Biomedical subjects

S Okada

Publications and source records attributed to S Okada.

At least 703 records · Page 39Linked to original sources

PubMed1994

Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus.

Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood. The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli. The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method. About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C. The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5. The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis. The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family. When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared. The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase. On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1994

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment.

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

Animals↗

PubMed1994

Possible mechanisms of airway hyperresponsiveness after late asthmatic response in guinea pigs.

To elucidate the mechanism of airway hyperresponsiveness after late asthmatic response (LAR), we analyzed bronchoalveolar lavage fluid (BALF) and examined the airway smooth muscle contractility to acetylcholine (ACh). On day 1 after LAR, there was a significant positive correlation between the number of neutrophils in BALF and the increase in airway responsiveness after LAR (r = 0.82, p < 0.01). In the in vitro study, the dose response curve to ACh was significantly shifted to the left after removal of the epithelium in control guinea pigs. However, in hyperresponsive animals after LAR, removal of the epithelium had no significant effect on ACh-induced response. These results indicate that infiltration of neutrophils and other inflammatory cells induce epithelial damage and hence the development of airway hyperresponsiveness after LAR.

Acetylcholine↗

PubMed1994

Therapeutic effect of reticuloendothelial system (RES)-avoiding liposomes containing a phospholipid analogue of 5-fluorouracil, dipalmitoylphosphatidylfluorouridine, in Meth A sarcoma-bearing mice.

Reticuloendothelial system (RES)-avoiding liposomes are known to accumulate in tumor tissues due to passive targeting. Dipalmitoylphosphatidylfluorouridine (DPPF), a potent antitumor agent readily incorporated into the lipid bilayer, was embedded in RES-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA). The therapeutic effect of DPPF in PGlcUA-liposomes was examined in tumor-bearing mice. Free or liposomal DPPF was injected intravenously into BALB/c mice bearing subcutaneously implanted Meth A sarcomas. The RES-avoiding liposomal formulation using PGlcUA was effective in reducing tumors, and prolonging survival time compared with free DPPF and also DPPF in conventional liposomes. Therefore, PGlcUA-liposomes might be of practical use as drug carriers for anticancer agents, especially their derivatives for embedding in liposomal membranes.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

[Sustained-release effect of the direct compressed tablet based on chitosan and Na alginate].

In the dissolution test of tablets prepared by the direct compression of chitosan with 10% sodium alginate (NaAlg), the release of theophylline was extended independently of the medium pH. The drug release mechanism of the tablet was investigated by measuring the dissolution behavior of such excipients as chitosan and NaAlg. It was shown that at the lower pH the drug release was accompanied with the dissolution of chitosan, while at the neutral pH it was controlled by the gelation and dissolution of NaAlg. Further more by the oral administration of the tablet to beagle dogs, the sustained release of theophylline was also confirmed by some bioavailability parameters. Thus the chitosan tablet with 10% NaAlg was expected to be a pH-independent sustained release dosage form.

Administration, Oral↗

PubMed1994

[Evaluation of anticoagulant activity for low molecular-weight heparin by chromogenic substrate. Measurement of factor Xa and thrombin activities].

We described an amidolytic method for determining the anticoagulant activity of commercially available low molecular-weight heparin (LMWH) with the use of factor Xa (FXa) and thrombin (FIIa), and a chromogenic peptidyl substrate, S-2222 or S-2238. The procedures were based on the photometric determination of the inactivation of FXa and FIIa after incubation with LMWH in the presence of antithrombin III (AT III). At first, the optimal conditions of FXa and FIIa activities with respect to pH, temperature, and the amounts of AT III and LMWH-International Standards (LMWH-IS) were determined. And then, the anticoagulant activities of various LMWHs were determined under the established conditions. In the comparative study with LMWHs, significant differences were found in anti FXa and anti FIIa activities, and their ratios. The anti FIIa activity was reconfirmed to decrease with decreasing molecular weight of LMWHs. On the other hand, the anti Xa activity was, however, less dependent on the molecular weight of LMWHs.

Anticoagulants↗

PubMed1994

Transfer action of alpha-D-xylosidases from Aspergillus flavus MO-5 on p-nitrophenyl-alpha-D-xylopyranoside.

The transfer action of alpha-D-xylosidase I and II from Aspergillus flavus MO-5 on p-nitrophenyl-alpha-D-xylopyranoside (alpha-p-NPX) was investigated, as compared with that of commercial alpha-D-xylosidase (from Bacillus sp.). In reaction mixtures with various concentrations of alpha-p-NPX, both enzymes (I and II) liberated p-nitrophenol and xylose at low concentrations, and showed transfer products in addition to hydrolyzates at high concentrations. IP-1 and IP-3, IIP-1 and IIP-2, and CP-1 and CP-3 were main transfer products by alpha-D-xylosidase I, alpha-D-xylosidase II, and commercial alpha-D-xylosidase, respectively. They were isolated by active charcoal column chromatography and silica gel column chromatography. Their structures were analyzed by enzymatic digestion, methylation analysis, and 13C-NMR analysis. As the results, IP-1, IIP-2, and CP-1 were found to be the same structure, p-nitrophenyl-4-O-(alpha-D-xylopyranosyl)-alpha-D-xylopyranoside. Then, it was proved that the structure of IIP-1 was p-nitrophenyl-3-O-(alpha-D-xylopyranosyl)-alpha-xylopyranoside. On the other hand, IP-3 and CP-3 were found to be the same structure, D-xylopyranosyl-alpha-1,4-D-xylopyranose without a p-nitrophenyl group. We found that alpha-D-xylosidase I showed alpha-1,4 xylosyl transfer action as well as commercial alpha-D-xylosidase, and alpha-D-xylosidase II showed alpha-1,3 xylosyl transfer action in addition to alpha-1,4 xylosyl transfer action on alpha-p-NPX.

Aspergillus flavus↗

PubMed1994

Classification of some alpha-glucosidases and alpha-xylosidases on the basis of substrate specificity.

Three alpha-glucosidases which passed under the names of transglucosidase (from Aspergillus niger), maltase (from Brewers yeast), and isomaltase (from Bakers yeast) for reasons of their substrate specificities and transfer actions, were purified to electrophoretically pure states. These purified alpha-glucosidases were made uniform in the hydrolyzing activities using p-nitrophenyl alpha-glucopyranoside (alpha-p-NPG) and were reacted with p-nitrophenyl alpha-xylopyranoside (alpha-p-NPX) or isoprimeverose (xylopyranosyl-alpha-1,6-glucopyranose), which are typical substrates of alpha-xylosidase. Only Asp. niger alpha-glucosidase among them hydrolyzed alpha-p-NPX and isoprimeverose. Further the substrate specificities of three alpha-glucosidases and two alpha-xylosidases (I and II from Asp. flavus MO-5) were investigated on maltose, isomaltose, alpha-p-NPG, isoprimeverose, and alpha-p-NPX in detail, and kinetic parameters [Km, Vmax, and molecular activity (Ko)] were estimated and compared with each other. In the comparison of kinetic parameters, Asp. niger alpha-glucosidase showed a broad specificity, that is, containing isoprimeverose in addition to maltose, isomaltose, and alpha-p-NPG. Though this enzyme barely hydrolyzed alpha-p-NPX too, the velocity was very slow. Though both yeast alpha-glucosidases barely hydrolyzed alpha-p-NPX or isoprimeverose too, these substrates were not good for yeast enzymes. On the other hand, two alpha-xylosidases showed narrow specificities, such that the substrates except for alpha-p-NPX and isoprimeverose were not hydrolyzed at all. The action on isoprimerose by Asp. niger alpha-glucosidase was completely the same as that on isomaltose at optimum pH, optimum temperature, inhibition pattern of hydrolyzing activity by 1-deoxynojirimycin, and transfer action pattern. Accordingly, we interpret these results as indicating that the hydrolyzations of isomaltose and isoprimeverose by Asp. niger alpha-glucosidase were catalyzed at the same active site. Asp. niger enzyme that has both alpha-glucosidase activity and alpha-xylosidase activity was shown to be classified in a middle position between alpha-glucosidase and alpha-xylosidase.

Aspergillus niger↗

PubMed1994

Effective production of glycosyl-steviosides by alpha-1,6 transglucosylation of dextrin dextranase.

Dextrin dextranase (EC 2.4.1.2; DDase), which is produced by Acetobacter capsulatus ATCC 11894, acted on a mixture of stevioside and starch hydrolysate with isoamylase, so that the enzyme was found to convert stevioside to predominantly mono-glucosyl-stevioside (SG1) and di-glucosyl-stevioside (SG2), and little of the stevioside initially added remained. SG1 was separated into two compounds (SG1a and SG1b) by reversed-phase high-pressure liquid chromatography. The structures of SG1a, SG1b, and SG2 were analyzed and concluded to be 13-O-(6-alpha-glucosyl-2-beta-glucosyl-beta-glucosyl)-19-O-beta-glucosyl -steviol, 13-O-[(6-alpha-glucosyl)(2-beta-glucosyl)-beta-glucosyl]-19-O- glucosyl-steviol, and 13-O-[(6-alpha-glucosyl)(6-alpha-glucosyl-2-beta- glucosyl)-beta-glucosyl]-19-O-beta-glucosyl-steviol, respectively. During the reaction for production of glycosyl-steviosides, DDase catalyzes transglucosylations from glucosyl donor compounds to stevioside to be SG1a and SG1b, and to SG1b to be SG2 rapidly forming alpha-1,6 glucosidic linkages. However transglucosylation to SG1a to be SG2 rarely occurred, and the conversions among stevioside and these glycosyl-steviosides were catalyzed by the action of DDase to transfer alpha-1,6 linked glucosyl residues, forming alpha-1,6 linkages.

Carbohydrate Conformation↗

PubMed1994

DNA damage induced in cultured human alveolar (L-132) cells by exposure to dimethylarsinic acid.

Gene damage in cultured human alveolar (L-132) cells induced by exposure to dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics in mammals, was studied. DNA single-strand breaks and DNA-protein cross-links were induced by the treatment of L-132 cells with 10 mM DMAA. These kinds of damage appeared at 8 hr after start of exposure to DMAA. As regards DNA-protein cross-links, the DNA was found to bind not only to core histone proteins but also linker histone (H1) and nonhistone proteins. Furthermore, the cross-links were formed by the binding to serine or threonine residues of H1 or nonhistone proteins through phosphate moieties of the DNA. The induction of the alkali-labile sites in DNA in DMAA-treated L-132 cells was observed prior to that of DNA single-strand breaks and DNA-protein cross-links. As one of the alkali-labile sites in DNA, we estimated apurinic/apyrimidinic (AP) sites in DNA. The present study suggests that the DNA single-strand breaks and DNA-protein cross-links induced by the treatment of L-132 cells with DMAA occurred via the formation of AP sites in the DNA and that the DNA-protein cross-links were produced by a Schiff-base reaction between amino groups of nuclear proteins and aldehyde groups of AP sites in the DNA and the DNA single-strand breaks, by a beta-elimination reaction on AP sites in the DNA.

Cacodylic Acid↗

PubMed1994

Induction of lung-specific DNA damage by metabolically methylated arsenics via the production of free radicals.

To clarify the genotoxicity of inorganic arsenics, we focused on the genotoxic effect of metabolically methylated arsenics in mammals. Oral administration to mice of dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics, induced lung-specific DNA damage, i.e., DNA single-strand breaks and the clumping of heterochromatin. The lung-specific strand breaks were not caused by DMAA itself, but by dimethylarsine, a further metabolite of DMAA. An in vitro experiment indicated that DNA single-strand breaks by dimethylarsine were suppressed by the presence of superoxide dismutase and catalase, suggesting that the strand breaks were induced via the production of free-radical species including active oxygens. Dimethylarsenic peroxyl radical [(CH3)2AsOO.] and superoxide anion radical produced from the reaction between molecular oxygen and dimethylarsine were detected by electron-spin resonance analysis using a spin-trapping agent and the cytochrome-c method, respectively. Of these two radicals, the dimethylarsenic peroxyl radical rather than the superoxide anion radical is assumed to play the dominant role in causing the DNA damage, at least for DNA single-strand breaks.

Administration, Oral↗

PubMed1994

Syalil-SSEA1(SLX) levels in supernatant of cultured human lung carcinoma cell lines.

SLX levels in the culture supernatant of the following 50 cell lines were measured by RIA: pulmonary carcinoma cell lines derived from 46 patients, cell lines of other human cancers derived from 4 patients, and 2 passaged human fibroblast cells as control. Of the 46 pulmonary carcinoma cell lines, 17 (37%) were SLX positive. When the SLX-positive rate was analyzed in relation to the histological type of pulmonary carcinoma, the positive rate was 71% (10/14) for adenocarcinoma, 27% (3/11) for squamous cell carcinoma, 33% (2/6) for large cell carcinoma, 0% (0/11) for small cell carcinoma and 50% (2/4) for adenosquamous cell carcinoma. Analysis of the relationship between tumor cell proliferation and SLX level in 20 patients revealed that the SLX level in the supernatant of SLX-producing cell lines becomes higher in proportion to the increase in the number of these cells. The SLX-positive rate did not differ significantly among different stages of pulmonary carcinoma at the time of tissue collection. There was no significant correlation between SLX production and prognosis. SLX production by each cell line was not correlated with the doubling time of the same cell line in vitro or in vivo (in nude mice). SLX production also showed no correlation with the duration of tumor cell passage.

Adult↗

PubMed1994

Physicochemical damage to liposomal membrane induced by iron- or copper-mediated lipid peroxidation.

A carboxyfluorescein (CF)-enveloping soybean phosphatidylcholine liposome was used as a model of physicochemical damage of biomembranes. The liposomes were exposed to a metal-chelate complex [2 mM of ferric nitrilotriacetate (FeNTA) or cupric nitrilotriacetate (CuNTA)] plus a reductant (2 mM of ascorbate or various concentrations of reduced glutathione), and CF release from damaged liposomal membranes and the generation of thiobarbituric acid-reactive substances (TBARS) were measured. In the presence of a reducing agent, both FeNTA and CuNTA stimulated markedly CF release and an increase in the TBARS level, while in the absence of a reducing agent both of the chelate complexes showed little CF release and TBARS. The effects of H2O2 addition to the reaction system containing liposome with FeNTA or CuNTA plus ascorbate were also examined. The CF release was slightly increased by the addition of a smaller dose (0.5 mM) of H2O2 and it was inhibited by 8 mM of H2O2. A similar result was obtained in the TBARS test. These results suggest that FeNTA- or CuNTA-mediated lipid peroxidation can damage liposomal membranes physicochemically, and the redox reaction of the chelated metal itself is more important than a Fenton-type reaction in the process.

Ferric Compounds↗

PubMed1994

Ultrastructure and microvasculature of the parotid duct in the cat.

Ultrastructure and microvascular pattern of the parotid duct (main excretory duct) in the cat were investigated by TEM and SEM. The duct was lined by a pseudostratified epithelium which was surrounded by a double-layered vascular sheath. Three kinds of principal cells were observed in this epithelium--tall columnar, light and basal cells. The tall columnar cells did not bear basement membrane specialization (infoldings) but well-developed lateral interdigitations. Microvilli were present on their luminal surface. Basal infoldings and lateral interdigitations did not develop in the light cells. Basal cells were located on the basement membrane of the epithelium. The basal surface of the duct epithelium was studded with half-desmosomes. The external sheath of the double-layered vascular sheath was composed of arterioles and venules, and the inner sheath was formed by a capillary network immediately beneath the epithelium. Meshes of the capillary network in the collecting ducts and the region of origin appeared round and in the superficial and deep regions became elongated along the duct axis. It can be concluded that the parotid duct in the cat not only transports the saliva into the oral cavity, but also actively builds up the primary saliva to the final saliva, owing to absorptive and secretory functions which were made by an important contribution of the double-layered vascular sheath surrounding the whole length of the duct.

Animals↗

PubMed1994

Microvasculature of the lingual papillae in primates and insectivores--fungiform, vallate and foliate papillae.

The microvascular architecture of the fungiform, vallate and foliate papillae was investigated under scanning electron microscope in man, common squirrel monkeys, common marmosets, common tree shrews (primates), large Japanese moles and dwarf shrews (insectivores) utilizing microvascular corrosion casts. The fungiform papilla of the lingual apex in man was supplied by an intrapapillary capillary network with a globular pattern. It was composed of 10-15 capillary loops in a circular arrangement and 5-7 thick capillaries in the center. The fungiform papilla of the lingual body was supplied by a dense capillary network on the top and lateral surfaces. That in other primates was supplied by a cylindrical capillary network and loop formation was seen on the top surface. That in insectivores was supplied by a thin cylindrical network with coarse meshes, at the tips of which were observed 2 or 3 capillary rings in the mole and only one in the dwarf shrew. The vallate papilla in primates was supplied by an intrapapillary capillary network with a globular pattern, and showed irregularly tortuous capillary loops on its top surface. The vallum was supplied by a capillary network in man and usually one or two rows of loops arranged in a circle in other primates. The vallate papilla in insectivores was supplied by a doughnut-like capillary network with a recess on the top surface, and an indistinctly low vallum by a low undulating network. The foliate papillae were most developed in man, and each lobule was supplied by capillaries passing longitudinal to it and capillary loops in 5-10 regular rows on the top, but 3-5 rudimentary lobules in the squirrel monkey and marmoset. In the two species, each lobule was supplied by one arteriole, one venule and a coarse capillary network continuing from the interpapillary network. No foliate papilla was observed, but large conical papillae were noted in the tree shrew and insectivores. In conclusion, the intrapapillary vasculature appeared most complicated in man, simplified in the squirrel monkey and marmoset, and was much more simplified in the insectivores, where it was quite different from that in the primates. The pattern of the intrapapillary vasculature in the tree shrew was transitional between primates and insectivores.

Aged↗

PubMed1994

Microvascular pattern of the retina in the Japanese monkey (Macaca fuscata fuscata).

The microvascular architecture of the retina in the Japanese monkey was revealed using scanning electron microscopy of microvascular corrosion casts. At the optic nerve disc, the central retinal artery radiated to four primary retinal arterioles: the superior and inferior temporal arterioles and superior and inferior nasal arterioles. A dense retinal capillary network showed regional differences in density. The capillary networks observed were divided in four parts: the optic nerve disc, macula, equator, and ora serrata. At the optic nerve disc, the network was a multi-laminar structure containing the radial peripapillary capillaries in parallel with the nerve fibers at the innermost layer. At the macula, the retinal capillaries formed a ring around the central fovea which is an avascular area. At the equatorial part, the network was observed to be double-layered: a superficial capillary network located in the nerve fiber layer and a deep capillary network located at the external nuclear lamina. The former showed a 30-50 microns wide capillary-free zone (CFZ) around arterioles. The latter was undulated in smaller capillaries without the CFZ. At the ora serrata, the network changed to a single layer with coarse ovoid meshes, and the CFZ developed to 50-70 microns in width around arterioles. Hence, the microvasculature of the retina in the Japanese monkey showed regional variations correlating with the fine structure of the retinal layers, e.g., the multi-laminar capillary network for the thick nerve fiber layer at the optic nerve disc part, and the central avascular fovea for the absence of any structure inside the external nuclear lamina. Capillary-free zone occurred since the retinal capillaries were located in the same level as the arterioles.

Animals↗

PubMed1994

[A case of pontine hematoma with Foville's syndrome in childhood].

We reported a surviving case of 6-year-old boy with pontine hematoma. He complained of headache as an initial symptom and developed progressively Foville's syndrome with impairments of the IX-XII cranial nerves. Although brainstem tumor was suspected initially using CT scan, MRI revealed the existence of hematoma in the ventromedial pons. During the first 4 months of his clinical course, Gd-DTPA did not demonstrate any enhancement in that lesion. However, hemangiomatous lesion was suspected by subsequent serial MRIs with positive Gd-DTPA enhancement. Using conservative treatment including oral corticosteroids, all the neurological deficits disappeared in several months and he did not show any recurrence of clinical signs for 3 years. It was suggested that MRI was very useful in the differential diagnosis and the follow-up of hematoma in the posterior fossa.

Accessory Nerve↗

PubMed1994

[A case of idiopathic plasmacytic lymphadenopathy with polyclonal hyperimmunoglobulinemia associated with chronic nephritis].

A 48-year-old female who had general fatigue was admitted to our hospital. She had swelling of the axillary, inguinal, and paraaortic lymph nodes and mediastinal lesions. Laboratory examinations showed anemia, polyclonal hyperimmunoglobulinemia with IgG 5570 mg/dl, renal dysfunction and interstitial changes of the lungs. Microscopic findings of hematoxylin-eosin staining in biopsy specimens of the left inguinal and axillary lymph nodes revealed increased levels of infiltration of mature plasma cells without evidence of malignancy. Immunoperoxidase staining showed intracytoplasmic polyclonal immunoglobulin. These findings were identical to those of idiopathic plasmacytic lymphadenopathy with polyclonal hyperimmunoglobulinemia (IPL) described by Mori et al. (1980). The specimens also showed evidence of chronic nephritis with infiltration of lymph cells and a slight invasion of plasma cells. Accordingly this case was diagnosed as IPL with renal involvement, which is associated with chronic nephritis. Recently, five cases of IPL with renal dysfunction have been reported. In particular, two cases of IPL with renal dysfunction, which included our case, revealed an increased level of IL6. These findings suggest that the occurrence of renal involvement with IPL may be related to changes in IL6, which is an important factor in the pathogenesis of IPL.

Chronic Disease↗