Biomedical subjects

K Kobashi

Publications and source records attributed to K Kobashi.

At least 91 records · Page 5Linked to original sources

PubMed1991

Urethanase of Bacillus licheniformis sp. isolated from mouse gastrointestine.

Trace levels of urethane, a cancer causing chemical, were detected in many kinds of wine, sherry, whisky, brandy and sake. Urethane formation from urea and ethanol in sake can be prevented by the treatment of acid urease, which is produced by Lactobacillus fermentum, but urethane, once formed, is very difficult to decompose. In order to keep the safety of alcoholic beverages, enzymatic removal of urethane has become an urgent problem. We found that Bacillus licheniformis sp., isolated from mouse gastrointestine, decomposed urethane to ethanol and ammonia. The enzyme showed higher urethanase activity at an acidic condition than at a neutral condition, and was resistant against ethyl alcohol of high concentrations. However, the enzyme had a low affinity to urethane for the industrial removal of the compound from alcoholic beverages.

Amidohydrolases↗

PubMed1991

Metabolism of aloesin and related compounds by human intestinal bacteria: a bacterial cleavage of the C-glucosyl bond and the subsequent reduction of the acetonyl side chain.

By anaerobic incubation with a bacterial mixture from human feces, aloesin (aloeresin B; 1) was converted to 2-acetonyl-7-hydroxy-5-methylchromone (aloesone; 3) and dl-7-hydroxy-2-(2'-hydroxypropyl)-5-methylchromone (aloesol; 4a + 4b) through a cleavage of the C-glucosyl bond, followed by reduction of the acetonyl side chain. An analogous compound, aloeresin A (2), was converted to p-coumaric acid and aloesin (1), the latter being subsequently transformed to aloesone (3) and dl-aloesol (4a + 4b). On the other hand, 7-O-methylated derivatives (7, 5a and 5b) of aloesin and of 8-C-glucosylaloesol were not cleaved to the corresponding aglycones, suggesting the importance of a free hydroxy group adjacent to the C-glucosyl group in the molecule for the bacterial cleavage of aloesin derivatives. This is the first report on the cleavage of the C-glycosyl bond of chromone C-glucosides by intestinal bacteria.

Acetone↗

PubMed1991

Induction and inhibition of novel sulfotransferase in a human intestinal bacterium, Eubacterium sp. A-44.

Induction and inhibition of a novel sulfotransferase produced by Eubacterium sp. A-44 isolated from human feces have been studied. Production of the enzyme was induced by phenylsulfate esters, sulfate donor substrates, but not by phenols, sulfate acceptor substrates, or inorganic sulfate. p-Nitrophenylsulfate (PNS), a good donor substrate, stimulated enzyme production more than 10-fold. Sulfotransferase production was strongly inhibited by phenylphosphate esters. Enzyme activity was competitively inhibited by phenylphosphate esters, but not by inorganic phosphate. High yields of sulfotransferase from sonicated cells were obtained when the bacteria were grown in a media containing 0.6% (w/v) or less of glycine.

Enzyme Induction↗

PubMed1991

Barbaloin stimulates growth of Eubacterium sp. strain BAR, a barbaloin-metabolizing bacterium from human feces.

Eubacterium sp. strain BAR, isolated from human feces, transformed barbaloin to aloe-emodin anthrone in a basal medium lacking carbohydrate. Barbaloin remarkably stimulated the growth of strain BAR in the basal medium, the stimulative extent of the growth depending on the amount of barbaloin added. The addition of D-glucose, D-galactose, maltose, cellobiose, sucrose or D-amygdalin to the basal medium containing barbaloin caused a decrease of the growth stimulated by barbaloin to the growth level with each sugar, resulting in a complete inhibition of the barbaloin transformation. On the other hand, the addition of D-fructose, which itself stimulated the growth of strain BAR, further increased the growth in the presence of barbaloin and little inhibited barbaloin transformation. Nojirimycin bisulfite, a specific inhibitor of glucosidases, potently inhibited the growth with barbaloin, but did not affect the growth with glucose or cellobiose. Also, nojirimycin bisulfite completely inhibited the transformation of barbaloin to aloe-emodin anthrone. These results indicate that a unique enzyme capable of cleaving the C-glycosyl bond is induced in strain BAR by barbaloin and, consequently, strain BAR grows by utilizing as a nutrient the carbohydrate liberated from barbaloin. It is further suggested that the barbaloin-cleaving enzyme is inhibited by nojirimycin bisulfite and that the induction of the enzyme is repressed with D-glucose and D-galactose.

Anthracenes↗

PubMed1991

Evaluation of effects of novel urease inhibitor, N-(pivaloyl)glycinohydroxamic acid on the formation of an infection bladder stone using a newly designed urolithiasis model in rats.

By using our new infection stone model of a rat, we evaluated the effect of a novel urease inhibitor, N-(pivaloyl)glycinohydroxamic acid (P-GHA), on the formation of an infection bladder stone. The oral dosing of P-GHA significantly inhibited the elevation of the urinary ammonia level of rats having the urinary tract infection with Proteus mirabilis. A short term regimen (7 d, 730 +/- 38 mg/kg) with P-GHA significantly inhibited the development of the infection bladder stone. Furthermore, a long term combination regimen (11 d) of P-GHA and aminobenzylpenicillin markedly inhibited the development of the infection bladder stone, and also caused a very slight renal impairment to the rats tested in contrast with the method of Vermeulen et al. Our infection stone model in rats, therefore, seems to be useful for the evaluation of therapeutic agents in long term examinations.

Ampicillin↗

PubMed1991

Effects of a novel urease inhibitor, N-(diaminophosphinyl)isopentenoylamide on the infection stone in rats.

We evaluated the effect of a novel potent urease inhibitor, N-(diaminophosphinyl)isopentenoylamide (IPA), on the development of an infection bladder stone using our urolithiasis model in rats. IPA was excreted into urine after oral administration to rats, and the cumulative urinary recovery rate of unchanged IPA reached about 29.6% within 24 h (50 mg/kg). The oral administration of IPA (6.25 mg/kg, b.i.d., 5 d) significantly inhibited the development of the infection bladder stone. The present result suggests that IPA is a very promising compound in the prevention of formation and recurrence of an infection stone owing to a high efficacy and a low toxicity of IPA in animals.

Animals↗

PubMed1991

High paraoxon-hydrolyzing activity in organophosphorus insecticide-resistant mosquitoes.

We found a strong paraoxon-hydrolyzing activity (23.4 +/- 8.50 nmol/h/individual and 137 +/- 86.2 nmol/h/mg protein) in the crude extract from larvae of Culex tritaeniorhynchus Toyama 89, which is markedly resistant to organophosphorous insecticides. The activity was higher than those from Cx. tritaeniorhynchus re-e-ae (0.175 +/- 0.0336 and 1.83 +/- 0.651), Anopheles omorii (0.112 +/- 0.0301 and 1.86 +/- 0.746) and An. stephensi (0.0651 +/- 0.0713 and 0.789 +/- 0.910), which are susceptible to organophosphorous insecticides. These facts suggest that the high paraoxon-hydrolyzing activity plays a role in the development of organophosphorous resistance in Cx. tritaeniorhynchus. The enzyme preparation obtained from Toyama 89 showed higher activity in the alkaline pH range and its Km values to paraoxon were 0.67 mM in larvae and 0.50 mM in adults. A calcium ion was strictly required for the hydrolysis of paraoxon. Fenitroxon was also hydrolyzed, in addition to paraoxon. However, it did not degradate parathion and fenitrothion at all. Dichlorvos and phenyl acetate competitively inhibited the enzyme. The phenyl acetate-hydrolyzing activity in the preparation of Toyama 89 was significantly (p less than 0.01) lower than those in susceptible strains, and was irreversibly inhibited by paraoxon. Therefore, the paraoxon-hydrolyzing activity belongs to the class of organophosphate compound hydrolases; it must be thus distinguished from bacterial phosphotriesterase.

Animals↗

PubMed1991

[Phase II study of 5'-DFUR treatment of the bladder and prostatic cancer].

Phase 2 study of 5'-DFUR in bladder and prostatic cancer was conducted at 15 collaborative institutions including Okayama University. 5'-DFUR was orally administered to patients at a daily dose of 800-1200 mg for more than 4 weeks. Forty-one patients with bladder cancer and 12 patients with prostatic cancer were evaluated. The response rate for bladder cancer was 31.7% (CR, 1 case: PR, 12 cases), against no response with prostatic cancer. Moreover, the concentration of 5-FU in bladder tumors was confirmed to be high. Adverse reactions such as diarrhea, anorexia, and nausea were observed. Thus, 5'-DFUR seems to be useful for the treatment of bladder cancer.

Administration, Oral↗

PubMed1991

Kinetic studies on a novel sulfotransferase from Eubacterium A-44, a human intestinal bacterium.

A novel sulfotransferase purified from a human intestinal bacterium stoichiometrically catalyzed the transfer of a sulfate group of phenylsulfate esters to phenolic compounds. Vmax values of the enzyme reaction were measured with various concentrations of a sulfate donor substrate, p-nitrophenylsulfate, and of a sulfate acceptor substrate, tyramine. Double reciprocal plots of the acceptor concentration and Vmax showed a linear correlation. One of the reaction products, tyramine O-sulfate, competitively inhibited the enzyme as to a donor substrate, p-nitrophenylsulfate (PNS), but the other reaction product, p-nitrophenol (PNP), noncompetitively inhibited it as to PNS. These kinetic data suggest that the sulfate transfer reaction proceeds according to a ping pong bi bi mechanism. The enzyme was activated by Mg2+ and inhibited by EDTA, which suggests that it is a metalloenzyme.

Binding, Competitive↗

PubMed1990

Characterization of NADP+: 3 beta-hydroxysteroid dehydrogenase from microsomes of rat liver.

A NADP(+)-dependent 3 beta-hydroxysteroid dehydrogenase activity was localized in the microsomal fraction of rat liver. This enzyme was solubilized and separated completely from 3 alpha-hydroxysteroid dehydrogenase by Matrex red A column chromatography. Partially purified 3 beta-hydroxysteroid dehydrogenase catalyzed the oxidation and reduction between the 3 beta-hydroxyl and 3-ketonic group of steroids or bile acids having no double bond in the A/B ring, but was inactive toward 3 alpha-hydroxyl group. The enzyme required NADP+ for oxidation and NADPH for reduction. The activity was inhibited by p-chloromercuribenzoic acid or p-chloromercuribenzenesulfonic acid at the concentration of 10(-4) M. The molecular weight of the enzyme was estimated to be about 43,000 by Sephadex G-200 column chromatography. From these results, it is concluded that the enzyme is a new type of microsomal NADP+:3 beta-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Acetylcholinesterase activities and monoamine metabolite levels in the cerebrospinal fluid of patients with Alzheimer's disease.

We measured cholinesterase (ChE) activity and monoamine metabolite levels in the cerebrospinal fluid (CSF) of 22 patients with early-onset Alzheimer type dementia (Alzheimer's disease; AD) and of 32 controls. Acetylcholinesterase (AChE) activity, 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) levels were significantly lower in AD patients than in controls. However, there was an overlap in values of each CSF parameter. The measurement of various CSF parameters rather than one alone was more useful as a diagnostic aid. CSF ChE activities correlated with scores on the GBS rating scale, Hasegawa dementia scale, and Wechsler Adult Intelligence Scale, but the monoamine metabolite levels did not. Although cholinergic and monoaminergic deficits may coexist in AD patients, cholinergic deficits tend to be more often associated with cognitive decline than the monoaminergic deficits.

Acetylcholinesterase↗

PubMed1990

Metabolism of glycyrrhetic acid by rat liver microsomes-II. 22 alpha- and 24-hydroxylation.

18 beta-Glycyrrhetic acid (GA, an aglycone of glycyrrhizin) is converted to 3-oxo-18 beta-glycyrrhetic acid (3-oxoGA) in the presence of NADP+ by rat liver homogenates, but GA was converted in the presence of NADPH to two other metabolites showing lower Rf values on thin-layer chromatography (TLC) than those of GA and 3-oxoGA by postmitochondrial supernatant of rat liver. The GA-metabolizing activity in the presence of NADPH was localized in microsomes, similar to localization of GA-oxidizing activity to 3-oxoGA. The GA-metabolizing activity required NADPH as a cofactor and O2 for full activity and was inhibited with CO, suggesting the hydroxylation reaction of GA by cytochrome P450. Two metabolites (I and II, lower and higher Rf values on TLC, respectively) were purified on preparative TLC. Mass spectral (MS) analyses of II and methyl ester of acetylated I indicated the formation of monohydroxylated metabolites. On the basis of 3H- and 13C-NMR assignments I and II were identified to be 22 alpha- and 24-hydroxy-18 beta-glycyrrhetic acids, respectively. 3-OxoGA and 3-epi-18 beta-glycyrrhetic acid (3-epiGA) seem to be also hydroxylated at C-22 and C-24. A metabolite of 3-oxoGA showing a lower Rf value was also identified as 22 alpha-hydroxy-3-oxo-18 beta-glycyrrhetic acid by MS and 3H- and 13C-NMR spectral analyses. In 22 alpha-hydroxylation the best substrate was 3-oxoGA, followed by GA and 3-epiGA. On the other hand, for 24-hydroxylation the best substrate was GA, then 3-oxoGA, and 3-epiGA in order. However, 18 alpha-glycyrrhetic acid (18 alpha-GA) was a poor substrate for both 22 alpha- and 24-hydroxylation.

Animals↗

PubMed1990

Metabolism of glycyrrhetic acid by rat liver microsomes: glycyrrhetinate dehydrogenase.

Glycyrrhetic acid, derived from a main component of liquorice, was converted to 3-ketoglycyrrhetic acid reversibly by rat liver homogenates in the presence of NADPH or NADP+. Glycyrrhetic acid-oxidizing and 3-ketoglycyrrhetic acid-reducing activities were localized in microsomes among the subcellular fractions of rat liver. Glycyrrhetic acid-oxidizing activity and 3-ketoglycyrrhetic acid-reducing activities showed pH optima at 6.3 and 8.5, respectively, and required NADP+ or NAD+ and NADPH or NADH, respectively, indicating that these activities were due to glycyrrhetinate dehydrogenase. The dehydrogenase was not solubilized from the membranes by the treatment with 1 M NaCl or sonication, indicating that the enzyme is a membrane component. The dehydrogenase was solubilized with detergents such as Emalgen 913, Triton X-100 and sodium cholate, and then separated from 3 beta-hydroxysteroid dehydrogenase (5 beta-androstan-3 beta-ol-17-one-oxidizing activity) by butyl-Toyopearl 650 M column chromatography. Partially purified enzyme catalyzed the reversible reaction between glycyrrhetic acid and 3-ketoglycyrrhetic acid, but was inactive toward 3-epiglycyrrhetic acid and other steroids having the 3 beta-hydroxyl group. The enzyme required NADP+ and NADPH for the highest activities of oxidation and reduction, respectively, and NAD+ and NADH for considerable activities, similar to the results with microsomes. From these results the enzyme is defined as glycyrrhetinate dehydrogenase, being quite different from 3 beta-hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestine, which is active for both glycyrrhetic acid and steroids having the 3 beta-hydroxyl group.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Urethane-hydrolyzing enzyme from Citrobacter sp.

Urethane, a cancer-causing chemical, was reported to contaminate alcoholic beverages such as whisky, liquor, wine and sake. Enzymatic removal of urethane would be a possible approach to remove this potentially hazardous chemical from alcoholic beverages. We found that Citrobacter sp. isolated from mouse feces stoichiometrically decomposed urethane to ethanol and ammonia. We named this enzyme "urethanase." Partially purified urethanase could hydrolyze several carbamates and some amides. However, urea, N-alkyl ureas and ethyl esters of organic acids were not hydrolyzed at all. These results suggest that urethanase belongs to the category of amidase. The enzyme was inactive in high concentrations of alcohol and at acidic pH and was practically ineffective for the elimination of urethane from alcoholic beverages.

Animals↗

PubMed1990

Pharmacological activities of synthetic human cholecystokinin-33 of which tyrosine was sulfated by arylsulfotransferase.

The pharmacological activities of synthetic human CCK-33, in which a tyrosine molecule was sulfated by arylsulfotransferase, were investigated in the rat and the guinea-pig. The activities were compared with those of non-sulfated CCK-33 (CCK-33NS), CCK-8 and CCK-4. CCK-33 was about 100 fold more potent than non-sulfated CCK-33(CCK-33NS) but was about 20 fold less potent than CCK-8 in the contraction of the isolated gallbladder of the guinea-pig. In rat pancreatic secretion, intravenous CCK-33 and CCK-8 showed almost the same activity. The potency of each was about 1000 fold more than the individual potency of CCK-33NS, non-sulfated CCK-8 (CCK-8NS) and CCK4. There were no significant differences in gastric acid stimulatory activities among CCK-33, CCK-8, CCK-4, but the activities of CCK-33NS and CCK-8NS were less than those of CCK-33 and CCK-8, respectively. CCK-33 and CCK-8 produced a reduction in the intake of powder chow in doses of 10(-8) and 3 x 10(-8) mol/kg i.p., but CCK-33NS, CCK-8NS and CCK-4 did not. In conclusion, the activities of synthetic human CCK-33 are almost the same as those of CCK-8 on exocrine pancreatic secretion, gastric acid secretion and food intake, but less than CCK-8 on isolated gallbladder contraction.

Animals↗

PubMed1990

Enzymatic sulfation of quercetin by arylsulfotransferase from a human intestinal bacterium.

A novel type of arylsulfotransferase was partially purified from human intestinal bacteria and its enzymatic properties were examined. Polyphenols such as chalcone, xanthone and flavonoid were found to be sulfated by the bacterial arylsulfotransferase though the sulfation activity varied depending upon the positions of the hydroxyl groups. Quercetin, as an example of a flavonol, was rapidly sulfated when p-nitrophenyl sulfate (PNS) was taken as a donor substrate. At a ten-fold molar excess of PNS over quercetin, two products, the 3,3'-disulfate and 3,3',7-trisulfate derivatives, were formed, but the 4'- and 5-hydroxyl groups were not sulfated. In the case of equimolar or two-fold molar excess of PNS to quercetin, only the 3,3'-disulfate was produced and no monosulfate was formed. The enzymatic procedure is useful as a specific and convenient method for the preparation of polyphenol sulfate esters.

Arylsulfotransferase↗

PubMed1990

Pharmacological activity of angiotensin-II modified by tyrosine sulfation.

An angiotensin-II analogue with a sulfated tyrosine residue was prepared by arylsulfotransferase treatment of synthetic human angiotensin-II. Its biological activities were studied in isolated smooth muscles, and its effect on blood pressure was determined. The sulfated angiotensin-II (AII-S) was about 15-30 fold less potent than angiotensin-II (AII) for ileum contraction and gallbladder contraction. The hypertensive potency of AII-S was about 30-fold less than that of AII.

Angiotensin II↗

PubMed1990

[Diffusion of ofloxacin (OFLX) into prostatic tissue in patients with benign prostatic hypertrophy].

Diffusion of ofloxacin (OFLX) into prostatic tissue was studied in 31 patients with benign prostatic hypertrophy. Concentrations of OFLX in the serum and prostatic tissue were measured at scheduled intervals after 200 mg OFLX oral administration. The mean OFLX level in prostatic tissue and tissue/serum ratio at 2 hours, 4 hours and 6 hours was 3.33 +/- 0.96 micrograms/g (1.25 +/- 0.28) in 10 patients, 2.21 +/- 0.55 micrograms/g (0.92 +/- 0.32) in 9 patients and 2.10 +/- 0.99 micrograms/g (1.01 +/- 0.23) in 12 patients, respectively. OFLX levels in prostatic tissue covered the minimum inhibitory concentration for several pathogenic bacteria detected from the infected prostatic fluid. Therefore, OFLX was thought to be a very useful drug for the treatment of bacterial prostatitis and postoperative infection of prostatic surgery.

Administration, Oral↗