Biomedical subjects

S Watabe

Publications and source records attributed to S Watabe.

At least 55 records · Page 3Linked to original sources

PubMed1998

[Mitochondrial benzodiazepine receptors (MBR) in association with neurological disorders].

Ro 5-4864, a specific agonist of the peripheral-type benzodiazepine receptor (PBR), elicited convulsions 2.6 times more potently in EL mice (an animal model of epilepsy) than in DDY mice (control animal). A binding assay revealed a 50% higher density of [3H] Ro 5-4864 binding sites in the mitochondrial fraction (i.e., mitochondrial benzodiazepine receptors; MBR) of the brain tissues in EL mice as compared with DDY mice. On an elevated plus-maze, EL mice showed fear responses similar to those increased in DDY mice after PBR stimulation, suggesting a hyperfunction of MBR underlying the abnormal behaviors of EL mice. In fluorometric studies using NG108-15 cells, Ro 5-4864 depolarized mitochondrial membranes and, possibly as a consequence of this, raised intracellular Ca2+. Finally, we propose that MBR could be a major target of therapy for various neurological disorders, so drugs such as "mitochondrial membrane stabilizers" should be developed.

Animals↗

PubMed1998

Molecular cloning and developmental expression patterns of the MyoD and MEF2 families of muscle transcription factors in the carp.

cDNA clones encoding the myogenic regulatory factors (MRFs) myogenin, MyoD and myf-5 were isolated by reverse-transcription polymerase chain reaction from larvae and embryos of the common carp (Cyprinus carpio L.). Myocyte-specific enhancer factor 2 (MEF2) cDNAs were identified from a cDNA library from adult carp. Northern blot analysis showed that MyoD, myf-5 and MEF2C transcripts were present in three-somite embryos, whereas myogenin and MEF2A transcripts were not detected until the 15-somite stage. Intense signals of myogenin and MyoD transcripts were observed even in 1-month-old juveniles. Levels of MyoD, myogenin and MEF2A transcripts declined between 1 and 7 months after hatching, and myf-5 gave only a weak signal in the oldest fish. In contrast, levels of MEF2C transcripts were considerably higher in 7-month-old juveniles than in 1-month-old larvae. mRNAs encoding carp myosin heavy chain and alpha-actin were first detected at approximately the time of the first heartbeat, and levels were maximal in juveniles 1 month post-hatching. The relatively high levels of MRF mRNA in juvenile fish probably reflect the recruitment of new muscle fibres from the satellite cell population. It was concluded that the relative importance of the different members of the MyoD and MEF2 families of transcription factors for muscle differentiation changes during ontogeny in the carp.

Amino Acid Sequence↗

PubMed1998

Two types of mRNA encoding myosin regulatory light chain in carp fast skeletal muscle differ in their 3' non-coding regions and expression patterns following temperature acclimation.

cDNA clones encoding the myosin regulatory light chain (RLC) were isolated from a cDNA library prepared from fast skeletal muscle of the carp Cyprinus carpio L. Two types of cDNA clone encoding carp RLC were found with identical deduced amino acid sequences. The two mRNAs differed in the number of polyadenylation signals prior to the poly(A) tail in the 3' non-coding region. The two mRNA species, with approximate sizes of 1.4 and 0.8 kilobases, were also observed in northern blot analysis. Carp were acclimated for a minimum of 5 weeks to either 10 degrees C or 30 degrees C (14h:10h light:dark photoperiod). The total levels of mRNA transcripts coding for the RLC and myosin heavy chain were, respectively, 3.3 and 3.9 times higher in cold-than in warm-acclimated fish. Differences in the levels of RLC in mRNA transcripts were largely due to the concentration of the 1.4 kilobase mRNA species.

Adaptation, Physiological↗

PubMed1997

A patient with a traumatic right diaphragmatic hernia occurring 4 years after sustaining injury--statistical observations of a delayed diaphragmatic hernia caused by uncomplicated injury in Japan.

We describe our experience with a patient in whom a traumatic right diaphragmatic hernia developed 4 years after sustaining injury and review cases of delayed diaphragmatic injury reported in Japan. The patient was a 28-year-old man who sustained a severe contusion of the right epigastric region and fractured a right rib in a traffic accident in September 1992. In August 1996, the patient presented with shortness of breath on effort or after meals. A chest roentgenogram revealed intestinal gas in the right side of the thoracic cavity. A right diaphragmatic hernia was diagnosed on the basis of a gastrointestinal series, and the patient was operated on. The hernial orifice extended anteriorly from the central tendon in an 11:00 direction and measured 11 x 6 cm. The small intestine, right side of the colon, and liver were herniated. A total of 297 cases of blunt traumatic diaphragmatic hernia were reported in Japan between 1981 and 1996, including 47 cases (left side, 32 cases; right side, 15 cases) of delayed diaphragmatic hernia, defined as occurring one month or more after injury. Diaphragmatic hernia should be considered as a possible diagnosis in patients with abnormal shadows in the thoracic region who have recently sustained injury or who have a past history of injury.

Abdominal Injuries↗

PubMed1997

SP-22 is a thioredoxin-dependent peroxide reductase in mitochondria.

SP-22 is a mitochondrial antioxidant protein in bovine adrenal cortex. The protein is homologous to thioredoxin peroxidase and other antioxidant proteins. It protects radical-sensitive enzymes from oxidative damage by a radical-generating system (Fe2+/dithiothreitol) in the presence of a small amount of serum. In this study we purified a second mitochondrial protein with Mr 11,777, which cooperates with SP-22 to protect glutamine synthetase and other proteins from Fe2+/dithiothreitol-mediated damage. Without SP-22, the protein had no protecting activity. We determined amino acid and nucleotide sequences of the protein and its cDNA, respectively, and found that it was a protein of the thioredoxin family. The protein, designated as mt-Trx (mitochondrial thioredoxin), had a presequence composed of 59 amino acids that seemed to be a mitochondrial targeting signal. Mitochondrial extract prepared from adrenal cortex contained NADPH-dependent 5,5'dithiobis(2-nitrobenzoic acid) (Nbs2) reductase activity. The enzyme was thought to have thioredoxin reductase activity, since the Nbs2-reducing activity was stimulated by mt-Trx. We partially purified the Nbs2 reductase from bovine adrenocortical mitochondria. In the presence of the partially purified reductase, mt-Trx, and NADPH, SP-22 showed the activity to protect oxyhemoglobin against ascorbate-induced damage. Furthermore, with the three protein components (Nbs2 reductase, mt-Trx, and SP-22) NADPH was oxidized in the presence of hydrogen peroxide or tert-butyl hydroperoxide. The oxidation of NADPH was concomitant with the disappearance of an equimolar amount of hydrogen peroxide. Without any one of the protein components no hemoglobin-protecting and peroxide-dependent NADPH-oxidizing activities were observed. From these results we concluded that SP-22 is thioredoxin-dependent peroxide reductase or so-called thioredoxin peroxidase in mitochondria from the adrenal cortex.

Adrenal Cortex↗

PubMed1997

Differential scanning calorimetry and CD spectrometry of acclimation temperature-associated types of carp light meromyosin.

Differential scanning calorimetry and CD spectrometry were employed to study the thermal unfolding of light meromyosin (LMM) prepared from carp acclimated to different temperatures. The transition temperatures given by the major peaks at pH 8.0 in 0.6 M KCl for LMM from carp acclimated to 10 degrees C were 32.5 and 39.5 degrees C with the calorimetric enthalpies (DeltaHcal) of 269 and 52 kcal/mol, respectively. LMM from carp acclimated to 20 degrees C exhibited three peaks of transition temperatures at 34.5, 40.2, and 46.9 with DeltaHcal of 152, 20, and 10 kcal/mol, respectively. On the other hand, LMM from carp acclimated to 30 degrees C showed two different patterns. The first experiment gave two transition temperatures at 39.2 and 47.3 degrees C with DeltaHcal of 231 and 39 kcal/mol, respectively. The second series of experiments resulted in showing three peaks of 34.4, 39.5, and 47.5 degrees C with DeltaHcal of 117, 123, and 28 kcal/mol, respectively. N-terminal amino acid sequence analysis revealed that LMM at the second series of experiments with the 30 degrees C-acclimated carp contained component(s) predominant in the 20 degrees C-acclimated carp. Thermal unfolding responsible for these transition temperatures was well explained by melting of alpha-helices which could be determined by far-ultraviolet CD spectroscopy. These results clearly demonstrate that the 30 degrees C-acclimated carp contained the most thermostable LMM.

Acclimatization↗

PubMed1997

Structural differences in the crossbridge head of temperature-associated myosin subfragment-1 isoforms from carp fast skeletal muscle.

We determined the primary structures of the three acclimation-temperature-associated isoforms of myosin subfragment-1 heavy chain from fast skeletal muscle of thermally acclimated carp. These isoforms were cloned by extending 5'-regions of cDNAs that encode the rod part of myosin heavy chain specifically expressed in 10 degrees C- and 30 degrees C-acclimated carp, together with the region that encodes an intermediate structure [Imai, J., Hirayama, Y., Kikuchi, K., Kakinuma, M. & Watabe, S. (1997) J. Exp. Biol. 200, 27-34]. These three isoforms generally resembled each other in primary structure, showing 94.8, 90.9, and 92% similarity between the 10 degrees C- and intermediate-type, between the 10 degrees C- and 30 degrees C-type, and between the intermediate- and 30 degrees C-type myosin heavy chains, respectively. However, isoform-specific differences were clearly observed between the 10 degrees C- and 30 degrees C-type heavy chains in the first 60 amino acid residues from the N-terminus, where the intermediate-type showed an intermediate feature in its sequence compared to the 10 degrees C- and 30 degrees C-type isoforms. Other striking differences were observed in two surface loops between the 10 degrees C- and 30 degrees C-type isoform. Five amino acid residues out of sixteen were different in loop 1 near the ATP-binding pocket, and six out of twenty were different in loop 2 on the actin-binding site. The loops connecting beta-sheets that are known to surround the ATP-binding pocket were highly conserved in primary structure for the three types. In northern blot analysis, the accumulated mRNA levels of the 10 degrees C- and intermediate-type isoforms were significantly higher in carp acclimated to 10 degrees C and 20 degrees C than carp acclimated to 30 degrees C, whereas the level of the 30 degrees C-type isoform was significantly higher in carp acclimated to 30 degrees C than those acclimated to 10 degrees C and 20 degrees C.

Amino Acid Sequence↗

PubMed1997

Structure of fucose branches in the glycosaminoglycan from the body wall of the sea cucumber Stichopus japonicus.

Fucose-branched chondroitin sulfate E was prepared from the body wall of sea cucumber Stichopus japonicus. The purified glycosaminoglycan (GAG) was chemically desulfated, followed by carboxyl reduction. Intact, desulfated, and desulfated/carboxyl-reduced GAG fractions were subjected to per-O-methylation. GC-MS analyses of the resultant partially methylated alditol acetates demonstrated that the fucose branch is formed by two fucopyranosyl residues linked glycosidically through position (1-->3), and that the fucose branch and glucuronic acid are almost equimolar. In addition, it was elucidated that about 20% of the branches stretch from O-3 position of a glucuronic acid moiety of the core chondroitin sulfate polymer, while remaining fucose branches are postulated to protrude from O-4 and/or O-6 position(s) of a N-acetylgalactosamine moiety. This fucose branch was also confirmed to be highly sulfated according to six kinds of substitution pattern in methylation analysis.

Acetylgalactosamine↗

PubMed1997

Cellular mechanisms underlying cognition-enhancing actions of nefiracetam (DM-9384).

We have studied cellular mechanisms underlying cognition-enhancing actions of nefiracetam (DM-9384), a newly developed cognitive enhancer, by biochemical experiments on cholinergic and GABAergic transmissions as well as electrophysiological experiments on neuronal Ca2+ channels. In behavioral experiments in rats, nefiracetam (3 mg/kg) ameliorated amnesia induced by basal forebrain (BF) lesion or treatment of scopolamine. Biochemical experiments revealed that nefiracetam increased uptake and release of transmitters in both cholinergic and GABAergic systems in rat brain. In electrophysiological studies, nefiracetam (1 microM) increased long-lasting (N/L-type) Ca2+ channel currents in NG108-15 cells. The nefiracetam action on Ca2+ channels was blocked by pertussis toxin (PTX). The results suggest that nefiracetam improves impaired memory by facilitating cholinergic and GABAergic transmissions in the brain. It is further suggested that PTX-sensitive G-proteins and Ca2+ channels associated with these G-proteins are responsible for the action of nefiracetam on neurotransmission.

Animals↗

PubMed1997

Phosphorylation of a high molecular weight (approximately 600 kDa) protein regulates catch in invertebrate smooth muscle.

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called 'catch'. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of approximately 600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the approximately 600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of approximately 600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the approximately 600 kDa protein regulates catch.

Animals↗

PubMed1997

Autolytic activation mechanism of Bombyx acid cysteine protease (BCP).

Acid cysteine proteinase in the eggs of the silkmoth, Bombyx mori, exists as an inactive proenzyme. This 47-kDa pro-BCP1 zymogen molecule can be processed in vitro into an enzymatically active 39-kDa BCP molecule. In this current study, the maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 37 degrees C under reducing conditions. The rate of conversion was not affected by increasing concentrations of pro-BCP. We prepared immobilized BCP bound to AH-Sepharose and examined the activation. Immobilized pro-BCP was autolysed, although the rate of processing was slow, indicating that the reaction might be an intramolecular one. Kinetic experiments suggest that the mechanism is likely to involve a stepwise reaction, in which pro-BCP is converted to an active enzyme through intermediate forms releasing small peptides stepwise. The results suggest that autocatalytic cleavage (intramolecular) is a major processing step in the early stage of pro-BCP activation.

Amino Acid Sequence↗

PubMed1997

Sister Joseph's nodule: a case report and review of the Japanese literature.

We report a case of metastatic carcinoma on the umbilicus, which is known as Sister Joseph's nodule. A 65-year-old man with the chief complaint of a painful nodule on the umbilicus was referred to our hospital. Histopathological examination revealed moderately differentiated tubular adenocarcinoma in the dermis. Endoscopic examination found gastric cancer of Borrmann type IV. The umbilical lesion represented a metastasis from the gastric cancer. We reviewed 80 cases of metastatic carcinomas to the umbilicus that have been reported in the Japanese literature.

Adenocarcinoma↗

PubMed1997

cDNA cloning of myosin heavy chain isoforms from carp fast skeletal muscle and their gene expression associated with temperature acclimation.

We have isolated cDNA clones encoding fast skeletal muscle myosin heavy chains of carp acclimated to 10, 20 and 30 degrees C for over 5 weeks. All clones covered at least the full length of L-meromyosin, the C-terminal part of the myosin molecule. Nucleotide sequence analysis on cDNA clones showed three types of 3' untranslated sequences, demonstrating that carp expresses at least three myosin heavy chain isoforms in fast skeletal muscle in an acclimation-temperature-dependent manner. cDNAs were identified which were the predominant types expressed in 10 degrees C- and 30 degrees C-acclimated fish, as well as an intermediate type present at all acclimation temperatures. Northern blot analysis using probes of three kinds of DNA fragments from the 3' untranslated region of carp acclimated to 10, 20 and 30 degrees C further confirmed the presence of acclimation-temperature-specific isoforms. In addition, it was found that mRNA levels of three isoforms were altered in an acclimation-temperature-dependent manner. When the deduced amino acid sequences of three types of carp L-meromyosin were compared with those of homoiotherms, the 30 degrees C-acclimated type was more similar to those of homoiotherms than was the 10 degrees C-acclimated type.

Acclimatization↗

PubMed1997

The two essential light chains of carp fast skeletal myosin, LC1 and LC3, are encoded by distinct genes and change their molar ratio following temperature acclimation.

cDNA libraries were constructed from fast skeletal muscles of carp acclimated to 10 and 30 degrees C for a minimum of 5 weeks and were screened for myosin alkali light chains, LC1 and LC3, using an anti-skipjack LC1 polyclonal antibody. Two types of LC1 cDNA clone were isolated and termed LC1a and LC1b: their nucleotide sequences showed 92% homology. The ratio of LC1a to LC1b cDNA clones isolated was approximately 3:1, showing no apparent changes following temperature acclimation. The occurrence of the two isoforms was further confirmed by N-terminal amino acid sequencing of purified LC1. No isoform was, however, detected for LC3, while homology in the overlapping region between LC1a and LC3 cDNAs was only 65% even after the most probable alignment. Southern blot analyses probed with cDNA clones specific to LC1a and LC3 showed different hybridization patterns from each other, demonstrating that carp LC1 and LC3 are encoded by different genes. These results are in marked contrast to those from higher vertebrates which express LC1 and LC3 from a single gene by alternative RNA transcription and two modes of splicing. Northern blot analysis showed that the ratios of LC3/LC1 mRNAs were significantly higher (3.93) in 30 degrees C-acclimated than in 10 degrees C-acclimated (3.10) carp.

Amino Acid Sequence↗

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↗