Biomedical subjects

K Kobashi

Publications and source records attributed to K Kobashi.

At least 19 recordsLinked to original sources

PubMed1997

Anticarcinogenic action of apple pectin on fecal enzyme activities and mucosal or portal prostaglandin E2 levels in experimental rat colon carcinogenesis.

Pectin is a partially methoxylated polymer of galacturonic acid obtained from fruits. Among pectin, apple pectin exerts stronger bacteriostatical action on Staphylococcus aureus, Streptococcus faecalis, Pseudomonas aeruginosa and Escherichia coli in comparison with citrus pectin. In this study, we used water-soluble methoxylated pectin from apple. The diet, supplemented by 20% apple pectin, significantly decreased the number of tumors and the incidence of colon tumor. PGE2 level in distal colonic mucosa in 20% apple pectin fed rats were lower than those in basal diet fed rats. Fecal beta-glucuronidase activities in the apple pectin fed group, which has been considered a key enzyme for the final activation of Dimethylhydrazine metabolism to carcinogens in the colonic lumen, were signifieantly lower than those in control group at initiation stage of carcinogenesis. In the case the concentrations of beta-glueosidase and azoreductase were also decreased. The effect of apple pectin on the colon carcinogenesis may partially depend on PGE, concentration decrease in colonic mucosa and on the type of pectin, also related to fecal enzyme activities.

Animals↗

PubMed1997

Measurement of the expiratory ammonia concentration and its clinical significance.

Although gaseous ammonia (NH3) can freely enter cells through the plasma membrane where NH3 is cyto(neuro)toxic, NH3 and ionic ammonia (NH4+) contents have not been studied in biological materials. We developed a new method for measurement of expiratory NH3 concentration, which may reflect blood NH3 concentrations. The method is a sensor tube type-gas assay system. Expiratory NH3 concentration in patients with chronic liver diseases increased when their blood ammonia (NH4(+)+NH3) concentrations increased above 90 micrograms/dl (normal range; 12-66 micrograms/dl). However, cirrhotic patients, who had relatively higher expiratory NH3 concentration compared to blood NH3 concentrations (calculated from Henderson-Hasselbalch formula), were found to have subclinical encephalopathy. Measurement of expiratory NH3 concentration may be of clinical significance for the diagnosis of encephalopathy associated with hyperammonemia.

Ammonia↗

PubMed1996

Structure-activity studies on C-terminal hirudin peptides containing sulfated tyrosine residues.

To clarify the role of the negative charge of the C-terminal region of hirudin, we chemically synthesized the C-terminal peptide of hirudin variant-1 (HV-1), HV-1-(54-65), and its analogs, [E61Y,E62Y]HV-1-(54-65) and [E62Y]HV-1-(54-65), and then sulfated the Tyr residue(s) in these peptides by both enzymic and chemical methods. Enzymic O-sulfation of Tyr residues in the peptides by use of sulfotransferase isolated from Eubacterium A-44 allowed us to produce four kinds of the sulfated peptide, whose C-terminal sequences were -PEY(SO3H)YLQ, -PYY(SO3H)YLQ, -PYYY(SO3H)LQ and -PYY(SO3H)Y(SO3H)LQ. On the other hand, all Tyr residues in the peptides were successfully sulfated by chemical reaction with N,N'-dicyclohexylcarbodiimide in the presence of sulfuric acid. Based on the analysis of structure-activity relationships of these sulfated peptides for thrombin inhibition, the Tyr62 and Tyr63 bisulfated peptide GDFEEIPEY(SO3H)Y(SO3H)LQ was found to be the most potent inhibitor of thrombin among the products tested. No increase in potency was observed by further substitution of Glu61 with Tyr(SO3H). The inhibitory activity by substitution with Tyr(SO3H) at position 63 was greater than that obtained by the substitution at position 62.

Amino Acid Sequence↗

PubMed1996

Bioavailability study of glycyrrhetic acid after oral administration of glycyrrhizin in rats; relevance to the intestinal bacterial hydrolysis.

To clarify the metabolic fate of glycyrrhizin when orally ingested, we investigated the bioavailability of glycyrrhetic acid, the aglycone of glycyrrhizin, after intravenous or oral administration of glycyrrhetic acid (5.7 mg kg-1, equimolar to glycyrrhizin) or glycyrrhizin (10 mg kg-1) at a therapeutic dose in rat. Plasma concentration of glycyrrhetic acid rapidly decreased after its intravenous administration, with AUC of 9200 +/- 1050 ng h mL-1 and MRT of 1.1 +/- 0.2 h. The AUC and MRT values after oral administration were 10600 +/- 1090 ng h mL-1 and 9.3 +/- 0.6 h, respectively. After oral administration of glycyrrhizin, the parent compound was not detectable in plasma at any time, but glycyrrhetic acid was detected at a considerable concentration with AUC of 11700 +/- 1580 ng h mL-1 and MRT of 19.9 +/- 1.3 h, while glycyrrhetic acid was not detected in plasma of germ-free rats at 12 h after oral administration of glycyrrhizin. The AUC value of glycyrrhetic acid after oral administration of glycyrrhizin was comparable with those after intravenous and oral administration of glycyrrhetic acid, indicating a complete biotransformation of glycyrrhizin to glycyrrhetic acid by intestinal bacteria and a complete absorption of the resulting glycyrrhetic acid from intestine. Plasma glycyrrhizin rapidly decreased and disappeared in 2 h after intravenous administration. AUC and MRT values were 2410 +/- 125 micrograms min mL-1 and 29.8 +/- 0.5 min, respectively. Plasma concentration of glycyrrhetic acid showed two peaks a small peak at 30 min and a large peak at 11.4 h, after intravenous administration of glycyrrhizin, with an AUC of 15400 +/- 2620 ng h L-1 and an MRT of 18.8 +/- 1.0 h. The plasma concentration profile of the latter large peak was similar to that of glycyrrhetic acid after oral administration of glycyrrhizin, which slowly appeared and declined. The difference of MRT values (19.9 and 9.3 h) for plasma glycyrrhetic acid after oral administration of glycyrrhizin and glycyrrhetic acid suggests the slow conversion of glycyrrhizin into glycyrrhetic acid in the intestine.

Administration, Oral↗

PubMed1996

Purification and characterization of beta-glucosidase from Bacteroides JY-6, a human intestinal bacterium.

A beta-glucosidase (EC 3.2.1.21.) was purified 2500-fold from Bacteroides JY-6, an intestinal anaerobic bacterium of human. The specific activity of the homogeneously purified enzyme was 210 mumol/min/mg protein. The enzyme (M(r) 75kDa) was an monomer whose pI and optimal pH values were 4.6 and 5.5-6, respectively. The best substrates were p-nitrophenyl beta-D-glucopyranoside and natural beta-bound glucosides, such as prunin and poncirenin. Puerarin, which is a C-glycoside, was weakly effective. However, cellobiose, alpha-bound glycosides and rhamnoglucosides were not effective. The apparent Kms for prunin and p-nitrophenyl-beta-D-glucopyranoside were determined to be 0.08 and 0.19 mM, respectively. The enzyme was strongly inhibited by p-chloromercuriphenylsulfonic acid and reaction products such as p-nitrophenol and glucose.

Bacterial Proteins↗

PubMed1996

A purgative action of barbaloin is induced by Eubacterium sp. strain BAR, a human intestinal anaerobe, capable of transforming barbaloin to aloe-emodin anthrone.

Orally administered barbaloin (100 mg/kg) did not induce any diarrhea in male Wistar rats, in spite of severe diarrhea with sennoside B (40 mg/kg). Also, in gnotobiote rats mono-associated with Peptostreptococcus intermedius, a human intestinal anaerobe capable of reducing sennidins to rhein anthrone, barbaloin did not induce diarrhea; the faecal water content (71.9%) 8 h after the administration of barbaloin was not increased, compared with that (73.9%) just before the treatment. However, severe diarrhea was induced with barbaloin in gnotobiote rats mono-associated with Eubacterium sp. strain BAR, another human intestinal anaerobe capable of transforming barbaloin to aloe-emodin anthrone; the faecal water content was significantly increased to 85.5% 8 h after the administration, from 73.2% before the treatment. At this time, barbaloin was transformed to aloe-emodin anthrone in the feces from the gnotobiote rats mono-associated with the strain BAR, but not in feces from the conventional rats or the gnotobiote rats mono-associated with P. intermedius. These facts indicate that barbaloin is inactive as a laxative itself but is activated to aloe-emodin anthrone, a genuine purgative component, by Eubacterium sp. strain BAR.

Animals↗

PubMed1996

Kinetic studies of Helicobacter pylori urease inhibition by a novel proton pump inhibitor, rabeprazole.

Urease is an important virulence factor of pathogenicity of gastric Helicobacter pylori. The inhibition of H. pylori urease by the novel proton pump inhibitor, rabeprazole, was investigated kinetically. It was found to act as an irreversible noncompetitive inhibitor of the enzyme. The inhibitory potency of rabeprazole was dependent on the pH of reaction mixture and its Ki values were 0.14 microM (pH 5.0), 0.34 microM (pH 7.0) and 6.10 microM (pH 8.5). Progressive inactivation of urease by rabeprazole initially proceeded according to pseudo-first-order kinetics with respect to the remaining enzymatic activity at pH 7.0 and 37 degrees C, with a second-order rate constant of 0.0017 microM-1 s-1. When the inactivation half-life was plotted versus the reciprocal of the rabeprazole concentration, a straight line was obtained with a slope of -3.12. From an Arrhenius-plot of the temperature-dependence of the inactivation (over the range of 5-37 degrees C), an activation energy of 13.2 kcal/mol was calculated. Recovery of activity was incomplete for H. pylori urease inhibited by rabeprazole, suggesting that the rabeprazole-urease complex is very stable.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1996

A sennoside-hydrolyzing beta-glucosidase from Bifidobacterium sp. strain SEN is inducible.

Bifidobacterium sp. strain SEN was isolated and characterized by hydrolytic conversion of sennosides to sennidins (Akao et al., Appl. Environ. Microbiol., 60, 1041 (1994)). The sennoside-hydrolyzing capacity of the strain SEN was disappeared following the addition of glucose to the media in spite of good bacterial growth and potent activity hydrolyzing p-nitrophenyl beta-D-glucopyranoside (pNPG). In a fructose-containing medium, no such suppressing effect was shown. Following a 10 h incubation in 50 mM potassium phosphate buffer (pH 7.4), the sennoside-hydrolyzing activity of the bacterium increased, dose-dependently, with the addition of sennoside B. Inhibition of the substrate-induced increase in sennoside-hydrolyzing activity was observed following the addition of some antibiotics (chloramphenicol, streptomycin, and rifampicin). In particular, chloramphenicol completely inhibited the increase of sennoside-hydrolyzing activity while 38% pNPG-hydrolyzing activity remained. It is suggested that the strain SEN produces two different beta-glucosidases of which the sennoside-hydrolyzing enzyme is inducible. In addition, the glucosides pNPG, esculin, salicin, or amygdalin stimulated the induction of the sennoside beta-glucosidase, but less markedly than sennoside. Sennidin A or sugars (glucose, fructose, cellobiose, or maltose) did not induce the enzyme.

Anthraquinones↗

PubMed1996

Purification and characterization of a novel sennoside-hydrolyzing beta-glucosidase from Bifidobacterium sp. strain SEN, a human intestinal anaerobe.

A novel beta-glucosidase, which is inducible and capable of catalyzing the hydrolysis of sennosides, was purified from Bifidobacterium sp. strain SEN with Triton X-100 solubilization and DEAE-cellulose column chromatography, by which hydrolytic activities toward sennoside B, 4-methylumbelliferyl beta-glucoside (MUG), and p-nitrophenyl beta-glucoside (pNPG) were obtained together in the same eluted fractions. The activity was stable against detergents such as sodium dodecyl sulfate (SDS) and Triton X-100, but was denatured by SDS and beta-mercaptoethanal when heated. The final preparation was shown to be nearly homogeneous on SDS-polyacrylamide gel electrophoresis (PAGE) either after the enzyme was denatured or when it was not denatured. In the non-denaturing SDS-PAGE, a single protein band hydrolyzed MUG on the gel. In the denaturing SDS-PAGE, the subunit mass of the enzyme was estimated to be 110 kDa. The enzyme was optimally active at pH 6.0 for hydrolysis of sennoside B and MUG. Km values for sennoside B and MUG are 0.94 and 0.53 mM, respectively. The enzyme also catalyzed the hydrolysis of pNPG, amygdalin, geniposide and salicin. It was less active against methyl beta-glucoside and incapable of hydrolyzing cellobiose. The beta-glucosidase activity was inhibited by deoxynojirimycin and p-chloromercuribenzenesulfonic acid, but was less susceptible to several metals (FeSO4, ZnCl2, and CuSO4), and 5,5'-dithio-bis(2-nitrobenzoic acid).

Anthraquinones↗

PubMed1996

[Experimental studies on intravesical instillation of SM-5887, a novel anthracycline derivative for treatment of bladder carcinoma].

SM-5887 is a novel anthracycline derivative. Experimental studies of its intravesical chemotherapy were carried out to elucidate its histopathological effect on the normal bladder mucosa and the pharmacokinetics in Beagle dogs. Forty mg (4,000 micrograms/ml), 60 mg (6,000 micrograms/ml) and 80 mg (8,000 micrograms/ml) of SM-5887 dissolved in 10 ml of physiological saline were instilled into the empty bladders of dogs with bilateral cutaneous ureterostomy, respectively. SM-5887 instilled intravesically scarcely passed into the blood. In only one dog of five instilled with 80 mg of SM-5887 intravesically, the serum level of 0.0248 micrograms/ml was detected 2 hours after instillation, but all the others were below the detection limit (0.020 micrograms/ml). Excretion of SM-5887 into the urine was also low. The highest urinary excretion was observed 6 hours after instillation of 80 mg of SM-5887, yet the concentrations of SM-5887 and its metabolites in the urine were extremely low. The urinary concentrations of SM-5887 and its active metabolite, 13-OH derivative, were 0.029 micrograms/ml and 0.131 micrograms/ml, respectively. Other metabolites were not detected. The distribution of SM-5887 in the bladder mucosa and muscular layer was almost equal, but the concentration of its active metabolite, 13-OH derivative, was 5 to 10 times higher in the bladder mucosa than in the bladder muscular layer. The distributions of SM-5887 in the organs other than the bladder, that is, the cortex and medulla of kidney, heart, lung, liver, and spleen, were very low, and those of a 13-OH active metabolite were even lower. In addition, SM-5887 barely affected the normal bladder mucosa. In dogs instilled with 80 mg of SM-5887, no histological change was observed in the bladder mucosa and submucosal layer even after 6-hour retention at the highest concentration of 8,000 micrograms/ml.

Administration, Intravesical↗

PubMed1995

Purification and reaction mechanism of arylsulfate sulfotransferase from Haemophilus K-12, a mouse intestinal bacterium.

A novel type of sulfotransferase, arylsulfate sulfotransferase [EC 2.8.2.22], was purified to homogeneity from Haemophilus K-12, a mouse intestinal bacterium. The purified enzyme (M(r) 290,000) is composed of four subunits (M(r) 70,000). The best donor substrate was 4-methylumbelliferyl sulfate, followed by beta-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. The best acceptor substrate was alpha-naphthol, followed by phenol and resorcinol. The apparent Km for PNS using phenol as an acceptor and that for phenol and resorcinol. The apparent Km for PNS using phenol as an acceptor and that for phenol using PNS as a donor substrate were determined to be 0.095 and 0.71 mM, respectively. One of the reaction products, p-nitrophenol inhibited the enzyme noncompetitively with respect to PNS, but competitively with respect to alpha-naphthol. The Ki values of PNP for PNS and alpha-naphthol were 0.89 and 0.12 mM, respectively. The other reaction product, alpha-naphthyl sulfate, inhibited the enzyme competitively with respect to PNS, but non-competitively with respect to alpha-naphthol. The Ki values of alpha-naphthyl sulfate for PNS and for alpha-naphthol were 2.72 and 1.7 mM. These results suggest that the sulfate transfer reaction proceeds according to a ping pong bi bi mechanism.

Animals↗

PubMed1995

Effects of apple pectin on fecal bacterial enzymes in azoxymethane-induced rat colon carcinogenesis.

Because of the potential significance of colonic bacteria in colon carcinogenesis, we investigated the effect of pectin of different types on fecal bacterial enzymes (beta-glucuronidase, beta-glucosidase and tryptophanase) at various periods of time after feeding rats with pectin-containing diets during azoxymethane-induced colon carcinogenesis. The diet supplemented with 20% apple pectin or 20% citrus pectin decreased the multiplicity of colon tumors, and the number of tumors was significantly decreased in the group fed apple pectin. The incidence of colon tumors in the apple pectin group was lower than that in the control group. The mean tumor size was similar among the three groups. Apple pectin feeding decreased fecal beta-glucosidase and tryptophanase levels. Furthermore, a significant decrease in the activity of beta-glucuronidase was observed in the apple pectin group during the initiation phase. These findings suggest that the protective effect of pectin on colon carcinogenesis may be dependent on the type of pectin and be related to the decrease of beta-glucuronidase activity in the initiation stage of carcinogenesis.

Adenocarcinoma↗

PubMed1995

Helicobacter pylori urease inhibition by rabeprazole, a proton pump inhibitor.

We investigated the inhibitory effects of four gastric proton pump inhibitors (PPIs): rabeprazole, a novel benzimidazole PPI, omeprazole, lansoprazole and AG-2000, on the urease activity of Helicobacter pylori (H. pylori). Their 50% inhibitory concentrations (I50s) were found to be 0.29, 5.4, 9.3 and 0.3 microM respectively. Rabeprazole and omeprazole were also potent inhibitors of Jack bean and Proteus mirabilis cellular ureases. The thioether derivative of rabeprazole, one of its metabolites, had no inhibitory effect on H. pylori urease, despite being reported as a more potent inhibitor of H. pylori growth than rabeprazole. The inhibitory effect of rabeprazole was prevented completely and reversed considerably by the addition of sulfhydryl compounds, such as beta-mercaptoethanol, glutathione and dithiothreitol. Moreover, the addition of beta-mercaptoethanol recovered the urease activity inhibited by rabeprazole. From these results, we expected that rabeprazole inhibited H. pylori urease activity by forming disulfide bonds between it and the active site of the enzyme.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1995

Purification and characterization of a geniposide-hydrolyzing beta-glucosidase from Eubacterium sp. A-44, a strict anaerobe from human feces.

A geniposide-hydrolyzing beta-glucosidase was discovered in Eubacterium sp. A-44, a human intestinal anaerobe. The enzyme was intracellularly distributed in the bacterium, and purified to homogeneity from the extract using Butyl-Toyopearl 650M, Sephacryl S-300, hydroxyapatite and chromatofocusing column chromatography. The enzyme was a single polypeptide chain with the molecular weight of 90 kDa and the N-terminal amino acid sequence initiated from methionine up to the 29th residue did not show more than 50% homology against known protein sequences. A broad substrate specificity was shown for the beta-glucosidase to hydrolyze aryl beta-D-glucosides (p-nitrophenyl beta-D-glucopyranoside-pNPG, esculin and salicin), alkyl beta-D-glucosides (geniposide and amygdalin) and cellobiose. The Km values (mM) for various beta-D-glucosides were 0.068 for geniposide, 0.10 for pNPG, 0.21 for esculin, 0.22 for salicin, 2.9 for amygdalin, and 0.91 for cellobiose. The pH optimum with pNPG and geniposide as the substrates was 6.0. The enzyme was inhibited by sulfhydryl reagents, Cu2+, and nojirimycin bisulfite.

Amino Acid Sequence↗

PubMed1995

Purification and characterization of beta-glucuronidase from Escherichia coli HGU-3, a human intestinal bacterium.

beta-Glucuronidase was purified 360-fold from Escherichia coli HGU-3, an human intestinal bacterium. The specific activity of the purified enzyme was 17.78 units/mg protein. The enzyme (M.W. 290000) is composed of four subunits (M.W. 72000) with a pI and optimal pH of 4.8 and 6-7, respectively. The apparent Km for p-nitrophenyl-beta-D-glucuronide was found to be 0.22 mM. The enzyme was inhibited by saccharic acid 1,4-lactone, glycyrrhizin, N-ethylmaleimide (NEM) and p-chloromercuriphenylsulfonic acid (PCMS). Using the bile containing bilirubin diglucuronide as a substrate, the purified beta-glucuronidase was able to hydrolyze it to bilirubin. This hydrolyzed bilirubin formed calcium bilirubinate with a reaction mixture containing CaCl2.

Bilirubin↗

PubMed1995

Inhibitory effect of glycine on ethanol absorption from gastrointestinal tract.

The oral administration of glycine remarkably decreased the blood ethanol level in mice which had ingested ethanol, and a large amount of ethanol was retained in their stomachs. These effects were observed by the oral administration of glycine previous to the ethanol ingestion, and depended on the dose of glycine. An intravenous injection of glycine did not affect the ethanol absorption at all. These findings indicate that glycine suppresses the rate of ethanol absorption from the gastrointestinal tract. Glycylglycine, glycylglycylglycine and alanine showed the same effects, but glucose did not. However, ethanol absorption from the ligated stomach of mouse was inhibited not only by glycine but also by glucose. On the other hand, the rate of ejecting a pigment from the stomach to the small intestine was lowered by glycine, but not by glucose. Thus, glycine lowers the gastric emptying rate, resulting in the suppression of ethanol absorption from the gastrointestinal tract.

Administration, Oral↗

PubMed1995

Inhibitory effect of beta-glucosyl-phenolic hydroxamic acids against urease in the presence of microfloral beta-glucosidase.

Three glucosyl-phenolic hydroxamates, 4-O-(beta-D-glucopyranosyl) benzohydroxamic acid, 4-O-(beta-D-glucopyranosyl)hippuric hydroxamic acid, and 3-[4-O-(beta-D-glucopyranosyl)phenyl]propionohydroxamic acid (Glc-PPHA), were hydrolyzed to their corresponding aglycones by beta-glucosidase of intestinal flora of rat without any major adverse hydrolysis in vitro. Inhibitory potency of these glucosyl-hydroxamates on urease was recovered to the same extent as that of the corresponding aglycone hydroxamates by preincubation for 2h with rat intestinal flora. p-Hydroxyphenylpropionohydroxamic acid inhibited noncompetitively jack-bean urease activity and its glucose-ligated form, Glc-PPHA inhibited it competitively. A single oral dose of Glc-PPHA tended to inhibit urease activity in proximal colon contents of rat at 6 h after administration (p = 0.06). After 14C-urea was orally administered to rat, 14CO2 was collected for to measure the ureolysis in vivo. Expired 14CO2 was limited to 40% by a single oral dose of Glc-PPHA during 6 h, and 75% of intestinal ureolysis was repressed during the first 1 h in the breath test.

Animals↗

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↗