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Biomedical subjects

S Watabe

Publications and source records attributed to S Watabe.

At least 19 recordsLinked to original sources
PubMed1996

Carnosine sustains the retention of cell morphology in continuous fibroblast culture subjected to nutritional insult.

L- Carnosine (beta-alanyl L-histidine), occurring abundantly in skeletal muscles, has been suggested to possess antioxidant and anti-aging properties. Using three different experimental approaches (microscopic, flow cytometric and ELISA for one of the markers of DNA oxidative damage) this study on rat embryonic fibroblasts demonstrates that L-carnosine at 30 mM concentration sustains the retention of cell morphology even during a nutritional insult for five weeks. Also, L-carnosine significantly reduces the formation of 8-hydroxy deoxyguanosine (8-OH dG) in the cells after four weeks of continuous culture. Thus it could be inferred that the anti-senescent effect of L-carnosine is probably linked to its inhibition of formation of intracellular 8-OH dG during oxidative stress.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1996

Peripheral-type benzodiazepine receptors in association with epileptic seizures in EL mice.

Peripheral-type benzodiazepine receptors (PBR) in the brain were studied in association with epileptic seizures using EL mice, an animal model of epilepsy, and DDY mice as controls. Ro 5-4864 (i.p.), a specific agonist for PBR, elicited tonic-clonic convulsions in EL mice 2.6-times more potently than in DDY mice with CD50s of 11.9 and 31.2 mg/kg for EL and DDY mice, respectively. In contrast, pentylenetetrazole (i.p.) exerted convulsant actions on EL and DDY mice in a less differential way with CD50s of 29.2 and 48.1 mg/kg for EL and DDY mice, respectively. PK 11195 (i.v.), a specific antagonist for PBR, raised seizure thresholds of EL mice at a dose of 2 mg/kg. Binding assay revealed a 50% higher density of [3H]Ro 5-4864 binding sites in the mitochondrial fraction isolated from the cerebrum of EL mice in comparison with DDY mice. Similarly, a 40% higher density of [3H]flunitrazepam binding was observed in the mitochondrial fraction of EL mice. The results support the hypothesis that PBR, particularly those associated with mitochondria, are involved in the pathogenesis of epileptic seizures in EL mice.

Animals↗
PubMed1996

Activation of cathepsin D by polyanionic compounds.

We examined the nature of the activation of cathepsin D by polyanionic compounds. Tripolyphosphate, a model compound for polyanions, decreased the Km value of porcine cathepsin D for bovine serum albumin without affecting VMAX. Half-maximal activation was achieved at 0.2 mM free tripolyphosphate. Electrophoretic mobility of cathepsin D decreased as the tripolyphosphate concentration was increased, and the enzyme had no net charge at 50 mM triP. The concentration for the half-maximal mobility change of cathepsin D (0.18 mM) was similar to that for half-maximal activation. These results suggest that tripolyphosphate increased affinity of the enzyme for its substrate by cancelling positive charges on cathepsin D and thus decreasing the electrostatic repulsion.

Adrenal Cortex↗
PubMed1996

Novel actin depolymerizing macrolide aplyronine A.

Aplyronine A is a macrolide isolated from Aplysia kurodai. By monitoring fluorescent intensity of pyrenyl-actin, it was found that aplyronine A inhibited both the velocity and the degree of actin polymerization. Aplyronine A also quickly depolymerized F-actin. The kinetics of depolymerization suggest that aplyronine A severs F-actin. The relationship between the concentration of total actin and F-actin at different concentrations of aplyronine A suggests that aplyronine A forms a 1:1 complex with G-actin. From these results, it is concluded that aplyronine A inhibits actin polymerization and depolymerizes F-actin by nibbling. Comparison of the chemical structure of aplyronine A and another actin-depolymerizing macrolide, mycalolide B, suggests that the side-chain but not the macrolide ring of aplyronine A may account for its actin binding and severing activity.

Actins↗
PubMed1996

Lower activation energy for sliding of F-actin on a less thermostable isoform of carp myosin.

We have examined the temperature-dependence of sliding velocity of fluorescent F-actin on myosins isolated from 10 degrees C- and 30 degrees C-acclimated carp. Activation energies for the sliding of F-actin were 63 and 111 kJ/mol for the 10 degrees C- and 30 degrees C-acclimated carp myosins, respectively. Arrhenius plots of the sliding velocity from 10 degrees C- and 30 degrees C-acclimated carp myosin were shown to intersect at high temperature (about 30 degrees C). The thermostability estimated by measuring the Ca(2-)-ATPase activity was less for myosin from 10 degrees C- than 30 degrees C-acclimated carp. We suggest that a less thermostable structure in cold-acclimated carp myosin results in a reduced activation energy for the contractile process, which allows the F-actin to slide fast even at low temperatures.

Acclimatization↗
PubMed1996

Cellular changes of rat embryonic fibroblasts by an actin-polymerization inhibitor, bistheonellide A, from a marine sponge.

Bistheonellide A, an inhibitor of actin polymerization from the marine sponge Theonella sp., was introduced at a concentration of 100 nM into rat fibroblast of 2.4 x 10(4) cells/ml. Within 1 h, it disrupted stress fibers, accompanied by a marked change of the cell morphology, resulting in the formation of processes from the cell surface. Further incubation for 24 h in the presence of 100 nM bistheonellide A led to binucleation in most cells and subsequent inhibition of cell cycle progression. When bistheonellide A was withdrawn from the culture medium, binuclear cells began to grow again within 20 h and reverted to mononuclear morphology. Flow cytometric analysis fluorescence-activated cell sorting showed that 2C diploid DNA content in G1 phase was changed into 4C content of tetraploid for the bistheonellide A treated-cells in G1 phase and into 8C content during G2 and M phase. Therefore, we suggested that the bistheonellide A treatment inhibited cytokinesis, but not mitosis in M phase, and that treated cells were arrested at the early G1 phase. These effects of bistheonellide A on the cell cycle progression of 3Y1 fibroblast were also observed more prominently in cells synchronized in S phase with hydroxyurea. Cells in G0 phase were then activated by the addition of fetal calf serum in the presence of 100 nM bistheonellide A. Cell cycle progression of the bistheonellide A-treated cells was obviously slowed down or completely inhibited during G1 phase. These results reveal that actin filaments are not only essential to cytokinesis but also for promoting the progression of cell cycle G1 to S phase.

Actins↗
PubMed1996

The cDNA sequence encoding bovine SP-22, a new defence system against reactive oxygen species in mitochondria.

We have isolated cDNA clones coding SP-22, an antioxidant protein in mitochondria, from a bovine adrenal medulla cDNA library constructed with (lambda)gt11. The largest clone contained the entire coding sequence for mature SP-22. Since the isolated cDNA clones lacked 5'- and 3'-ends, we determined the sequences of both ends by the "Rapid Amplification of cDNA Ends (RACE)" tecnique. The deduced amino acid sequence of the mature protein region was the same as that determined by protein sequencing. Since SP-22 had a mitochondrial targetting signal, its mitochondrial localization was confirmed.

Adrenal Medulla↗
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↗