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Biomedical subjects

S Sano

Publications and source records attributed to S Sano.

At least 19 recordsLinked to original sources

A direct demonstration of the catalytic action of monodehydroascorbate reductase by pulse radiolysis.

To elucidate the catalytic mechanism of monodehydroascorbate (MDA) reductase from cucumber, its interaction with MDA radical was investigated by the use of pulse radiolysis. When approximately equimolar MDA radical to the fully reduced MDA reductase was generated, the fully reduced enzyme reacted first with MDA radical to form the red semiquinone, and the semiquinone further reacted with MDA radical to form the oxidized enzyme. At a low ratio (< 20) of MDA radical to enzyme concentration, the fully reduced enzyme reacted quantitatively with MDA radical to form the semiquinone with a second-order rate constant of 2.6 x 10(8) M-1 s-1 at pH 7.4. At excess MDA radical to enzyme concentration, a similar rate constant was obtained from the decay of MDA radical. These results suggest that the reaction of the semiquinone with MDA radical occurs at the same rate or rate-limiting step of the oxidation of the fully reduced enzyme by MDA radical. The rate constants decreased with an increase in NaCl concentration, suggesting that the localization of cationic groups of amino acid residue near the active site may provide electrostatic guidance to the anionic substrate of MDA radical.

Catalysis

Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in Escherichia coli.

Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is the first enzyme to be identified whose substrate is an organic radical and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA has been cloned from cucumber seedlings (Sano, S., and Asada, K. (1994) Plant Cell Physiol. 35, 425-437), and a plasmid was constructed in the present study that allowed a high level expression in Escherichia coli of the cDNA-encoding MDA reductase using the T7 RNA polymerase expression system. The recombinant MDA reductase was purified to a crystalline state, with a yield of over 20 mg/liter of culture, and it exhibited spectroscopic properties of the FAD similar to those of the enzyme purified from cucumber fruits during redox reactions with NADH and MDA. The red semiquinone of the FAD of MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the reduced enzyme to MDA. The specificity of electron acceptors of the recombinant enzyme appeared to be similar to that of MDA reductase, even though the amino acid sequence encoded by the cDNA was somewhat different from that of the enzyme purified from cucumber fruits. The Km values for NADH and NADPH of the recombinant enzyme indicated a high affinity of the enzyme for NADH. The reaction catalyzed by the enzyme did not exhibit saturation kinetics with MDA up to 3 microM. A second order rate constant for the reduction of the enzyme-FAD with NADH was 1.25 x 10(8) M-1 s-1, as determined by a stopped-flow method, and its value decreased with increases in ionic strength, an indication of the enhanced electrostatic guidance of NADH to the enzyme-FAD.

Cloning, Molecular

Effects of bidisomide (SC-40230), a new class I antiarrhythmic agent, on ventricular arrhythmias induced by coronary artery occlusion and reperfusion in anesthetized rats; comparison with mexiletine and disopyramide.

We investigated the antiarrhythmic effects of bidisomide (SC-40230), a new class I antiarrhythmic drug, in early-phase ventricular arrhythmias induced by coronary artery occlusion and reperfusion in anesthetized rats. The effects of bidisomide were compared with those of mexiletine (MXT) and disopyramide (DSP), established class I antiarrhythmic drugs. Drugs were administered intravenously, 5 min before induction of coronary occlusion. Bidisomide (5 mg/kg) reduced the number of premature ventricular complexes and the incidence of ventricular tachycardia and ventricular fibrillation similarly to MXT and DSP in rats with ventricular arrhythmias induced by coronary artery occlusion. In rats with ventricular arrhythmias induced by coronary artery reperfusion following a 5-min coronary occlusion, the antiarrhythmic effects of 5 mg/kg of bidisomide were similar to those of the same doses of MXT and DSP. All three drugs significantly slowed the heart rate. Our results suggest that bidisomide may effectively reduce the severity of life-threatening ventricular arrhythmias that occur during acute coronary syndrome.

Animals

Enhancement in gastric mucosal EGF and PDGF receptor expression with ulcer healing by sulglycotide.

1. The effect of an antiulcer agent, sulglycotide, on mucosal expression of EGF and PDGF receptors with chronic ulcer healing was investigated. 2. Rats with experimentally-induced gastric ulcers were treated twice daily for 14 consecutive days, either with sulglycotide at 200 mg/kg or vehicle, and at different stages of treatment used for quantitation of gastric mucosal EGF and PDGF receptors. 3. The ulcer healing was accompanied by an increase in mucosal expression of both types of receptors. A 1.8-fold increase in EGF and 3.1-fold increase in PDGF receptors occurred by the 4th day following the development of ulcer and reached a maximum of 2.4-3.9-fold increase by the 10-14th day. 4. Treatment with sulglycotide caused accelerated ulcer healing accompanied by a significant enhancement in the receptors expression. A 2.3- and 3.6-fold increase in EGF and PDGF receptor expression occurred by the 4th day of sulglycotide treatment, reaching a 5.5- and 5.6-fold respective increase by the 10th day when the ulcer essentially healed. 5. The results attest to the ability of sulglycotide to stimulate the gastric mucosal proliferative activities associated with ulcer healing.

Acetates

Quality of peptic ulcer healing induced by lansoprazole and roxatidine.

This study reports preliminary results of a controlled, multicenter trial on the quality of ulcer healing induced by lansoprazole (LPZ) or roxatidine (R) in gastric ulcer (GU) or duodenal ulcer (DU) patients. Group A received LPZ 30 mg q.d. and group B received R 75 mg b.i.d. All drugs were given for 8 weeks in GU and for 6 weeks in DU. Endoscopy and gastric biopsy were performed to detect Helicobacter pylori before and on completion of treatment. The healing rates of groups A and B were 100 and 69.2%, respectively, in GU and 100 and 70.0%, respectively, in DU. This difference (p < 0.01) was significant between the two groups in GU. There was no significant difference between the two groups in the S2 stage shift rate in GU and DU. The H. pylori clearance rates of groups A and B were 33.3 and 20.0%, respectively, in GU and 62.5 and 33.3%, respectively, in DU. The differences in treatment response (healing rates and S2 shift rates) between the LPZ group and the R group may be related to the differences in suppression of acid secretion and in bactericidal effects on H. pylori.

2-Pyridinylmethylsulfinylbenzimidazoles

[Fontan-type procedure for an adult case of double-outlet right ventricle (S, D, L)].

The double-outlet right ventricle with L-malposition (DORV (S, D, L)) is one of the rare congenital heart diseases. Intracardiac rerouting with a internal conduit has been indicated in principle to it as the radical operation. DORV (S, D, L) is often combined with the pulmonic stenosis (PS), and Rastelli's operation is indicated to the case in which release of PS is difficult. We report a case of DORV (S, D, L) combined with PS and the hypoplastic right ventricle which is treated by the Fontan-type procedure. The patient was a 35-year-old female who had undergone Glenn's operation at 10 years of age under the diagnosis of transposition of the great arteries. Recently she complained of aggravation of the cyanosis and dyspnea on exertion. After thorough examinations, the disease was diagnosed as DORV (S, D, L) with doubly committed VSD which was combined with severe PS, hypoplastic right ventricle (29% of normal) and left-sided juxtaposition of atrial appendages. The development of her distal pulmonary arteries was estimated well enough (PA-index 448 mm2/m2). We judged that intracardiac rerouting with an internal conduit could not be applied due to her severe pulmonic stenosis, and that Rastelli's operation as the biventricular repair was also impossible because of her hypoplastic right ventricle. Thus we adopted the Fontan-type procedure with a new septation in the right atrium, namely oblique partition, and attained a good result. To our knowledge, this case is the fourth case report in Japan of DORV (S, D, L) treated with the Fontan-type procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult

Biosynthesis of mucin derived from a 60-kDa precursor protein in the human stomach.

We studied the biosynthesis of mucin in the human stomach using an anti-mucin core peptide monoclonal antibody, 3G12. Human stomach mucosa was labeled with [35S]methionine, and chased for 3 h. An approximately 60-kDa subunit of human gastric mucin precursor protein was detected in the intracellular product. Under nonreducing conditions, dimer, trimer, and tetramer mucin precursor protein (120, 180, 240 kDa) were detected. Treatment with tunicamycin or endo-beta-N-acetylglucosaminidase H had no effect on the 60-kDa subunit and its oligomers. Extracellular products contained only the high molecular weight mucin, and the secretion was not affected by tunicamycin. By treatment with monensin or brefeldin A, the mature mucin was not secreted extracellularly. These findings suggested that a 60-kDa subunit of the mucin precursor protein was biosynthesized into mature mucin after oligomerization to tetramers, and that neither the oligomerization nor the intracellular transport of the mucin in the human stomach was associated with N-glycosylation.

Antibodies, Monoclonal

Cytokeratin patterns of normal middle ear epithelia in humans, cats, and chinchillas.

We describe the cytokeratin patterns of epithelia from the tympanic orifice, tympanic cavity, and mastoid cavity of humans, cats, and chinchillas, and compare these findings with those of tracheal epithelium and external canal epidermis. Our findings are as follows: 1) middle ear epithelium from all locations demonstrates some type of cytokeratin staining, 2) broad-spectrum cytokeratin antibodies stain epithelia of middle ear cleft, tracheal epithelium, and external canal epidermis in all species, 3) specific cytokeratin antibodies reveal species-related differences in middle ear and tracheal epithelia, 4) middle ear and tracheal epithelia usually have the same pattern, and 5) none of the monospecific cytokeratin antibodies have a positive reaction with external canal epidermis. These findings suggest that the cytokeratin patterns of middle ear epithelium are useful in studying the hyperplastic and metaplastic changes in otitis media; however, caution must be exercised when making interspecies comparisons.

Animals

Different subsets of CNS neurons express different glycosaminoglycan epitopes on large perineuronal proteoglycans.

Proteoglycans are known to occur in the central nervous tissue, but their function is not well understood. We made a biochemical study of four perineuronal antigens on different subsets of rat brain neurons and found that three antigens recognized by antibodies against glycosaminoglycan epitopes were large proteoglycans. Molecular masses estimated by immunoblotting were 700 kDa for 473, 376 and 1B5 antigens and 600 kDa for the 374 antigen. Reactivities of the antigens on immunoblots to monoclonal antibodies (MAbs) 473 and 376 were lost, and that to MAb 1B5 uncovered, after chondroitinase ABC treatment as in the brain sections. The elution profiles from ion-exchange and gel-filtration column chromatography of 473, 376 and 1B5 antigens were quite similar, but those of 374 antigen were slightly different. The immunoaffinity-purified 473 antigen migrated at 700 kDa on sodium dodecylsulfate gel electrophoresis, and chondroitinase ABC treatment decreased the molecular mass to 600 kDa. The 473 antigen was recognized by MAbs 376 and 1B5 although these MAbs also reacted with 473-negative 700 kDa molecules. These results suggest that neuronal subset-specific glycosylation may occur on the 700 kDa proteoglycans, and that the glycosaminoglycan structures may be involved in the pericellular diversity of CNS neurons.

Animals

Bacterial tympanogenic labyrinthitis, meningitis, and sensorineural damage.

Pathologic changes (sensorineural hearing loss, labyrinthitis, meningitis) can follow otitis media. Various macromolecular substances demonstrably enter the inner ear via the round window membrane, but its permeability to bacteria is less known. We inoculated Streptococcus pneumoniae type 7F bilaterally into the middle ears of two groups of chinchillas, with and without grafted round window membranes. Inner ears of inoculated animals were observed by light and electron microscopy. None with continuous grafts had labyrinthitis. Bacteria penetrated all three layers of nongrafted round window membranes and into all cochlear turns, entering Schuknecht's channels and following neuronal pathways; nerves were often degenerated, hair cells were damaged or missing, and the stria vascularis was edematous and hemorrhagic. The neural damage suggests a mechanism for the hearing loss that can follow otitis media. Absence of labyrinthitis and meningitis in grafted animals suggests a tympanogenic pathway for the bacteria.

Animals

Primary cultures of middle ear epithelial cells from chinchillas.

A reproducible method is presented for primary cultures of middle ear epithelial cells (MEEC) from chinchillas. The MEEC were first dissociated with protease and grown on collagen-coated membrane using a culture medium containing equal volumes of Dulbecco's modified Eagle medium and Ham's F12 supplemented with 0.5% fetal bovine serum. Outgrowth of cells was first noted within 24 h, reaching confluency in 6-7 days. These cells grew in a monolayer and appeared to be ovoid or polygonal. By immunofluorescence microscopy, these cells stained for cytokeratin, but not for type III collagen. In contrast, fibroblasts stained for type III collagen, but not for cytokeratin. Based on growth characteristics, morphology, and immunofluorescent findings, these cells were determined to be epithelial cells. To retard the outgrowth of fibroblasts, 5 mM putrescine was added to the culture medium on the 2nd day of explant. Contamination with fibroblasts was consistently less than 5% when defined as type III collagen-positive cells. Establishment of a method for the primary culture of MEEC will provide a new approach for studying the role of epithelial cells in the pathogenesis of various types of otitis media.

Animals

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid.

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.

Acid Anhydride Hydrolases

Tetralogy of Fallot: favorable outcome of nonneonatal transatrial, transpulmonary repair.

This report describes our experience with 366 patients who had a transatrial, transpulmonary repair of tetralogy of Fallot between December 1980 and December 1991. Included in this group are patients with tetralogy of Fallot plus atrioventricular septal defect as well as patients displaying all degrees of aortic override (in the presence of subaortic ventricular septal defect and right ventricular outflow tract obstruction). Median age was 15.3 months and median weight, 12.3 kg. Of the 366 patients, 72% required a pericardial patch to reconstruct the main pulmonary artery or right ventricular outflow tract. Serious coronary anomalies were seen in 11 patients, without influencing surgical approach. There were two hospital deaths (0.5%; 70% confidence limits, 0.2% to 1.2%). Actuarial survival was 97.5% at 42 months (95% confidence limits, 95% to 99%) reflecting four late deaths over 1,129 patient-years of follow-up. Postoperative cardiac catheterization studies were performed in 61 patients at a mean follow-up interval of 23 months. Mean right ventricular/left ventricular systolic pressure ratio after repair was 0.46 (standard deviation, 0.28), and mean gradient across the right ventricular outflow tract was 15 mm Hg (standard deviation, 24 mm Hg). Actuarial freedom from reoperation for any reason has been 95% (95% confidence limits, 92% to 97%) at 5-year and 10-year follow-up. These early and medium-term results encourage us to continue with transatrial, transpulmonary repair of tetralogy of Fallot. We believe that this approach has an operative risk similar to or lower than transventricular repair, and that it will result in better preservation of right ventricular function in the long term.

Child, Preschool

Repair of hypoplastic or interrupted aortic arch via sternotomy.

Herein we describe our experience with repair of interrupted aortic arch and coarctation plus hypoplastic aortic arch in 55 consecutive infants (1984 to 1990). Median age at operation was 6 days and median weight 3.1 kg. Associated severe intracardiac anomalies were the rule. All patients had significant congestive cardiac failure, and the majority required prostaglandin E1 resuscitation and inotropic support (with or without ventilation) before the operation. All operations were performed via sternotomy with core cooling and circulatory arrest. Isolated myocardial perfusion was used in 13 patients during arch repair. A complete intracardiac (biventricular) repair was performed except in patients expected to require a Fontan operation as definitive treatment. The operative mortality overall was 14.5% (confidence limits 10% to 22%). For arch repair plus biventricular intracardiac repair, the operative mortality was 9% (confidence limits 5% to 15%), and for arch repair plus palliative intracardiac repair, 40% (confidence limits 22% to 60%). The mortality in the isolated myocardial perfusion group was 0% (confidence limits 0% to 14%), which may be related to reduced myocardial ischemic time (p less than 0.05). Actuarial survival was 75% (confidence limits 65% to 83%) at 12 months, with no subsequent deaths over 1294 patient-months (mean 28 months) of follow-up. Actuarial freedom from recurrent arch obstruction was 69% (confidence limits 48% to 85%) at 46 months' follow-up. Primary repair of interrupted aortic arch and coarctation plus hypoplastic arch compares favorably with a staged approach and is recommended even when complex intracardiac anatomy is present.

Aorta, Thoracic

[Congenital lobar emphysema successfully treated by right upper lobectomy at five hours after delivery: a case report].

Lobar emphysema is a rare disease and one of the causes of respiratory disturbance in the newborn and infancy. A case report is presented and compared with related data in the literature in Japan. Maternal echographic findings indicated the cystic lung disease of the fetus. The cystic space was punctured and aspirated three times. The baby was delivered by caesarean section after having taken sufficient precaution to prevent respiratory failure. Since the baby developed dyspnea gradually, at five hours following the delivery, right upper lobectomy was performed and the major symptoms were eliminated. The pathological diagnosis was congenital lobar emphysema and the etiology was concluded to be bronchiectasis.

Cystic Adenomatoid Malformation of Lung, Congenita

Purification and characterization of phytase from rat intestinal mucosa.

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8 or 3.1.3.26) was purified from rat intestinal mucosa. The purified enzyme preparation exhibited two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular masses of 70 kDa and 90 kDa. Rabbit antisera prepared against the 90K subunit cross-reacted with the 70K subunit on immunoblotting. The peptide maps of the 70K and 90K subunits were similar, and the N-terminal amino acid sequences of the two subunit proteins were almost identical. Treatments to remove sugar moieties from the proteins showed that the two subunit proteins had different oligosaccharide chains, although the difference in their molecular masses was not due to the difference in their oligosaccharide compositions. The purified enzyme also showed activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1), but the properties of the two enzyme activities were different; the optimum pH for phytase activity was 7.5, while that for alkaline phosphatase was 10.4. Phytase activity did not necessarily require divalent cations, while Mg2+ was essential for alkaline phosphatase activity. Phenylalanine, a specific inhibitor of intestine-type alkaline phosphatase had no effect on the phytase activity.

6-Phytase