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Biomedical subjects

S Watabe

Publications and source records attributed to S Watabe.

At least 91 records · Page 5Linked to original sources
PubMed1994

Enhancement of neuronal calcium channel currents by the nootropic agent, nefiracetam (DM-9384), in NG108-15 cells.

Effects of nootropic agents on neuronal calcium channels were studied in NG108-15 cells using the whole-cell patch-clamp technique. Nefiracetam (DM-9384) at a concentration of 1 microM increased a long-lasting component of calcium channel currents two-fold without affecting a transient component. The dose-response relationship yielded a bell-shaped curve with a peak at 1 microM. Similar, but slightly less potent effects were observed by aniracetam. Dibutyryl cyclic AMP (1 mM) also enhanced the currents, which were not further increased by nefiracetam, or vice versa. The currents enhanced by nefiracetam were markedly reduced by nifedipine (10 microM), an 'L-type' calcium channel blocker. Cells treated with pertussis toxin (PTX; 500 ng/ml, > 20 h) to inactivate inhibitory G-proteins were apparently insensitive to nefiracetam. The results suggest that the nootropic agents may enhance the activity of neuronal L-type calcium channels under the regulation of inhibitory G-proteins and possibly, cyclic AMP-dependent processes.

Animals↗
PubMed1994

Carp parvalbumin binds to and directly interacts with the sarcoplasmic reticulum for Ca2+ translocation.

Interaction between two relaxing factors, the sarcoplasmic reticulum and parvalbumin, in carp fast skeletal muscle was investigated. Immunoblotting using an anti-parvalbumin antibody revealed that parvalbumin bound to the light sarcoplasmic reticulum isolated from carp fast skeletal muscle in the presence of Ca2+. Parvalbumin enhanced Ca2+ uptake activity of the light sarcoplasmic reticulum. Furthermore, using a photoreactive cross-linker, we detected a protein in the light sarcoplasmic reticulum which bound to parvalbumin in a Ca(2+)-dependent manner. These results suggest that parvalbumin may directly interact with the sarcoplasmic reticulum in contraction-relaxation cycle of carp fast skeletal muscle.

Animals↗
PubMed1994

Purification and characterization of a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex.

We have purified SP-22, a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex. Native SP-22 showed an M(r) of 350,000 +/- 20,000, and was composed of more than 10 molecules of an M(r) 21,600 subunit. Subcellular and submitochondrial fractionation of adrenocortical tissues revealed that SP-22 was localized in the mitochondrial matrix, suggesting that SP-22 is a natural substrate for ATP-dependent protease, a matrix enzyme. The concentration of SP-22 in adrenocortical mitochondrial fractions was 16 +/- 3 micrograms/mg proteins (mean +/- SD, n = 6) as determined by radioimmunoassay using specific anti-SP-22 antibody. Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla. We determined the amino acid sequence of SP-22, which is composed of 195 amino acids. Amino acid 47 was not identified by the sequencer. FAB-mass spectrometry of AA47-AA55 fragment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H). By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% homologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation. SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium, thiol-specific antioxidant in Saccharomyces cerevisiae, and some other proteins. Since a segment around AA47 was highly conserved, this residue may be important for the biochemical functions of SP-22.

ATP-Dependent Proteases↗
PubMed1994

Myosin subfragment-1 isoforms having different heavy chain structures from fast skeletal muscle of thermally acclimated carp.

Three heavy chain isoforms of chymotryptic myosin subfragment-1 (S1) with different molecular sizes of 96 kDa (H1), 94 kDa (H2), and 92 kDa (H3), were detected in the fast skeletal muscle from thermally acclimated carp. In total, six S1 isoforms were present, including two S1 isoforms for each heavy chain due to associated A1 and A2 light chains. H1 heavy chain was dominant in the 10 degrees C-acclimated carp and responsible for high acto-S1 Mg(2+)-ATPase activity and low thermostability. In contrast, H3 heavy chain predominating in the 30 degrees C-acclimated carp showed low acto-S1 Mg(2+)-ATPase activity and high thermostability. H2 heavy chain was found in the 10- and 20 degrees C-acclimated fish. H3 heavy chain featured three tryptic fragments with normal molecular masses of 25, 50, and 20 kDa in order from the N-terminus. However, H1 heavy chain contained an unusual, longer "20 kDa" peptide whose molecular size was estimated to be about 23 kDa.

Adenosine Triphosphatases↗
PubMed1994

Cloning and sequencing of an ice nucleation active gene of Erwinia uredovora.

An ice nucleation activity gene, named inaU, of the bacterium Erwinia uredovora KUIN-3 has been sequenced. This gene encodes a protein of 1034 amino acid residues, and its expression product, inaU protein, has an 832-amino acid residue segment consisting of 52 repeats of closely related 16-amino acid motifs (R-domain), flanked by N- and C-terminal sequences (N- and C-domains, respectively). The primary structure of the inaU protein is similar to those of the inaA, inaW, and inaZ gene products of Erwinia ananas, Pseudomonas fluorescens, and Pseudomonas syringae, respectively, but is smaller than any of these products in terms of the size of the R-domain.

Amino Acid Sequence↗
PubMed1994

Improvement of impaired brain monoamine metabolism by the cognition-enhancing agent nefiracetam after microsphere-induced cerebral embolism in rats.

Nefiracetam (N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide DM-9384, CAS 77191-36-7), a pyrrolidone (cyclic GABA) derivative, is a newly developed cognition-enhancing (nootropic) agent. In the present study, the effects of nefiracetam on cerebral monoamine metabolism in rats after microsphere-induced cerebral embolism have been examined. For cerebral embolization, microspheres were injected into the left internal carotid artery. Nefiracetam (3, 10 and 30 mg/kg p.o.) was administered daily to the animals for 13 days from the 9th day after the operation. The levels of DA, DOPAC and HVA in the control (embolism-induced, but untreated) group were significantly decreased compared with those of the normal (non-operated) group. In animals treated with nefiracetam (3 mg/kg p.o.), these monoamine levels were higher than controls in the cortex and hippocampus, whereas they were not significantly changed in the striatum. 5-HT contents in the control group significantly decreased from normal levels in the cortex and hippocampus, on which nefiracetam at various doses had no effect. The levels of 5-HIAA in the control group also decreased, which were, however, significantly increased by 3 mg/kg of nefiracetam. The results suggest that nefiracetam has improving actions on the dysfunction of the dopaminergic and serotonergic systems induced by cerebral embolism.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

DM-9384, a new cognition-enhancing agent, increases the turnover of components of the GABAergic system in the rat cerebral cortex.

DM-9384 (nefiracetam) (N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide), a pyrrolidone derivative (or a cyclic derivative of gamma-aminobutyric acid (GABA)), is a newly developed nootropic (or cognition-enhancing) agent. In the present study, we examined the biochemical effect of DM-9384 on GABAergic neurons in adult rat brains. DM-9384, when administered orally at a daily dose of 10 mg/kg for 7 days, significantly increased GABA turnover and glutamic acid decarboxylase activity in the cortex and hippocampus, and stimulated Na(+)-dependent high-affinity GABA uptake in cortical synaptosomes. In in vitro experiments, the K(+)-evoked release of [14C]GABA from cortical slices was markedly increased by low concentrations (10(-8), 10(-9) M) of DM-9384. The binding of GABAA and benzodiazepine to their receptors in the brain was not affected by DM-9384 (10(-10)-10(-3) M). The results suggest that DM-9384 increases the turnover of components of the GABAergic system by influencing presynaptic sites rather than postsynaptic sites.

Administration, Oral↗
PubMed1993

Differential inhibition of transient and long-lasting calcium channel currents by benzodiazepines in neuroblastoma cells.

The effects of diazepam, nitrazepam, clonazepam, and Ro5-4864 on transient (type I) and long-lasting (type II) calcium channels associated with low-affinity benzodiazepine receptors were investigated using the whole-cell patch-clamp technique. Clonazepam (100 microM), a specific agonist for the central-type benzodiazepine receptor, reduced transient currents through the type I calcium channel by 40% without affecting long-lasting currents through the type II calcium channel. Diazepam and nitrazepam (100 microM), non-specific agonists for both the central- and peripheral-type benzodiazepine receptors, reduced both transient and long-lasting currents equally by 25-30%. A similar non-selective inhibition was observed by Ro5-4864 (1-10 microM), a specific agonist for the peripheral-type benzodiazepine receptor. It is concluded that the two calcium channel types are regulated differentially by two different kinds of benzodiazepines; central-type for type I channel and peripheral-type for both type I and type II channels.

Animals↗
PubMed1993

Subunit structure of multiple hemoglobins in carp.

Three hemoglobin components in carp designated CI, CII, and CIII, were isolated by DEAE-Tokyo-pearl ion-exchange chromatography. Constituent globin chains, alpha 1, alpha 2, beta 1, and beta 2, were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography with a reverse-phase column. Tryptic peptide mapping indicated that the alpha-globin chains of the three hemoglobin components have slightly different structures. In addition, N-terminal amino acid sequence analysis indicated that the beta 1-globin chain has a primary structure different from that of the beta 2-chain. A series of hybridization experiments between isolated hemoglobins, together with such structural properties of globin chains, suggested that the three hemoglobins have the following compositions: CI (alpha 1 alpha 2 beta 1(2)), CII (alpha 1 alpha 2 beta 1 beta 2), and CIII (alpha 1 alpha 2 beta 2(2)). Hemoglobin CII was a hybrid between the two types each of alpha- and beta-chain and could be constructed in vitro from two hemoglobin components CI and CIII.

Amino Acid Sequence↗
PubMed1993

In vitro degradation of mitochondrial proteins by ATP-dependent protease in bovine adrenal cortex.

We have examined in vitro degradation of mitochondrial proteins by ATP-dependent protease in bovine adrenal cortex. Purified ATP-dependent protease degraded at least five proteins in vitro in the presence of ATP and Triton X-100 using mitochondrial fraction as a substrate. Two of the degraded proteins were identified as P-450scc and adrenodoxin reductase by immunoblotting. No degradation of P-45011 beta or adrenodoxin was detected. Purified P-450scc and adrenodoxin reductase were also degraded. By analyzing degradation products of P-450scc we identified 49 peptides and 59 cleavage sites. The size of the products was between 3 and 18 amino acid residues. The protease preferentially cleaved the carboxy-side of Leu, Phe, Ala, Val, Met, and some other amino acids. Since the protease (a matrix enzyme) is accessible to P-450scc and adrenodoxin reductase localized on the matrix side of inner membrane, the present results suggest that the protease might participate in the turnover of these two proteins and some other mitochondrial proteins.

ATP-Dependent Proteases↗
PubMed1993

Large-scale production and purification of an Erwinia ananas ice nucleation protein and evaluation of its ice nucleation activity.

The ice nucleation-active protein of Erwinia ananas IN-10 (inaA protein) was over-expressed as inclusion bodies in Escherichia coli in a yield of 15.3 mg of inaA protein from 60 mg of bacterial cells on a dry-matter basis. The inaA protein was purified from the inclusion bodies by solubilization with detergents to obtain a protein preparation free from sugar and lipid. This preparation had a distinct ice nucleation activity, indicating that the inaA protein per se is able to act as a nucleus.

Amino Acid Sequence↗
PubMed1992

Fast skeletal myosin isoforms in thermally acclimated carp.

Fast skeletal myosins were isolated from carp acclimated to 10 and 30 degrees C, and their structural and enzymatic properties were compared. Myosins in 0.5 M KCl were subjected to limited proteolysis by using various proteases including alpha-chymotrypsin, trypsin, and papain, and different SDS-PAGE patterns were seen for the 10- and 30 degrees C-acclimated myosins in all cases. Myosin subfragment-1 (S1) prepared from the 10 degrees C-acclimated myosin by alpha-chymotryptic digestion in 0.12 M NaCl showed higher acto-S1 Mg(2+)-ATPase activity and lower thermostability than S1 from the warm-acclimated myosin. The peptide maps and ATP-induced spectral changes of tryptophan fluorescence also showed an obvious difference between the two types of S1. Temperature acclimation further caused changes in the rod region of myosin, since the apparent sizes of light meromyosin were different from each other for the two types of myosin. Myosin from carp acclimated to 20 degrees C showed intermediate properties between those of the 10- and 30 degrees C-acclimated myosins. Myosin isoforms might be expressed in a temperature-dependent manner to compensate for the effect of seasonal environmental temperature variation on swimming ability.

Acclimatization↗
PubMed1992

[A case of gastric carcinoma with peritonitis carcinomatosa responding remarkably to etoposide, adriamycin and cisplatin (EAP) therapy].

The patient was a 62-year-old male who had Borrmann 4 type gastric cancer. He presented massive ascites due to peritonitis carcinomatosa and the cytology of ascites was class V. He was treated with 3 courses of EAP (etoposide, adriamycin, cisplatin) therapy. Computed tomography showed ascites nearly disappeared. Remarkable improvement was observed by barium meal study and endoscopic examination, and partial remission (PR) was achieved. As for toxicity bone marrow suppression, alopecia and elevation of BUN were observed.

Adenocarcinoma↗
PubMed1992

Effects of the new cognition-enhancing agent nefiracetam in rats with cerebral embolism.

The effects of nefiracetam (DM-9384, CAS 77191-36-7) on the learning behavior and cholinergic and GABAergic neuronal transmitter systems of rats with experimentally-induced cerebral embolism were investigated. Cerebral embolisms were induced in male Wistar rats by injection of 800 microspheres 50 microns in diameter via the left internal carotid artery under 2% halothane anesthesia. Daily oral administration of nefiracetam (30 mg/kg/d) was started 9 days after embolization. Nefiracetam caused significant (p < 0.05) improvement of deficits in the learning of both water maze and passive avoidance tasks beginning 22 days after embolization of the rats. The drug also significantly restored decreases in cortical choline acetyltransferase (p < 0.05) and hippocampal glutamic acid decarboxylase activities (p < 0.01) in the embolized cerebral hemisphere and significantly increased cortical choline acetyltransferase (p < 0.05) and acetylcholinesterase activities (p < 0.05) in the contralateral cerebral hemisphere 21 days after embolization. These results demonstrate that nefiracetam improves cognitive dysfunction in the late phase in embolized rats and suggest that the effect is at least partly due to the increase in glutamic acid decarboxylase, choline acetyltranseferase and acetylcholinesterase activities.

Acetylcholinesterase↗
PubMed1991

Calyculin-A increases the level of protein phosphorylation and changes the shape of 3T3 fibroblasts.

Calyculin-A, an inhibitor of type 1 and 2A phosphatases, was applied extracellularly to 3T3 fibroblasts. At 0.1 microM, calyculin-A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phosphorylation following treatment with calyculin-A was observed in vimentin, the 20-kD myosin light chain, and an unidentified 440-kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells. Calyculin-A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin-A (0.1 microM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin-A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin-A an unusual "ball-like" structure was observed with transmission electron microscopy. This unique structure was 2-3 microM in diameter and was located close to the nucleus. The use of calyculin-A adds further support to the idea that cell shape is controlled, at least in part, by concerted actions of a kinase-phosphatase couple.

Animals↗
PubMed1991

Purification and characterization of a calsequestrin-like calcium-binding protein from carp (Cyprinus carpio) sarcoplasmic reticulum.

1. A calsequestrin-like calcium-binding protein was purified from carp sarcoplasmic reticulum by column chromatographies using DEAE-cellulose and Butyl-Toyopearl 650S. 2. The mol. wt was estimated to be 50 kDa, which was larger than that of rabbit calsequestrin (42 kDa). 3. Carp calsequestrin-like protein bound Ca2+ with a higher affinity (apparent Kd = 400 microM) and lower capacity (25 mol/mol) compared with rabbit calsequestrin (1 mM and 40-50 mol/mol, respectively). 4. Anti-carp calsequestrin-like protein rabbit antiserum reacted with rabbit calsequestrin in immunoblotting analysis. 5. Carp calsequestrin-like protein was rich in acidic amino acids, as was rabbit calsequestrin.

Amino Acids↗
PubMed1991

Improvement of postoperative hypocalcemia by repeated allotransplantation of parathyroid tissue without anti-rejection therapy.

Long lasting postoperative hypocalcemia, an uncomfortable complication of a thyroid operation for hyperthyroidism, was treated with allotransplantation of parathyroid tissue. Small pieces of the parathyroid tissue offered from two unrelated donors were transplanted to an 18-year-old male with severe postoperative hypoparathyroidism. Prednisolone was given for 2 days, but no other immunosuppressive drugs were used. The remaining tissue was stored in frozen for the repeat transplantation. The functional activity of the frozen tissue was determined by the production of parathyroid hormone in the tissue culture medium adjusted to appropriate concentration of calcium. Loss of the graft function, probably due to rejection, was supplemented with repeated grafting. Hypocalcemia was improved by three times of transplantation using frozen tissue (once) and fresh tissue (twice). This preliminary trial demonstrates that the tissue transplantation of the parathyroid gland is effective to lessen the symptoms and medication of postoperative hypoparathyroidism.

Adolescent↗