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Biomedical subjects

A Ohara

Publications and source records attributed to A Ohara.

At least 55 records · Page 3Linked to original sources
PubMed1994

Effect of radical scavengers on the inactivation of papain by ascorbic acid in the presence of cupric ions.

Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu2+ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm.Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu(2+)-AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu(2+)-AsA system, although hydrogen peroxide (H2O2) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H2O2 serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu2+ or Cu+) at the specific site of inactivation.

Ascorbic Acid↗
PubMed1994

Hydroxylation of phenylalanine and salicylate by stimulated polymorphonuclear leukocytes and the accelerating effect of glutathione on their hydroxylation.

Incubation of phorbol-myristate acetate-stimulated human polymorphonuclear leukocytes (PMNs) with phenylalanine and salicylate induced significant levels of formation of o- and m-tyrosines, and 2,3- and 2,5-dihydroxybenzoates (DHBAs), respectively, dependent on reaction time. Aromatic hydroxylation reactions were not inhibited by desferrioxamine, nor were they affected by the removal of trace ion contamination from the buffer solution used by treatment with conalbumin. Hydroxylation reactions were largely blocked by superoxide dismutase and hydroxyl radical (OH.) scavengers. The results of the present study suggest that the generation of OH. by human PMNs occurs during the respiratory burst. Hydroxylation of both phenylalanine and salicylate by stimulated human PMNs were significantly accelerated by incubation in the presence of the reduced form of glutathione (GSH). Hydroxylation of phenylalanine by stimulated guinea pig PMNs in the presence of GSH was significantly inhibited by desferrioxamine, although the same hydroxylation in the absence of GSH was not affected. Hydroxylation of phenylalanine by the hypoxanthine (HX)-xanthine oxidase (XO) system by intact PMNs was significantly accelerated by the addition of GSH, although that in the absence of PMNs was largely inhibited. Desferrioxamine showed an inhibitory effect on hydroxylation by the HX-XO system in the presence, but not in the absence, of intact PMNs. The results suggest that the formation of OH. by stimulated PMNs is accelerated by GSH, based on the occurrence of the Harber-Weiss reaction catalyzed by transition metal ions liberated and reduced by GSH from PMNs, and by the effective accumulation of H2O2 by the GSH-induced inhibition of catalase.

Animals↗
PubMed1994

On the mechanism of inactivation of active papain by ascorbic acid in the presence of cupric ions.

An inactivation mechanism of active papain (EC 3.4.22.2) by the Cu(2+)-ascorbic acid (AsA) system was examined. Incubation of active papain, which contains an active sulfhydryl (SH) group, with the Cu(2+)-AsA system under aerobic conditions resulted in an irreversible loss of enzyme activity. The enzyme was not inactivated at a molar ratio of enzyme to Cu2+ of 1:< 1, whereas at a molar ratio of 1:1-2, the extent of inactivation showed the same dependence on the extent of oxidation of AsA. Saturation kinetics were observed with respect to the concentration of AsA. The degree of inactivation was dependent on the decrease in SH content of the enzyme. Catalase at a low concentration partially protected the enzyme from inactivation, but did not affect the oxidation of AsA. In addition, catalase at a high concentration completely protected both the enzyme from inactivation and AsA from oxidation. The present results suggest that an additional function of H2O2, besides producing hydroxyl radicals (.OH), is to promote the conversion of Cu+ into Cu2+, and that an active SH group of papain is site-specifically modified by the .OH, resulting in inactivation of the enzyme.

Amino Acids↗
PubMed1994

Study on the acceleration in healing of ethanol-induced gastric lesions in rats by methylprednisolone.

This study was designed to determine the influences of methylprednisolone (MP, CAS 2375-03-3) on the genesis and healing of gastric lesions induced by 50% ethanol in relation to the changes in gastric mucosal prostaglandin (PG) and leukotriene (LT) contents and vascular permeability. Intragastric administration of 1 ml of 50% ethanol induced hemorrhagic lesions in the fundic portion, and these lesions were not affected by 20 mg/kg of MP injected subcutaneously 30 min before ethanol administration. In untreated rats, 4 kinds of PGs, i.e., 6-keto-PGF1a, PGF2a, PGE2, and PGD2, were determined in gastric mucosa by high performance liquid chromatography (HPLC), but no LTs were detected. Administration of ethanol significantly reduced all PG contents and increased production of peptide-LTs in gastric mucosa. Pretreatment with MP did not influence ethanol-induced changes in PG and peptide-LT contents. Ethanol-induced lesions required 96 h for total healing. Ulcer healing was not affected by MP, which was injected subcutaneously twice daily from 1 h after ethanol administration until the end of the experiment, and complete ulcer healing was observed after 72 h. MP did not affect ethanol-induced decrease in PG contents, their recoveries, or peptide-LT contents. Ethanol increased vascular permeability and MP reduced this increase. These results suggest that MP does not have a hazardous effect, but is rather beneficial with regard to healing of ethanol-induced gastric lesions through prevention of the increase in vascular permeability caused by ethanol, and that it affected neither gastric mucosal PG nor peptide-LT contents.

Animals↗
PubMed1993

Kallidin-releasing enzyme from Bitis arietans (puff adder) venom.

A kallidin-releasing enzyme with arginine ester hydrolytic activity was isolated from Bitis arietans venom by Sephadex G-75, DEAE-cellulose, and Sephadex G-100 column chromatography. This enzyme was shown to be homogeneous as demonstrated by a single band on polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH2-terminal sequence analysis. The molecular weight was determined to be 58,000 Da with an isoelectric point of 4.4. Kallidin-releasing enzyme possesses proteolytic activity demonstrated by the hydrolysis of Gly(8)-Ser(9), Ala(14)-Leu(15), Tyr(16)-Leu(17), and Phe(25)-Tyr(26) bonds of oxidized insulin B chain. This enzyme did not convert fibrinogen to fibrin, yet it did hydrolyze the A alpha, B beta, and gamma chains of fibrinogen. The enzyme was shown to cleave a kininogen analog with the release of kallidin. Arginine ester hydrolytic activity of the preparation was inhibited by diisopropyl fluorophosphate and benzamidine hydrochloride, suggesting that serine and glutamic acid or aspartic acid are involved in this activity. This protein was stable to heat and over the pH range of 3-12.

Amino Acid Sequence↗
PubMed1993

Effect of bilineobin, a thrombin-like proteinase from the venom of common cantil (Agkistrodon bilineatus).

A thrombin-like proteinase, named bilineobin, was isolated from Agkistrodon bilineatus venom by Sephadex G-75, DEAE-Sephacel and Heparin-Sepharose CL-6B column chromatography. The purified enzyme has a mol. wt of 57,000 and catalysed the hydrolysis of arginine esters and thrombin substrates Boc-Val-Pro-Arg-MCA and Boc-Asp(OBz)-Pro-Arg-MCA. Although bilineobin converted fibrinogen into fibrin resulting in the production of fibrinopeptides, the activity was relatively low (0.65 NIH units/mg). Fibrinopeptides released upon hydrolysis by this proteinase were identified as fibrinopeptide A (FpA) and fibrinopeptide B (FpB) by measuring fast atom bombardment (FAB) mass spectra and amino acid sequence. This indicates that bilineobin hydrolyses the Arg(19)-Gly(20) bond in the A alpha chain and the Arg(21)-Gly(22) bond in the B beta chain of the bovine fibrinogen molecule. Kinetic study of FpA and FpB release reveals that bilineobin has a preference for cleaving the B beta chain. In addition, bilineobin is resistant to thrombin inhibitors such as hirudin. These suggest that the mechanism of action of bilineobin is similar but not identical to that of thrombin. It was demonstrated that the NH2-terminal region of bilineobin has significant similarities in sequence with thrombin-like proteinases from other snake venoms; however, only three residues were common with thrombin up to residue number 24.

Amino Acid Sequence↗
PubMed1993

Tyrosine kinase regulates epithelial sodium transport in A6 cells.

Insulin increases epithelial Na+ reabsorption, and many of its actions involve tyrosine kinase. We used tyrosine kinase inhibitors to examine the role of tyrosine kinase in the action of insulin. Pretreatment of Na+ transporting cells with tyrosine kinase inhibitors attenuates the subsequent action of insulin, suggesting that the action of insulin on epithelial Na+ transport involves tyrosine kinase activity. In addition to their effect on insulin-induced Na+ transport, the tyrosine kinase inhibitors also significantly reduce Na+ transport in Na(+)-transporting epithelial cells, suggesting that there is a significant tonic tyrosine kinase activity that modulates epithelial Na+ transport. Using patch-clamp methods, we found that one inhibitor, genistein, reduces the number of active Na+ channels in cell-attached patches without significantly affecting the open probability of any remaining channels. The effects of the tyrosine kinase inhibitors are not due to inhibition of protein kinase A (PKA), since H89, a PKA inhibitor, does not affect Na+ transport of control cells (as the tyrosine kinase inhibitors do), and the tyrosine kinase inhibitor, genistein or tyrphostin 23, does not alter the stimulation of ion transport by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue (as H89 does).

Animals↗
PubMed1993

G protein activation inhibits amiloride-blockable highly selective sodium channels in A6 cells.

Single-channel methods were used to examine the regulation of amiloride-blockable highly selective sodium channels by guanine nucleotide-binding proteins (G proteins). A6 cells (a renal cell line derived from Xenopus laevis kidney) were cultured on permeable collagen films, and patch recordings were made from the apical membranes of confluent cells. The predominant channel in the apical membranes is a highly selective, 4-pS, amiloride-blockable sodium channel (the Na(+)-to-K+ permeability ratio is > 30). In inside-out patches, application to the cytosolic surface of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which deactivates G proteins, increased sodium channel activity. GDP beta S produced a sevenfold increase in channel activity. In contrast, GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) decreased sodium channel activity to about one-twentieth of the untreated value. The effect of GTP (but not GTP gamma S) was reversible. In cell-attached patches, a 3- to 4-h exposure of the apical membrane to pertussis toxin (PTX) increased the mean open time of sodium channels approximately 2.7 times and the open probability approximately 1.6-fold, but pretreatment of apical membranes with cholera toxin (250 ng/ml) for 3-4 h had no effect on open probability or mean open time. These results imply that a PTX-sensitive G protein regulates amiloride-blockable highly selective sodium channels in the apical membranes of A6 cells and that the G protein in a GTP-bound, activated state inhibits sodium channel activity.

Amiloride↗
PubMed1993

Site-specific inactivation of papain by ascorbic acid in the presence of cupric ions.

The mechanism of inactivation of papain (EC 3.4.22.2) by ascorbic acid (AsA) in the presence of cupric ions (Cu2+) was investigated. The aerobic combination of Cu2+ and AsA resulted in an irreversible loss of enzyme activity. The inactivation was found to be an apparent first order reaction. The prior mixing of Cu2+ and AsA caused the complete disappearance of the inactivation. The addition of iron ions led to significant suppression against the inactivation. Cu2+ was bound to the enzyme in a molar ratio of 1:1. At lower concentrations of Cu2+ (molar ratio of enzyme to Cu2+ of 1: < 1), the extent of inactivation showed the same dependence against the extent of oxidation of AsA. The rate of inactivation increased as the concentration of AsA was increased. Saturation kinetics were observed with respect to the concentration of AsA. Changes in the concentration of Cu2+ had no effect on the dissociation constant of the enzyme-AsA complex (KI), though the rate constant of inactivation (k2) showed a linear relationship with the concentration of Cu2+. At various pH values tested, no change of k2 was found, whereas the value of KI increased when the pH bacame lower. At higher concentrations of Cu2+, the rate of inactivation fell beyond a certain concentration of AsA. The present results suggest that both Cu2+ and AsA bind to the enzyme to form a ternary complex and that free radicals are site-specifically formed and react preferentially with the enzyme, at the site of their formation, impairing its activity.

Ascorbic Acid↗
PubMed1993

Formation of a hydroxyl radical by the myeloperoxidase-NADH-oxygen system.

When phenylalanine was incubated with myeloperoxidase (MPO) and NADH in citrate buffer (pH 4.5), o-, m-, and p-tyrosines were identified as hydroxylated products. Tyrosine formation was dependent on the reaction time and MPO concentration. No significant quantities of tyrosines were formed on if MPO was omitted and inactivated MPO was added instead of active MPO. The tyrosine formation by the MPO-NADH system was greatly reduced under anaerobic conditions, and significantly inhibited by hydroxyl radical scavengers. Superoxide dismutase was a potent inhibitor, but catalase was less effective. Even though the superoxide radical (O2-)-producing ability of the MPO-NADH system was about 29% of that of the hypoxanthine-xanthine oxidase system, under the experimental conditions employed, the rate of tyrosine formation from phenylalanine by two systems was found to be a similar. The above results suggest that the formation of a hydroxyl radical (OH.) may occur in the MPO-NADH system under aerobic conditions and a superoxide radical may be involved in the OH. formation, with MPO promoting the OH. formation from O2-.

Aerobiosis↗
PubMed1993

Purification and characterization of zinc-dependent acid phosphatase from bovine brain.

Zn(2+)-Dependent acid phosphatase (Zn(2+)-APase) was purified to homogeneity from bovine brain. The apparent molecular weight of the enzyme was estimated to be about 62000 by gel filtration and 31000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme exhibited an isoelectric point of approximately 4.8. The enzyme required Zn2+ ions for catalytic activity, but other cations had little or no effect. The maximum enzyme activity was obtained in the presence of about 5 mM of Zn2+ at pH 5.5 in 50 mM 2-(N-morpholino)ethanesulfonic acid-NaOH buffer. The enzyme significantly catalyzed the hydrolysis of p-nitrophenyl phosphate, phenyl phosphate, and phosphotyrosine. The enzyme was also active for myo-inositol-2-monophosphate and adenosine 2'-monophosphate of the other common phosphate esters tested, though significantly less active than for p-nitrophenyl phosphate. The optimum activity pH of the enzyme was around 5.5 with p-nitrophenyl phosphate and myo-inositol-2-monophosphate. The enzyme was resistant to fluoride ions. Two types of Zn(2+)-APases, a high molecular weight (molecular weight, M(r)., about 110,000) and a low molecular weight (M(r), about 62,000) type, were found to exist in various tissues of rat. Brain, lung, spleen, stomach, heart, skeletal muscle, and erythrocytes contained only the lower molecular weight type. On the other hand, liver and kidney contained mainly the higher molecular weight type, and the small intestine contained significant quantities of the both types.

Acid Phosphatase↗
PubMed1993

[Preoperative diagnosis of unroofed coronary sinus: a case report].

A 54-year-old man was admitted to our hospital because of exertional dyspnea. A second heart sound with fixed splitting and a systolic ejection murmur along the left sternal border was audible. The chest roentgenogram showed increased pulmonary vascularity, and the electrocardiogram showed incomplete right bundle branch block. Two-dimensional echocardiography in the parasternal view demonstrated a partition defect between the left atrium and the coronary sinus. Furthermore, transesophageal echocardiography revealed a left-to-right shunt flow into the coronary sinus through the defect. At these points, the patient was diagnosed as having a partially unroofed mid-portion of the coronary sinus. Unroofed coronary sinus is a cardiac anomaly rarely diagnosed prior to surgical operation. Two-dimensional echocardiography, especially transesophageal echocardiography, is useful for the preoperative diagnosis of unroofed coronary sinus.

Coronary Vessel Anomalies↗
PubMed1992

Regulation of renal epithelial sodium channels.

The high selectivity, low conductance, amiloride-blockable, sodium channel of the mammalian distal nephron (i.e. cortical collecting tubule) is the site of discretionary regulation which allows maintainance of total body sodium balance. In order to understand the physiological events that participate in this regulation, we have used the patch-clamp technique which allows us to measure individual Na+ channel currents and permits access to the cytosolic side of the channel-protein as well as its associated regulatory components. Most of our experiments have utilized the A6 amphibian renal cell line, which when grown on permeable supports is an excellent model for the mammalian distal nephron. Different mechanisms have been examined: (1) regulation by hormonal factors such as Anti-Diuretic Hormone (ADH) and aldosterone, (2) regulation by G-proteins, (3) modulation by protein kinase C (PK-C), and (4) modulation by products of arachidonic acid metabolism. Consistent with noise analysis of tight epithelial tissues, ADH treatment increased the number of active channels in apical membrane patches of A6 cells, without any apparent change in the open probability (Po) of the individual channels. Agents that increased intracellular cAMP mimicked the effects of ADH. In contrast, aldosterone was found to act through a dramatic increase in Po rather than through changes in channel density. Inhibition of methylation by deazaadenosine antagonizes the stimulatory effect of aldosterone. In excised inside-out patches GTP gamma S inhibits channel activity, whereas GDP beta S or pertussis toxin stimulates activity suggesting regulatory control by G-proteins. PK-C has been shown to contribute to 'feed-back inhibition' of apical Na+ conductance in tight epithelia.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1992

Age-associated decreases in prostaglandin contents in human gastric mucosa.

This study was designed to clarify effects of ageing on human gastric mucosal prostaglandin (PG) contents. Forty examinees were divided into 5 age groups of 8 persons each, as follows: age under 40, age 40-49, age 50-59, age 60-69, and age over 70. PG contents in human gastric mucosa were measured by microcolumn high performance liquid chromatography (HPLC) with helium/cadmium laser induced fluorescence detection using biopsy samples obtained by endoscopy. The contents of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and PGD2 in the under 40 group were 638 +/- 39, 97 +/- 16, 468 +/- 68, 497 +/- 86 (pg/mg tissue), respectively. No significant differences in PG contents among groups aged under 70 were observed. In contrast, significantly low PG contents in the over 70 group were observed, i.e., the contents of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and PGD2 were 311 +/- 58, 36 +/- 8, 196 +/- 48, 171 +/- 40, respectively, and their contents were significantly lower than those in other age groups. In conclusion, gastric mucosal PG contents decrease significantly in over 70 years-old and this might be a contributing factor in the pathogenesis of gastric ulcers in elderly people.

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Isolation of permanent clonal bone marrow stromal cell lines derived from "viable moth-eaten" and "severe combined immunodeficiency" mutant mice.

Long-term bone marrow cultures (LTBMCs) were established from bone marrow of mev/mev mice with severe combined immunodeficiency (scid) and their normal littermates, and permanent stromal cell lines were derived. In cultures from mutant mev/mev mice, an adherent layer containing around 10% of fibro-endothelial cells and 90% macrophage-like cells was observed. A permanent cell line actively inhibited hematopoiesis resulting in an 80-fold decrease in production of cumulative colony-forming unit culture units (CFU) including granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) and granulocyte-macrophage (CFU-GM). To confirm active inhibition, we performed coculture experiments using permanent cell lines and engrafted each in vitro with hematopoietic progenitors from normal mouse LTBMCs. "Cobblestone island" and nonadherent cell production were observed adding hemopoietic progenitors to coculture of fibroblastic cells of mev/mev cultures with lines from normal littermates. In contrast, macrophage cell line Mev #5 suppressed hemopoiesis and itself had a proliferative advantage. Thus, bone marrow stromal cell lines from mev/mev mice reproduce some characteristics of the hematopoietic microenvironmental defect in vitro.

Animals↗
PubMed1992

Mechanism and prevention of chronic colonic inflammation with trinitrobenzene sulfonic acid in rats.

1. The role of prostanoids in experimental colitis with trinitrobenzene sulfonic acid (TNBS) in rats was investigated. The effects of cyclosporine A (CsA) on the development of experimental colitis were also examined. 2. Five kinds of prostanoids were detected in rat colonic tissue by high performance liquid chromatography. These were 6-keto-prostaglandin (PG) F1 alpha, PGF2 alpha, PGE2, PGD2 and thromboxane B2. 3. In TNBS-induced experimental colitis, all prostanoid concentrations except PGD2 increased, although the time courses differed from each other. 4. Medication with indomethacin markedly reduced prostanoid concentrations in TNBS-induced colitis. However, indomethacin did not show any effect on damage scores. 5. Cyclosporine A reduced damage scores 14 days after TNBS treatment, and the protective effects were observed, whereas CsA did not affect colonic tissue prostanoid concentrations. 6. Prostanoids might be produced secondarily in the genesis of TNBS-induced colitis, although they may attenuate the inflammatory response. It was also suggested that CsA was likely to have therapeutic effects on experimental colitis by inhibiting the immune reaction with TNBS, which induced the chronic inflammation.

Animals↗
PubMed1992

Effect of duodenal mucosal blood flow on duodenal alkaline secretion in rats.

To investigate the role of duodenal mucosal blood flow (DMBF) in the regulation of duodenal alkaline secretion (DAS), both parameters were measured before and after the administration of various drugs in rats. The DMBF was determined using an electrolytically generated hydrogen gas clearance technique, and the DAS was measured by the perfusion method. The administration of dulcerozine, a potent duodenal ulcerogenic agent, at a dose of 250 mg/kg and serotonin at a dose of 20 mg/kg, which produces duodenal ulcerations with an acid load, decreased both DMBF and DAS. On the other hand, the administration of secretin at a dose of 10 U/kg increased both parameters. There were parallel changes in DMBF and DAS. It is concluded, therefore, that DAS may be regulated by DMBF and that both parameters may be involved in the defense mechanism of duodenal mucosa.

Alkalies↗