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Biomedical subjects

S Watabe

Publications and source records attributed to S Watabe.

At least 73 records · Page 4Linked to original sources
PubMed1995

Possible function of SP-22, a substrate of mitochondrial ATP-dependent protease, as a radical scavenger.

SP-22 was found to be a substrate protein of a mitochondrial ATP-dependent protease in bovine adrenal cortex. Its amino acid sequence was homologous to that of some prokaryotic and eukaryotic proteins such as thioredoxin peroxidase (formerly called thiol-specific antioxidant) in yeast and mammalian brains and the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium. In the present study, we found SP-22 to have the ability to scavenge reactive oxygen species, thus protecting radical-sensitive proteins such as tryptophan hydroxylase, glutamine synthetase and hemoglobin from oxidation. The protecting activity was enhanced by the addition of horse serum. The "serum factor(s)" seemed to be protein(s), since the physiological roles of SP-22 in adrenocortical mitochondria are discussed.

ATP-Dependent Proteases↗
PubMed1995

The warm temperature acclimation-related 65-kDa protein, Wap65, in goldfish and its gene expression.

cDNAs encoding a warm temperature acclimation-related protein (Wap65) were cloned from the muscle and hepatopancreas cDNA libraries of the warm temperature-acclimated goldfish Carassius auratus, and their nucleotide sequences containing 5'- and 3'-noncoding regions together with their polyadenylation signal were determined. The deduced amino acid sequence of Wap65 was 31% homologous to rat hemopexin. However, goldfish Wap65 lacked a few possible glycosylation sites and presumed functional histidine residues, implying that it may have different functions from hemopexin. Wap65 contained a leader peptide of 30 amino acids and a mature protein region of 415 amino acids. Southern blot analysis demonstrated that the protein is expressed by a single copy gene in the goldfish haploid genome. In RNA blot analysis using isolated cDNA clones, a single transcript of about 2.0 kilobases was detected in the hepatopancreas but not in brain, muscle, or hemocytes. The abundancy of this transcript markedly increased in the hepatopancreas as a result of warm temperature acclimation. Electrophoretic analysis of plasma proteins revealed a good correlation of plasma Wap65 levels to those of the corresponding transcript in the hepatopancreas, suggesting that serum Wap65 concentrations are regulated mainly by transcript levels in the hepatopancreas via the secretion process.

Acclimatization↗
PubMed1995

Temperature acclimation induces light meromyosin isoforms with different primary structures in carp fast skeletal muscle.

Carp acclimated to 10 degrees C gave 69k, 66k, and 62kDa light meromyosin (LMM) fragments in SDS-PAGE, while fish acclimated to 30 degrees C gave 74k, 69k, 66k, and 62kDa fragments. The microsequence analysis revealed that the 69k and 66kDa components from the 10 degrees C-acclimated carp contained an N-terminal amino acid sequence different from that of 62kDa. The four fragments from the 30 degrees C-acclimated carp showed the same sequence as that of the 69k and 66kDa components from the 10 degrees C-acclimated carp, except that the 2nd amino acid, Ala, of the 10 degrees C-acclimated LMM was replaced by Thr. DNA fragments encoding an N-terminal region of LMM were amplified by PCR or reverse transcriptase-PCR, demonstrating that the two acclimated groups further contained several amino acids substituted.

Acclimatization↗
PubMed1995

Differences in the thermal stability of acclimation temperature-associated types of carp myosin and its rod on differential scanning calorimetry.

Differential scanning calorimetry (DSC) was employed for studying the thermal unfolding of myosin and its rod part prepared from carp acclimated to 10 and 30 degrees C. Differences in the thermal stability reflecting structural properties were clearly demonstrated by the DSC data obtained at pH 8.0 in 0.6 M KCl for the two types of carp myosin and rod. The transition temperatures on myosin and rod given by the major peaks for the 10 degrees C-acclimated carp were 33.9 and 47.4 degrees C and 33.0 and 44.0 degrees C, respectively, assuming two endotherms for this type. Since the shape of the first peaks at 33.9 and 33.0 degrees C was not symmetrical, two peaks having similar transition temperatures overlapped in this temperature range. When the data were analyzed using three endotherms, the three transition temperatures obtained for myosin and rod were 32.8, 34.9, and 47.4 degrees C and 32.9, 33.4, and 44.1 degrees C, respectively. Thus, the position of the first peak for the 10 degrees C-acclimated carp myosin did not change even after removal of the large subfragment-1 part, but the transition of the second peak shifted to a lower temperature by about 3 degrees C. The myosin and rod from carp acclimated to 30 degrees C showed three distinct peaks at 35.9, 39.7, and 49.1 degrees C and 34.5, 39.7, and 46.7 degrees C, respectively. The position of the largest peak for myosin remained unchanged, and the shift of the peak position of the highest temperature was about 3 degrees C, as obtained for the 10 degrees C-acclimated carp.(ABSTRACT TRUNCATED AT 250 WORDS)

Acclimatization↗
PubMed1995

In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses.

By the use of recombinant baculoviruses, the trans-cleavage of hepatitis C virus (HCV) non-structural polyprotein was studied. The viral serine proteinase encoded by the NS3 gene was expressed efficiently in insect cells infected with a baculovirus recombined with HCV cDNA corresponding to amino acids 1046-1243 and the signal sequence of the rabies virus G protein. Coinfection studies showed the in vivo trans-cleavage activity of the expressed protein by the use of a recombinant producing NS5 as a substrate. We also found that the partially purified NS3 serine proteinase prepared from the recombinant-infected cells could cleave NS5A/5B substrate. Characterization of the proteinase obtained wil provide basic knowledge on processing of the HCV polyprotein.

Amino Acid Sequence↗
PubMed1995

Relationship between molecular weights of pectin and hypocholesterolemic effects in rats.

Hypocholesterolemic activities and other properties of three different molecular weight pectin were examined. The low-molecular-weight pectin (M(r) not equal to 66,000) obtained by decomposition of original pectin (M(r) not equal to 750,000) had the properties of low viscosity and high solubility, but it lost hypocholesterolemic activities in rats. On the other hand, the medium-molecular-weight pectin (M(r) not equal to 185,000) had characteristics of both low viscosity and hypocholesterolemic activities.

Animals↗
PubMed1994

Mycalolide B, a novel actin depolymerizing agent.

We investigated the effects of a novel marine toxin, mycalolide B, on actin polymerization and actin-activated myosin Mg(2+)-ATPase activity using purified actin and myosin from rabbit skeletal muscle. The results were compared with cytochalasin D which inhibits actin polymerization by binding to the barbed end of F-actin. By monitoring fluorescent intensity of pyrenyl-actin, mycalolide B did not accelerate actin polymerization but quickly depolymerized F-actin, whereas cytochalasin D accelerated actin nucleation and depolymerized F-actin at slower rate. The kinetics of depolymerization suggest that mycalolide B severs F-actin. The relationship between the concentration of total actin and F-actin at different concentration of mycalolide B suggests that mycalolide B forms 1:1 complex with G-actin. Viscometry and electron microscopic observation further suggest that actin filament was depolymerized by mycalolide B. Unlike cytochalasin D, furthermore, mycalolide B suppressed actin-activated myosin Mg(2+)-ATPase activity. We concluded that mycalolide B severs F-actin and sequesters G-actin and may serve as a novel pharmacological tool for analyzing actin-mediated cell functions.

Actins↗
PubMed1994

Structure and expression of mRNA for vitellogenin in Bombyx mori.

Vitellogenin, a precursor of major yolk protein of the silkworm, Bombyx mori is a tetramer composed of each two molecules of heavy and light subunits. We cloned mRNA sequence for the B. mori vitellogenin and analyzed its structure. Sequence alignment of several overlapping cDNA clones indicated that the vitellogenin mRNA is approx. 5.7 kb, containing an open reading frame for a peptide with 1782 amino acid residues. By comparing the deduced amino acid sequence with the amino-terminal primary structures of vitellogenin subunits, it is suggested that the heavy and light subunits of the B. mori vitellogenin are encoded by a single contiguous mRNA. The primary translation product of the vitellogenin mRNA was detected in the microsomal fraction prepared from the fat body of vitellogenic females. Northern blot analysis of the fat body RNA demonstrated that the biosynthesis of vitellogenin in B. mori is regulated in a tissue-, sex- and stage-specific manner at the level of mRNA. Possible cause for discrepancy between the present results and our previous proposal (Izumi, S. and Tomino, S. (1983) Insect Biochem. 13, 81-85) on the biosynthesis of B. mori vitellogenin is also discussed.

Amino Acid Sequence↗
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↗