonium sulfate fractionation, and DEAE-cellulose, butyl-Toyopearl and Sephacryl S-300 column chromatographies. The enzyme (molecular weight (M.W.) 232000) was composed of three identical subunits (M.W. 75000) whose NH2-terminal amino acids were threonine. The enzyme was stable at pH 6-8, having the optimum activity at pH 6.0-6.2. The enzyme activity was stable under 40 degrees C and the optimal temperature was 55 degrees C. This enzyme catalyzed the hydrolysis of sucrose, 1-kestose, nystose, inulin and raffinose at the relative velocities of 100, 297, 365, 140 and 3.8, respectively, but did not catalyze the hydrolysis of maltose or cellobiose. These results indicated that this fructooligosaccharide hydrolyzing enzyme is a novel type of beta-fructofuranosidase.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of iron-containing urethanase from Bacillus licheniformis.

Urethane is potentially carcinogenic and teratogenic to human and has been reported to be a contaminant of various kinds of alcoholic beverages. Enzymatic removal of urethane is one possible approach to remove this cancer-causing chemical from alcoholic beverages. Among Bacillus licheniformis strains, IFO 12107 showed the highest urethane hydrolyzing activity when cultivated in a urethane-containing white medium. The enzyme was purified about 300-fold by means of several chromatographic steps to homogeneity. The enzyme activity was strongly inhibited by batho- and o-phenanthroline. The complete loss of enzyme activity following treatment with bathophenanthroline was fully restored by the addition of Fe3+ at a ratio of 4 iron atoms to 1 mol apoenzyme. This result was obtained by the incorporation of 59Fe3+ into apourethanase. ESR spectroscopy showed that the enzyme contained a typical high-spin Fe3+. The urethanase hydrolyzed carbamyl ester derivatives more rapidly than amide derivatives. The molecular weight of the native enzyme was about 160 kDa (gel-filtration), and that of the subunit was 42 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE). This shows the enzyme to be a homotetramer. The pI and Km values were 5.5 and 0.17 mM, respectively. The enzyme was considerably resistant to high concentrations of ethanol, which is a great advantage for the industrial removal of urethane from alcoholic beverages.

Amidohydrolases↗

PubMed1993

Cleavages of the O- and C-glucosyl bonds of anthrone and 10,10'-bianthrone derivatives by human intestinal bacteria.

A strictly anaerobic bacterium, Bifidobacterium sp. SEN, capable of hydrolyzing the O-glucosyl of sennosides was isolated from human feces. The bacterium stepwisely hydrolyzed sennoside B to sennidin B through sennidin-8-monoglucoside in PYF medium but not in GAM broth. Addition of D-glucose to PYF medium resulted in loss of the hydrolyzing activity in culture but addition of D-fructose did not affect the activity. Coculture of this bacterium with Peptostreptococcus intermedius led to rapid accumulation of rhein anthrone in the medium. Similarly, a bacterium, Eubacterium sp. BAR, capable of cleaving the C-glucosyl of barbaloin was isolated from human feces. This bacterium grew in PYF medium containing barbaloin and produced enzyme(s) that cleave(s) the C-glucosyl. The induction of the enzymes was completely inhibited in the presence of D-glucose. Nojirimycin inhibited the enzyme activity induced by barbaloin but it did not inhibit the bacterial growth in the presence of D-glucose.

Aloe↗

PubMed1993

L-gulono-gamma-lactone oxidase is the enzyme responsible for the production of methylguanidine in the rat liver.

A methylguanidine-synthesizing enzyme localized in rat liver microsomes produces methylguanidine via the intermediates creatone A and creatone B from the substrate, creatol, a substance produced from creatinine mainly by reaction with hydroxyl radicals. This enzyme has been identified as L-gulono-gamma-lactone oxidase (EC 1.1.3.8). However, no corresponding activity was found in extracts from human livers.

Amino Acid Sequence↗

PubMed1993

Purification of methylguanidine synthase from the rat kidney.

Methylguanidine (MG)-synthesizing enzyme was purified from rat kidney lysosomes and peroxisomes. The enzyme was a flavoprotein with a molecular weight of about 37,000 and oxidized creatol to produce MG. The present results suggest that the reaction mechanism of this enzyme is different from that of L-gulono-gamma-lactone oxidase (EC 1.1.3.8) isolated from rat liver microsomes.

Amino Acid Sequence↗

PubMed1993

[Statistical analysis of recurrent factors in superficial bladder cancer. Natural history of superficial bladder cancer].

Three hundred thirty-six patients with superficial bladder cancers underwent their initial surgery at the Department of Urology of Okayama University School of Medicine from 1961 to 1985. They have been followed-up until December 1988 (the median: 68 months). Among the 336 cases, excluding 9 cases which were treated initially with total cystectomy or incomplete resection, the remaining 327 cases were reviewed in order to study the pattern of their local recurrences in the bladder. Major factors affecting recurrences of superficial bladder cancer were evaluated by the multivariate analysis using Cox's proportional hazard model. The examined factors included age on admission, sex, tumor's number, size, stage, histological grade and shape, operative method, intravesical chemotherapy, systemic chemotherapy, radiotherapy, frequency of recurrences and the date of operation. During the follow-up period, 179 cases out of the 327 cases did not show any recurrences. The remaining 148 cases recurred from once to 11 times, therefore, a total number of recurrences was counted to be 406. In the 406 recurrences, 338 recurrences were treated by TUR, SVR, or SR. The remaining 68 recurrences were treated with total cystectomy or untreated. Fifty-seven cases out of the 148 cases demonstrated recurrences more than 3 times. Only 4 cases were free from recurrences more than 24 months following the surgery for their last recurrences. The analysis in the 327 primary cases showed that the number of tumors was the most important factor. On the other hand the analysis in 338 recurrences showed that the second most important factor was the frequency of recurrences, following the number of tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Transitional Cell↗