Search PubMed⌕ Search

Biomedical subjects

T Masaki

Publications and source records attributed to T Masaki.

At least 487 records · Page 27Linked to original sources
PubMed1987

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin.

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.

Amino Acid Sequence↗
PubMed1987

Recognition of 5' and 3' splice site sequences in pre-mRNA studied with a filter binding technique.

A nuclear extract from HeLa cells was fractionated by DEAE-Sepharose chromatography. The obtained fractions were assayed for binding to an RNA transcript carrying a splice site sequence of 9-16 nucleotides by a filter binding technique. The U1 RNA-rich small nuclear ribonucleoprotein (snRNP) fractions, which showed binding activities for both 5' and 3' splice site RNAs, were studied for the sequence specificity of their binding. Results indicate that the U1-rich snRNP fraction can recognize both 5' and 3' splice site sequences. The U1 RNP, which was highly purified from the snRNP fractions, bound to at least some 5' splice site sequences, but not to a consensus 3' splice site sequence. Therefore, purified U1 RNP can directly recognize a 5' splice site, but not a 3' splice site. The binding activity for the 5' splice sites was lost either by digestion with micrococcal nuclease or by digestion of the 5' end of U1 RNA with RNase H and a complementary oligodeoxynucleotide, indicating the involvement of U1 RNA. Involvement of a protein moiety as well in this binding was suggested by the loss of binding activity upon heating at 60 degrees C. The binding activity to a 3' splice site sequence was not sensitive to digestion by micrococcal nuclease and was removed by protein A-coupled anti-Sm antibody. This activity was found in sucrose gradient fractions of about 8 S.

Base Sequence↗
PubMed1987

Amino acid sequence of thermostable direct hemolysin produced by Vibrio parahaemolyticus.

The complete amino acid sequence of the subunit of thermostable direct hemolysin, a dimeric protein composed of identical subunits isolated from Vibrio parahaemolyticus, was determined by sequencing BrCN-peptides, their tryptic peptides, and overlaps obtained by Achromobacter protease I digestion. The subunit consists of 165 amino acid residues with the sole disulfide bond between Cys 151 and Cys 161. It is deduced that the biologically active hemolysin is formed by noncovalent association of subunits which are not linked together by disulfide bonds. The primary structure of hemolysin elucidated in the present study is essentially the same as that deduced from the nucleotide sequence of a gene encoding the protein but differs in 9 amino acid residues, suggesting the possibility of the presence of multiple genes for the thermostable direct hemolysin in Vibrio parahaemolyticus.

Alcaligenes↗
PubMed1986

3-Methylhistidine content and pH dependency of ATPase activity of adult and embryonic chicken cardiac ventricular myosins.

A distinct difference in the 3-methylhistidine (3-MeHis) content and the pH-dependency curve for calcium-activated adenosine triphosphatase (Ca-ATPase) activity was observed between chicken and mammalian cardiac ventricular myosins. The 3-MeHis content and pH dependency of the Ca-ATPase activity of myosins from adult and embryonic chicken cardiac ventricular muscles and chicken fast white and slow red muscles were almost the same.

Animals↗
PubMed1986

Stoichiometry of actin and phalloidin binding: one molecule of the toxin dominates two actin subunits.

Sonication of F-actin in the absence of added ATP in the solvent induces denaturation of the actin. When phalloidin is added to actin at the molar ratio of 1:2, the denaturation is completely inhibited. More directly, pelleting experiments have indicated that the binding of phalloidin to actin subunits is saturated at the same molar ratio. The protection of F-actin from heat denaturation or depolymerization with 0.6 M KI is also complete with one molecule of the toxin over two actin subunits. Therefore, it is concluded that the binding ratio of phalloidin and the actin subunit is not 1:1 but 1:2.

Actins↗
PubMed1986

Pharmacological analysis of autonomic innervation of the right atria of rats and guinea pigs: demonstration of nonadrenergic noncholinergic nerves.

Transmural nerve stimulation (TNS) of the right atria in vitro produced positive and negative chronotropic responses in rats and guinea pigs. The negative component appeared only temporarily soon after the cessation of TNS and was usually masked by the positive component. The positive and negative chronotropic responses were accompanied by an increase and decrease in contractile force, respectively. Atenolol (3 X 10(-6) M) decreased and atropine (10(-6) M) potentiated the TNS-induced positive chronotropic response. In the presence of both atenolol and atropine, TNS accelerated the heart rate markedly in the right atria of guinea pigs and slightly in those of rats. TNS-induced acceleration of the heart rate was also observed in surgically sympathectomized or reserpine-pretreated (5 mg/kg/48 hr and 2.5 mg/kg/24 hr i.p.) right atrium, in which tyramine (up to 10(-4) M) exerted no appreciable effect. Inasmuch as tetrodotoxin (10(-6) M) almost abolished the TNS-induced chronotropic response, this atenolol- and atropine-resistant response is likely mediated by nonadrenergic noncholinergic (NANC) nerve(s). The NANC nerve-mediated positive chronotropic response was not affected by diphenhydramine (10(-6) M), cimetidine (10(-6) M), methysergide (10(-6) M) and hexamethonium (3 X 10(-6) M). The NANC nerve-mediated response was relatively slow at the onset yet long-lasting compared with adrenergic and cholinergic responses, suggesting that the neurotransmitter of the NANC nerve is a certain substance which may be inactivated more slowly than biogenic amines. Histochemical studies demonstrated the presence of vasoactive intestinal polypeptide-like and substance P-like immunoreactive nerves in the right atrium, as well as catecholamine-fluorescence and acetylcholinesterase-positive nerves.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1986

Suppression of myocardial protein degradation by the protease inhibitor bis[ethyl(2R,3R)-3-[(S)-methyl-1-[4-(2,3,4-tri-methoxy-phenyl-methyl) piperazine-1-ylcarbonyl]butyl-carbonyl]oxiran-2-carboxylat e]sulfate under hypoxia.

The synthetic protease inhibitor bis[ethyl(2R,3R)-3-[(S)-methyl-1-[4-(2,3,4-tri-methoxyphenyl-methyl) piperazine-1-ylcarbonyl]butyl-carbonyl]oxiran-2-carboxylate] sulfate (NCO-700) suppressed both activities of calcium-activated neutral protease and cathepsin B isolated from cardiac muscle, showing 50% inhibition at 46 and 0.8 mumol/l, respectively. A kinetics study using 14C-labelled NCO-700 suggested its incorporation into cultured myocardial cells, demonstrating the half-maximal saturation time at 17 min. Under both aerobic and hypoxic conditions, the reagent inhibited the peptide release from cultured myocardial cells dose-dependently. The amino acid release from heart slices of adult rabbit was also blocked by the drug under hypoxic and glucose-depleted condition. These data and the myocardial infarction size reducing action of NCO-700 might support the view that NCO-700 sensitive protease(s) - possibly, calcium-activated neutral protease and/or cathepsin B - is (are) working to induce an irreversible proteolysis in the process of myocardial cell degradation.

Aerobiosis↗
PubMed1986

Temporary salvage of ischemic myocardium by the protease inhibitor bis[ethyl(2R,3R)-3-[(S)-methyl-1-[4-(2,3,4-trimethoxyphenyl-methyl) piperazin-1-ylcarbonyl]butyl-carbonyl]oxiran-2-carbo xylate]sulfate.

The administration (i.v., 20, 40 and 60 mg/kg) of the thiol protease inhibitor bis[ethyl(2R, 3R)-3-[(S)-methyl-1-[4-(2,3,4-trimethoxyphenyl-methyl) piperazin-1-ylcarbonyl] butyl-carbonyl] oxiran-2-carboxylate]sulfate (NCO-700), before and/or after the coronary ligation significantly reduced the necrotic mass in the rabbit left ventricle and also prevented the creatine phosphokinase loss in the ischemic myocardium up to 3 h and not at 6 h after the ligation. The activities of both calcium-activated neutral protease and cathepsin B in the subendo- and subepicardial layers of the ischemic, marginal or control myocardium of dog were inhibited by the NCO-700 administration (20 mg/kg i.v.) after the coronary ligation. A hemodynamic study using heart-lung preparation of the dog demonstrated a dose-dependent coronary dilatation with weak and transient negative ino- and chronotropic effects. These data suggested that NCO-700 sensitive protease(s) is(are) included in the step of myocardial cell degradation and that NCO-700 temporarily salvages it, as is important for the recent PTCR (percutaneous transluminal coronary reperfusion) therapy.

Animals↗
PubMed1985

Analysis of the metal-induced conformational change in myosin with a monoclonal antibody to light chain two.

A monoclonal antibody capable of detecting a conformational change in myosin light chain two (LC2) was characterized in detail. The antibody was shown to bind only to myosin LC2 when tested against fast skeletal myosin (chicken pectoralis muscle). With cardiac or slow muscle myosins, the antibody exclusively recognized their first light chains (LC1). Staining of myofibrils by the monoclonal antibody could be observed only after their irreversible denaturation by acetone or ethanol, or after incubation of the myofibrils in divalent metal chelators. This latter effect was shown to be fully reversible. The metal effect was independent of ionic strength although the affinity of the antibody for myosin was depressed at high salt concentrations. Similar metal effects were detected in the binding of antibody to cardiac or slow myosins. Neither the metal nor the ionic strength-related inhibition of antibody binding were detected with denatured myosin. The antibody binding site overlaps one of the alpha-chymotryptic sites in LC2 protected by divalent metals. Electron microscopic observations of myosin-antibody complexes demonstrated that the antibody binding site is located near the head-rod junction of myosin. Since the binding site of this monoclonal antibody has been mapped by recombinant DNA methods to the junction of the first alpha-helical domain with the calcium binding site of LC2, the location of the calcium binding site must also be located near the head-tail junction of myosin. A model for conformational changes at the myosin head-tail junction is proposed to account for the metal-induced blockage of antibody binding and the inhibition of alpha-chymotryptic digestion of LC2.

Animals↗
PubMed1985

Acute effect of triiodothyronine on the dynamics of thyrotropin release from superfused anterior pituitary cells.

The effects of triiodothyronine (T3) on the dynamics of thyrotropin (TSH) release induced by TSH-releasing hormone (TRH) were examined in the presence or absence of a protein synthesis inhibitor, cycloheximide (CX), in a superfusion system using primarily cultured cells of the rat anterior pituitary gland on microcarrier beads. When the cells were continuously stimulated with TRH (10 nM, 180 min), TSH release occurred in a biphasic manner and the profile of TSH release was characterized by an initial sharp peak (phase I), followed by a lower plateau form phase (phase II). Both phase-I and phase-II releases were significantly suppressed in the presence of T3 (1 ng/ml), which was added to the superfusion medium 1 h before initiation of TRH stimulation. The biphasic nature of the release profile was maintained in the presence of T3, suggesting that the site of the T3 action may be common between phase-I and phase-II release. We have already suggested that phase-I release is protein synthesis-independent and phase-II release protein synthesis-dependent using CX in TRH-stimulated cells. In the presence of CX, phase-I release was not suppressed by T3, while phase-II release was still suppressed by T3. The inability of CX to reverse the T3-induced suppression of phase-II release may be masked by the direct CX effect on phase-II release of TSH. The present study indicates that each component (phase I and phase II) of the biphasic release of TSH induced by TRH stimulation was acutely suppressed by T3 and suggests that the T3 action is mediated through protein synthesis.

Animals↗
PubMed1985

Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

Animals↗
PubMed1985

Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A-bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross-bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15-nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.

Animals↗