Biomedical subjects

M Yazawa

Publications and source records attributed to M Yazawa.

At least 37 records · Page 2Linked to original sources

PubMed1999

Acid-labile ATP and/or ADP/P(i) binding to the tetraprotomeric form of Na/K-ATPase accompanying catalytic phosphorylation-dephosphorylation cycle.

The Na/K-ATPase has been shown to bind 1 and 0.5 mol of (32)P/mol of alpha-chain in the presence [gamma-(32)P]ATP and [alpha-(32)P]ATP, respectively, accompanied by a maximum accumulation of 0.5 mol of ADP-sensitive phosphoenzyme (NaE1P) and potassium-sensitive phosphoenzyme (E2P). The former accumulation was followed by the slow constant liberation of P(i), but the latter was accompanied with a rapid approximately 0.25 mol of acid-labile P(i) burst. The rubidium (potassium congener)-occluded enzyme (approximately 1.7 mol of rubidium/mol of alpha-chain) completely lost rubidium on the addition of sodium + magnesium. Further addition of approximately 100 microM [gamma-(32)P]ATP and [alpha-(32)P]ATP, both induced 0.5 mol of (32)P-ATP binding to the enzyme and caused accumulation of approximately 1 mol of rubidium/mol of alpha-chain, accompanied by a rapid approximately 0.5 mol of P(i) burst with no detectable phosphoenzyme under steady state conditions. Electron microscopy of rotary-shadowed soluble and membrane-bound Na/K-ATPases and an antibody-Na/K-ATPase complex, indicated the presence of tetraprotomeric structures (alphabeta)(4). These and other data suggest that Na/K-ATP hydrolysis occurs via four parallel paths, the sequential appearance of (NaE1P:E.ATP)(2), (E2P:E.ATP:E2P:E. ADP/P(i)), and (KE2:E.ADP/P(i))(2), each of which has been previously referred to as NaE1P, E2P, and KE2, respectively.

Adenosine Diphosphate↗

PubMed1999

Mental stress test is an effective inducer of vasospastic angina pectoris: comparison with cold pressor, hyperventilation and master two-step exercise test.

BACKGROUND: Cold pressor, hyperventilation and exercise stress tests were usually used for inducing an angina attack in patients with vasospastic angina pectoris. We induced vasospastic angina attack using the mental calculation stress test, and compared the results with those using other stress tests. SUBJECTS AND METHODS: Subjects were 29 patients with vasospastic angina pectoris. Their ages were 60.8+/-8.4 years. Coronary vasospasm was induced by an acetylcholine infusion test during coronary angiography. The mental stress test was performed as follows; after memorizing six digits numbers, they repeated these numbers in reverse for 5 min, and performed serial subtraction of 17 from 1000 for 5 min. Blood pressure, heart rate and ECG were recorded every 1-5 min during the mental stress test. The serum concentrations of epinephrine and norepinephrine were measured before and during the mental stress test. We compared these results with those obtained using cold pressor, hyperventilation and the Master two-step exercise stress test. RESULTS: (1) Eight of the 29 patients (28%) showed ischemic ST-T change, which was caused by the mental stress test. (2) The increase in norepinephrine was greater in patients with an ST-T change than without an ST-T change (0.11+/-0.06 vs. 0.04+/-0.04 ng/ml, P<0.01). (3) The incidence of the ST-T change caused by the mental stress test (28%) was similar to the cold pressor test (27%) and greater than that caused by the hyperventilation test (13%). The incidence of ST-T change caused by the Master two-step test was 55%. CONCLUSIONS: The mental stress test is an effective inducer of vasospastic angina attack, and attack may be induced by neurohumoral vasoconstrictive reflex and/or increased left ventricular afterload.

Acetylcholine↗

PubMed1999

Calcium binding induces interaction between the N- and C-terminal domains of yeast calmodulin and modulates its overall conformation.

Calmodulin from the yeast Saccharomyces cerevisiae binds 3 mol of Ca2+ cooperatively. We report here lines of evidence supporting the intramolecular interaction between the N- and C-terminal domains which modulates the Ca2+ binding properties of yeast calmodulin. First, the sum of the Ca2+ binding curves of the N-terminal and the C-terminal half-molecule did not yield the Ca2+ binding curve of yeast calmodulin. Second, the mean residue CD of yeast calmodulin at 222 nm (-Delta epsilon222) decreased with increases in the concentration of Ca2+, whereas those of each half-molecule increased. Finally, the C2 proton of His107 in the C-terminal domain of yeast calmodulin showed three resonance peaks with increases in the concentration of Ca2+, each corresponding to the apo, the intermediate, and the Ca2+-saturated state. The intermediate peak could not be observed in the C-terminal half-molecule of yeast calmodulin. Computer simulation considering the macroscopic Ca2+ binding constants assigned this intermediate to a species consisting of the apo C-terminal domain and the N-terminal domain with at least one of the two sites occupied by Ca2+. Peptide segments spanning the defective fourth Ca2+ binding site may be involved in the interdomain interaction and the yeast-specific function of calmodulin.

Binding Sites↗

PubMed1999

Identification of trimeric myosin phosphatase (PP1M) as a target for a novel PKC-potentiated protein phosphatase-1 inhibitory protein (CPI17) in porcine aorta smooth muscle.

CPI17, a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1), is dominantly expressed in smooth muscle, and the inhibitory activity is potentiated by protein kinase C and its related enzymes [Eto, M. et al. (1997) FEBS Lett. 410, 356-360]. In order to identify its physiological target in smooth muscle, the myofibrillar extract from porcine aorta media was analyzed by affinity chromatography on CPI17-conjugated Sepharose. The binding of phosphatases to the resin depended on thiophosphorylation of CPI17, and about 90% of the phosphatase activities toward phosphorylated myosin (p-myosin) and phosphorylase-a were bound to the resin and could be eluted with 0.5 M NaCl. The IC50 values of thiophosphorylated CPI17 toward phosphatases bound to the resin were in the range of 0.5-3 nM, as expected for the PP1 holoenzymes sensitive to CPI17. The CPI17-sensitive fraction was further separated into several peaks of phosphatase activity by column chromatography on Mono Q, which suggested multiple functions of CPI17 as a mediator of the protein kinase C-related signal transduction pathway in aorta smooth muscle. The major activity toward p-myosin was identified as the myofibril-bound PP1 (PP1M), and its subunit composition (140, 37, and 20 kDa) was consistent with that of PP1M from chicken gizzard and porcine bladder. The purified PP1M was completely inhibited by phosphorylated and thiophosphorylated CPI17. Kinetic analysis showed mixed inhibition of PP1M by CPI17 (Ki = 1.9 nM and Ki' = 5.1 nM). The concentration of CPI17 in aorta smooth muscle cells was estimated to be at least 0. 3 microM from the result of Western analysis. This concentration appears to be sufficient to suppress the in situ PP1M in aorta smooth muscle, and PP1M is thus identified as a target of CPI17 in vascular smooth muscle.

Amino Acid Sequence↗

PubMed1999

Conformations of vertebrate striated muscle myosin monomers in equilibrium with filaments.

Porcine cardiac myosin monomers in equilibrium with filaments under physiological conditions were observed to have two conformations, extended and folded forms, upon electron microscopy and gel filtration HPLC. The conformational state was independent of ATP and the phosphorylation of regulatory light chain. The folded monomers of cardiac myosin were mainly in an open conformation with only one bend in the tail, and may not trap the hydrolysis products of ATP, as assessed by single turnover experiments. These properties are similar to those of the folded monomers of rabbit skeletal myosin [Katoh, T., Konishi, K., and Yazawa, M. (1998) J. Biol. Chem. 273, 11436-11439]. The conformational states of skeletal and cardiac myosin monomers were not affected by pH between 7.0 and 8.5. Although significant disassembly of filaments and thus an increase in the monomer concentration were observed with an increase in pH. The results indicate that the pH-dependent change in filament assembly is due to a shift of equilibrium between the filaments and extended monomers toward filament disassembly. The Mg2+-ATPase activity of these myosin monomers decreased with a decrease in the salt concentration below approximately 0.1 M, suggestive of the formation of a closed conformation similar to the conformation of 10S smooth myosin. The results suggest that the conformational change from the extended to the folded form is a common property of various myosin IIs.

Actin Cytoskeleton↗

PubMed1999

Measurement of telomerase activity in dog tumors.

Telomeres are specific structures present at the end of liner chromosomes. DNA polymerase can not synthesize the end of liner DNA and, as a result, the telomeres become progressively shortened by successive cell divisions. To overcome the end replication problem, telomerase adds new telomeric sequences to the end of chromosomal DNA. The enzyme activity is undetectable in most normal human adult somatic cells, in which shortening of the telomere is thought to limit the somatic-cell life span. In contrast to normal somatic cells, many human tumors possess telomerase activity. The present study looked at whether telomerase activity might serve as a marker for canine tumors. Telomerase activity was measured using the telomeric repeat amplification protocol assay. Normal dog somatic tissues showed little or no telomerase activity, while normal testis exhibited a high level of telomerase activity. We measured telomerase activity in tumor samples from 45 dogs; 21 mammary gland tumors, 16 tumors developed in the skin and oral cavity, 7 vascular tumors and 1 Sertoli cell tumor. Greater than 95% of the tumor samples contained telomerase activity (3-924 U/2 micrograms protein). The results obtained in this study indicated that telomerase should be a useful diagnostic marker for a variety of dog tumors, and it may serve as a target for antitumor chemotherapy.

Adenocarcinoma↗

PubMed1999

A strange calmodulin of yeast.

Calmodulin of Saccharomyces cerevisiae has different Ca2+ binding properties from other calmodulins. We previously reported that the maximum number of Ca2+ binding was 3 mol/mol and the fourth binding site was defective, which was different from 4 mol/mol for others. Their macroscopic dissociation constants suggested the cooperative three Ca2+ bindings rather than a pair of cooperative two Ca2+ bindings of ordinary calmodulin. Here we present evidence for yeast calmodulin showing the intramolecular close interaction between the N-terminal half domain and the C-terminal half domain, while the two domains of ordinary calmodulin are independent of each other. We will discuss the relationship of the shape and the shape change caused by the Ca2+ binding to the enzyme activation in yeast. The functional feature of calmodulin in yeast will also be considered, which might be different from the one of vertebrate calmodulin.

Animals↗

PubMed1999

[A case of neurofibromatosis with intrathoracic meningocele presenting respiratory failure caused by pleural effusion].

We reported a 58-year-old man with neurofibromatosis and an intrathoracic meningocele. He was admitted to our hospital because of left-sided chest pain and dyspnea on exertion. He presented with severe kyphoscoliosis and showed a round, well circumscribed mass lesion in the paravertebral region of the left upper lung on a chest roentgenogram. Just before admission, pleural effusion accumulated in the left thoracic cavity, which had caused the respiratory symptoms. The mass was diagnosed as an intrathoracic meningocele by MRI and iotrolan CT myelography. The pleural effusion was transudate fluid and no leakage from the meningocele to pleural cavity was demonstrated. Posterolateral extradural approach with laminectomy was done and dural plasty to close the connection between the meningocele and the subarachnoidal space was carried out. After the operation, both the intrathoracic meningocele and the pleural effusion disappeared with remarkable improvement in the respiratory function. Intrathoracic meningocele is known to be seen in association with neurofibromatosis and scoliosis, but it is very rare to see an intrathoracic meningocele which causes respiratory failure due to massive pleural effusion like this report.

Humans↗

PubMed1998

Skeletal muscle myosin monomer in equilibrium with filaments forms a folded conformation.

Rabbit skeletal myosin forms stable filaments under physiological conditions, and only a small amount stays as a monomer in equilibrium with filaments. The myosin monomers were observed in two conformational states, as extended and folded forms upon electron microscopy and gel filtration high performance liquid chromatography. The fraction of monomers in the folded conformation increased with a decrease in the concentration of NaCl below 0.2 M, and the conformational state was affected neither by the presence of ATP nor by the phosphorylation of regulatory light chain. In most of the folded monomers, the tail bent back toward the heads at one region, 45 nm apart from the head-tail junction, and the remaining tail portion containing the C-terminal tip appeared to interact with the head-tail junction. Only a small percentage of the folded monomers was in a more compact conformation close to the 10 S conformation of vertebrate smooth muscle and non-muscle myosins. The folded monomers, however, may not trap the products of ATP hydrolysis as assessed by single turnover experiments. The percentage of monomers in the 10 S-like conformation was increased by the exchange of a regulatory light chain with the smooth muscle light chain, indicating the participation of head-tail junction, including the regulatory light chain in the formation of folded conformation. The folded conformation may be common to various myosin IIs, suggestive of common roles for the folded monomers.

Animals↗

PubMed1998

Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle.

1. CPI-17 has recently been identified as a novel protein in vascular smooth muscle. In vitro , its phosphorylation and thiophosphorylation by protein kinase C (PKC) specifically inhibits the type 1 class of protein phosphatases, including myosin light chain (MLC) phosphatase. 2. Both of the phosphorylated CPI-17 states dose-dependently potentiated submaximal contractions at constant [Ca2+] in beta-escin-permeabilized and Triton X-100-demembranated arterial smooth muscle, but produced no effect in intact and less intensely permeabilized (alpha-toxin) tissue. Thiophosphorylated CPI-17 (tp-CPI) induced large contractions even under Ca2+-free conditions and decreased Ca2+ EC50 by more than an order of magnitude. Unphosphorylated CPI-17 produced minimal but significant effects. 3. tp-CPI substantially increased the steady-state MLC phosphorylation to Ca2+ ratios in beta-escin preparations. 4. tp-CPI affected the kinetics of contraction and relaxation and of MLC phosphorylation and dephosphorylation in such a manner that indicates its major physiological effect is to inhibit MLC phosphatase. 5. Results from use of specific inhibitors in concurrence with tp-CPI repudiate the involvement of general G proteins, rho A or PKC itself in the Ca2+ sensitization by tp-CPI. 6. Our results indicate that phosphorylation of CPI-17 by PKC stimulates binding of CPI-17 to and subsequent inhibition of MLC phosphatase. This implies that CPI-17 accounts largely for the heretofore unknown signalling pathway between PKC and inhibited MLC phosphatase.

Animals↗

PubMed1998

Endoscopic ligation for non-esophageal variceal upper gastrointestinal hemorrhage.

BACKGROUND: Endoscopic ligation can be used for bleeding lesions in non-fibrotic tissue; however, only small numbers of patients with non-esophageal variceal upper gastrointestinal hemorrhage have been treated in this way. To evaluate the utility of the technique, we performed the procedure to treat hemorrhage not from esophageal varices during emergency endoscopy. PATIENTS AND METHODS: Bleeding was identified from gastric Dieulafoy's ulcers (n=4), duodenal ulcers (n=3), gastric angiodysplasia (n=2) and Mallory-Weiss tears (n=3). The bleeding points were aspirated and controlled by endoscopic ligation and complete hemostasis was achieved in all cases. RESULTS: Although these lesions were located in difficult areas where endoscopic injection therapy and clipping sometimes fail, endoscopic ligation was performed easily and effectively without complications. Six of the patients had severe underlying disease, including acute and chronic myelogenous leukemia, liver cirrhosis and chronic renal failure; none suffered deterioration in their general condition after endoscopic ligation. CONCLUSIONS: Our findings suggest that endoscopic ligation is an easy and effective method of treatment for patients with gastrointestinal hemorrhage not from esophageal varices, and is safe even in patients with poor general health.

Adult↗

PubMed1998

Conformation, filament assembly, and activity of single-headed smooth muscle myosin.

Single-headed myosin was prepared by digestion of porcine aorta smooth muscle myosin with Staphylococcus aureus V8 protease in the presence of actin. The single-headed myosin preparation contained intact light chains, a rod fragment of a heavy chain, and a heavy chain of which only a minor fraction contained a nick in the head segment. Below 0.2 M NaCl, the single-headed myosin showed a decrease in Ca2+-ATPase activity and an increase in the elution time on gel filtration HPLC in a phosphorylation-dependent manner, indicating a phosphorylation-dependent conformational transition between the extended and folded forms. These conformations were confirmed by electron microscopic observation of rotary-shadowed samples of single-headed myosin. However, the conformational transition of single-headed myosin occurred in a narrower range with lower salt concentrations than that of double-headed myosin. The filament assembly of single-headed myosin was thus facilitated and phosphorylation-independent. The single-headed myosin also showed high actin-activated ATPase activity independent of phosphorylation. These results indicate that the two-headed structure of smooth muscle myosin is not essential for the conformational transition, but is required for the phosphorylation-dependent regulation of enzymatic activity and filament assembly.

Actin Cytoskeleton↗

PubMed1998

Reactivities of Cys707 (SH1) in intermediate states of myosin subfragment-1 ATPase.

To detect structural changes around the reactive Cys707 (SH1) in the myosin heavy chain during the ATPase reaction, the reactivity of SH1 in rabbit skeletal myosin subfragment-1 (S-1) was measured using a fluorescent reagent, 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, in the presence of various ATP analogs: adenosine 5'-(3-thiotriphosphate) (ATPgammaS), ADP-vanadate (ADP-Vi), ADP-BeFx, and ADP-AlF4. The SH1 reactivities in the S-1 complexes with ATPgammaS and ADP-BeFx, analogs of the E-ATP state, were very high, as well as that in the E-ADP state. In contrast, the SH1 reactivities in the S-1 complexes with ADP-Vi and ADP-AlF4, analogs of the E-ADP-P state, were extremely low. The structural changes around SH1 can be correlated to changes in the structure of the gamma-phosphate of ATP during the ATPase reaction or to the structure of the corresponding part of ATP analogs at the active site of ATPase. This is consistent with the crystal structure of S-1 in which the heavy chain structure around SH1 of S-1-ADP-BeFx is significantly different from those of S-1-ADP-Vi and S-1-ADP-AlF4 [Fisher et al. (1995) Biochemistry 34, 8960-8972; Smith and Rayment (1996) Biochemistry 35, 5404-5417].

Adenosine Diphosphate↗

PubMed1997

Essential light chain exchange in smooth muscle myosin.

The exchange of 17-kDa essential light chain (LC17) in smooth muscle myosin was carried out by incubating myosin with a 10-fold molar excess of exogenously added LC17 over the corresponding endogenous light chain in the presence of trifluoperazine and 4.5 m ammonium chloride. Porcine aorta myosin contains two kinds of LC17 isoform, LC17nm and LC17gi, while chicken gizzard myosin contains only one kind of LC17 isoform. As LC17gi can be separated from LC17nm and gizzard LC17 by urea-gel electrophoresis, LC17nm in aorta myosin and LC17 in gizzard myosin were exchanged with LC17gi and LC17gi in aorta myosin was exchanged with LC17nm, and the degree of exchange was estimated by urea-gel electrophoresis. Under the optimal conditions (6 and 10 degrees C for aorta and gizzard myosin, respectively), nearly 90% of exchange, which is close to the theoretical value, was achieved for the former combinations, and a slightly lower exchange was obtained for the latter. The LC17-exchanged myosins contained stoichiometric amounts of the heavy and light chains and retained the original nature in the phosphorylation-dependent actin-activated ATPase activity, 6S-10S conformational transition, and filament assembly.

Actin Cytoskeleton↗

PubMed1997

Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle.

The cDNA encoding a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147-residue polypeptide, which we termed CPI17, a 17-kDa PKC-potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC50 = 0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non-muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.

Amino Acid Sequence↗

PubMed1997

Sarcoid reaction in thyroid diseases: report of a case of thyroid carcinoma demonstrating sarcoid reaction in regional lymph nodes.

We describe an extremely rare case of thyroid carcinoma accompanied by sarcoid reaction in regional lymph nodes. The patient, a 40-year-old woman, was found to have anterior neck swelling during a routine medical examination. Physical examination revealed a bad movable and firm nodule measuring 3.5 x 2.5 cm in the right thyroid lobe. Examination of a fine-needle aspiration cytology of the nodule suggested papillary carcinoma of class V. Right lobectomy and isthmectomy with neck dissection were performed. Histopathological examination of the resected specimens revealed papillary carcinoma accompanied by metastatic foci in right perithyroidal lymph nodes and non-caseous sarcoid-like granulomas in pretracheal, right upper internal deep cervical and submandibular lymph nodes. We concluded that these granulomas were due to a sarcoid reaction associated with thyroid carcinoma, after performing examinations to rule out the possibility of systemic sarcoidosis.

Adult↗

PubMed1997

Crosslinking of telokin to chicken gizzard smooth muscle myosin.

Telokin, which is a candidate for one of the factors stabilizing dephosphorylated myosin filaments in smooth muscle cells, was subjected to a chemical crosslinking study to determine its binding location on the myosin molecule. Telokin labeled with 5-iodoacetamidofluorescein (Fl-telokin) retained the nature of unlabeled telokin: it bound to dephosphorylated gizzard myosin with a stoichiometry of 1 mol/mol myosin and induced filament assembly. The carboxyl groups of Fl-telokin were activated with 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide and N-hydroxysuccinimide and then crosslinked to myosin. The production of three fluorescent peptides was observed on an SDS-gel, accompanying a decrease in the amount of regulatory light chain (LC20). The molecular weights of these products estimated to be > 200, 62, and 41 kDa. When unlabeled telokin was crosslinked to myosin of which LC20 was exchanged with 5-[[2-[(iodoacetyl)amino]ethyl]amino]-naphthalene-1-sulfonic acid-labeled LC20, the 62- and 41-kDa bands were also fluorescent. These results suggest that the > 200, 62, and 41-kDa species are telokin crosslinked with a heavy chain, with 2 LC20, and with 1 LC20, respectively. Myosin crosslinked with unlabeled telokin showed an extra structure with a small projection at the head-rod junction on electron microscopy and this structure was proved to be telokin by decorating it with anti-telokin antibodies. In addition, dephosphorylated myosin crosslinked with Fl-telokin was incapable of folding into the 10S conformation at 0.2 NaCl in the presence of MgATP, and assembled into filaments at 0.15 M NaCl in the presence of MgATP. Thus, telokin may bind to LC20 and a heavy chain region at the head-rod junction and suppress folding into the 10S conformation, leading to the assembly of myosin into filaments.

Adenosine Triphosphate↗

PubMed1996

Chimeras of yeast and chicken calmodulin demonstrate differences in activation mechanisms of target enzymes.

Various chimeric proteins were constructed from yeast (Saccharomyces cerevisiae) and chicken calmodulin (CaM), and regions essential for target activation and responsible for the specific features of the yeast CaM were identified. The chimeric CaMs were designed so that each Ca2+ binding site of the yeast CaM was replaced in series from the C-terminus. Resulting CaM proteins showed Ca2+ binding properties inherent to the original Ca2+ binding site. Cooperative Ca2+ binding and a suitable rearrangement of the two EF-hand sites in each half-molecular domain were shown to be important for high-affinity interaction with CaM-dependent cyclic nucleotide phosphodiesterase (PDE). Residues in chicken CaM sequences 129-148 and 88-128, respectively, were required for low values of Kact (the concentration of CaM required for the half-maximal activation) in the activation of PDE and myosin light chain kinase (skMLCK and smMLCK). The difference in the structural requirements indicated different manners of the interaction. While PDE was activated to similar levels by different chimeras, the maximum activity (Vmax) given by chicken CaMs was not achieved by any chimeric CaMs in MLCKs. Residues in chicken CaM sequences 1-50 and 88-129, in addition to Ca2+ binding to the fourth site, were important for high values of Vmax of skMLCK. On the other hand, Met51 and residues in chicken CaM sequence 88-129 were critical for the high Vmax of smMLCK. These residues may work to form the active structure of the catalytic site of each MLCK, while simple binding of CaM seems sufficient to expose the active site of PDE.

Amino Acid Sequence↗