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K Ebisawa

Publications and source records attributed to K Ebisawa.

At least 19 recordsLinked to original sources
PubMed1998

Molecular identification of the ryanodine receptor Ca2+ sensor.

We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca2+ by using site-directed mutagenesis together with functional assays consisting of Ca2+ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca2+ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca2+ sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca2+ sensor in the tetrameric structure corresponding to RyR.

Amino Acid Sequence↗
PubMed1997

Functional characterization of the recombinant type 3 Ca2+ release channel (ryanodine receptor) expressed in HEK293 cells.

To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca2+ release measurements using the fluorescence Ca2+ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine- and ryanodine-sensitive Ca2+ release channel in HEK293 cells. Functional properties of the cloned RyR3 were further characterized by using single channel recordings in lipid bilayers. The cloned RyR3 channel exhibited a K+ conductance of 777 picosiemens in 250 mM KCl and a Ca2+ conductance of 137 picosiemens in 250 mM CaCl2 and displayed a pCa2+/pK+ ratio of 6.3 and an open time constant of about 1.16 ms. The response of the cloned RyR3 to cytoplasmic Ca2+ concentrations was biphasic. The channel was activated by Ca2+ at about 100 nM and inactivated at about 10 mM. Ca2+ alone was able to activate the cloned RyR3 fully. Calmodulin activated the cloned RyR3 at low Ca2+ concentrations but inhibited the channel at high Ca2+ concentrations. The cloned RyR3 was activated by ATP, caffeine, and perchlorate, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Cyclic ADP-ribose did not seem to affect single channel activity of the cloned RyR3. The most prominent differences of the cloned RyR3 from the rabbit skeletal muscle ryanodine receptor were in the gating kinetics, extent of maximal activation by Ca2+, and sensitivity to Ca2+ inactivation. Results of the present study provide initial insights into the single channel properties of the mammalian RyR3.

Adenosine Diphosphate Ribose↗
PubMed1996

Hydrogen bonding effects on amine rotation rates in crystalline amino acids.

Rates of rotation for amines in a variety of crystalline environments are reported, and the trends are explained in terms of the strengths of local hydrogen bonding interactions. Proton spin-lattice relaxation times (T1) and deuterium broad-line NMR spectra have been measured for D-, DL- and L- aspartic acid, two polymorphs of glycine, alanine, and leucine in the temperature range from -40 to 110 degrees C. The energy barriers for amine rotation are 27 +/- 2 kJ mol-1 for D- or L-aspartic acid and 22 +/- 2 kJ mol-1 for DL-aspartic acid; these energies are slightly lower than the previously reported value for the L from based on direct proton T1 measurements at 60 MHz. The values for the alpha and gamma forms of glycine were 24 +/- 2 and 30 +/- 2 kJ mol-1 respectively, that for L-alanine was 40 +/- 2 and that for L-leucine was 49 +/- 3 kJ mol-1. These are all in rough agreement with previously reported values (although the differences for the polymorphs of glycine and for L- vs. DL-aspartic acid were not reported). Crystal structures of these amino acids indicate differences in hydrogen bonding environments around the R-NH3+ groups that are probably responsible for the different activation barriers. A molecular mechanics calculation of the rotation energy barriers for L- and DL-aspartic acid based on the crystal structures gave satisfactory agreement with experimental results if a uniform (and arbitrarily chosen) dielectric constant of 2.5 was assumed. Differences between L- and DL-aspartic acids and between two polymorphs of glycine were well represented qualitatively. Including additional neighboring molecules not involved in the hydrogen bonding or including periodic boundary conditions to describe the crystal packing did not significantly affect these results. If vacuum dielectric constants are used, the barriers are uniformly overestimated, and if the experimental macroscopic dielectric constant values are used, the barriers are generally underestimated. Dielectric constants differ substantially from one amino acid to another and significantly affect the estimated barriers; in fact, the bulk dielectric constants appear to be the major difference between the highest and the lowest values. The difficulty of accurately including dielectric relaxation into molecular mechanics calculations resulted in the disagreement between experimental measurements and theoretical calculations.

Amines↗
PubMed1995

Cardiac electrophysiologic effects of parathyroid hormone in the guinea pig.

The cardiac electrophysiologic effects of parathyroid hormone (PTH) were studied in three different experimental models. Purified synthetic human parathyroid hormone (hPTH 1-84), at a dose of 30 ng/g body weight (BW), prolonged the QT interval of surface ECGs in intact guinea pigs. To further clarify the mechanisms of the QT prolongation caused by hPTH, the action potentials of guinea pig papillary muscle were recorded by conventional microelectrode techniques. hPTH (10(-7)M) prolonged the action potential duration without changing the action potential amplitude, resting potential, maximal upstroke velocity of the action potential, or maximal repolarization rate. hPTH affected only the plateau phase, in a concentration-dependent fashion. The half-maximum concentration was 6.8 x 10(-8)M for action potential duration at 30% repolarization (APD30), 3.2 x 10(-8)M for APD50, and 2.1 x 10(-8)M for APD90. In isolated single guinea pig ventricular cells, the patch clamp method was used to record the slow inward calcium current (ICa) and delayed rectifier potassium current (IK) responsible for the action potential plateau phase. hPTH increased the peak amplitude of ICa at + 10mV by an average of 42%, and increased IK at + 10mV by 85%. These results indicate that, in ventricular muscle, hPTH prolongs the action potential duration and QT interval by the balance of the prolongation effect on APD by enhancing the slow inward calcium current and the shortening effect on APD by enhancing the delayed rectifier potassium current.

Action Potentials↗
PubMed1995

Familial atrioventricular heart block of adult onset: electrocardiogram and HLA typing analysis.

A family was investigated because of heart block and sinus arrhythmia. The electrical characteristics were: (1) adult onset in all members; (2) complete heart block with atrial fibrillation in 2, and first- or second-degree heart block in 7 members; and (3) sinus arrhythmia in 3 members. Human leukocyte antigen (HLA) typing was performed. HLA A2, B39, Cw7, and DR12 were positive in 4 of 5 members in the heart block group. In the sinus arrhythmia group, HLA DR12 was positive in all members. In the normal group, none of these HLA typings was positive except one. These findings indicate a tighter relationship between heart block and the HLA locus than previously thought.

Adult↗
PubMed1994

Irreversible cardiomyopathy due to thyrotoxicosis.

We report 4 adult patients with thyrotoxicosis accompanied by irreversible low-output heart failure. Each patient showed elevated plasma levels of thyroid hormone and prolonged low-output heart failure even after thyroid function returned to normal. Specimens of the right ventricular myocardium stained with anti-beta myosin heavy-chain MAb showed a normal staining pattern with a predominance of the beta-form. Our observations suggest that thyrotoxicosis may be one possible cause of irreversible cardiomyopathy.

Aged↗
PubMed1994

Follow-up laparoscopy in patients with ovarian cancer.

Between 1982 and 1990, two hundred and fourteen consecutive laparoscopies were performed on 75 patients with epithelial ovarian cancer when the disease appeared to be clinically in complete remission according to simultaneous CT scan or ultrasonography. Evaluation were based on macroscopic findings through the laparoscope, by cytologic examination of ascitic and peritoneal washings and by intraperitoneal biopsy. The results were positive in 34 laparoscopies for 19 patients, suspicious in 15 laparoscopies for 13 patients and negative in 164 laparoscopies. the intraperitoneal recurrence rate after negative second-look laparoscopy was 14.0% as a whole, and 4.5% in cases with more than two consecutive negative evaluations. Serum CA-125 levels exhibited and elevation in 28 of 34 positive laparoscopies before treatment and remained within the normal range in 22 (78.6%) of the 28 cases after treatment despite positive follow-up laparoscopies. The repeated laparoscopies offer the advantage of lowering the false negative rate. This will enable early detection of intraperitoneal recurrence, better prognoses and reduction of unnecessary laparotomies.

Female↗
PubMed1994

[Retrospective analysis of relationship between progression and regression of dysplasia and existence of HPV DNA by in situ hybridization].

We investigated the relationship between the progression and regression of cervical dysplasia and the existence of HPV DNA. The materials we analyzed were 80 biopsied specimens obtained from 22 cases of dysplasia which were followed-up for a long time at our out-patient clinic, and 47 specimens of squamous cell carcinoma. Detection of HPV DNA was done by in situ hybridization with biotinylated HPV DNA probes types 6/11, 16/18 and 31/33/35. The analysis of several specimens obtained at long term follow-up showed that the dysplasia-progression group had a higher incidence and more frequent detection of HPV DNA than the regression group. In the progression group, HPV 16/18 were detected more frequently than HPV 31/33/35 at the dysplasia lesion, but HPV 6/11 was detected in neither the dysplasia nor the squamous cell carcinoma. Fourteen of the 47 cases of squamous cell carcinoma (29.8%) were positive for HPV DNA, and 13 of them (92.9%) were detected in the lesions of dysplasia adjacent to carcinoma in situ or invasive cancer. Comparison of the detection rate for HPV DNA was done in the 3 dysplastic areas, i.e. the dysplastic areas in the dysplasia progression group (A), dysplasia regression group (B) and in the area adjacent to squamous cell carcinoma (C). The detection rates for HPV DNA were 43.5% in A, 27.7% in C and 15.0% in B. From these results we drew the following conclusions: 1) The continuous existence of HPV DNA, especially type 16/18 in the dysplasia lesions, may progress from lesions to carcinoma in situ or invasive cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Squamous Cell↗
PubMed1993

[Two adult cases with corrected transposition of the great arteries treated with permanent endocardial pacemaker implantation].

[Case 1] A 32 year-old male had (I, D, D) type corrected transposition of the great arteries (cTGA) associated with ventricular septal defect and pulmonary stenosis. [Case 2] A 69 year-old male had (S, L, L) type cTGA associated with tricuspid regurgitation. Both cases showed advanced atrioventricular (AV) block due to HV block. Both patients had endocardial DDD pacemakers implanted, with no complication. (I, D, D) type cTGA is rarely accompanied with AV block, while the incidence of AV block is high in (S, L, L) type cTGA. We speculate that this results from the difference in the position of the AV node between these two types.

Adult↗
PubMed1992

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.

Animals↗
PubMed1991

Application of immunocytochemistry to the cytologic study of peritoneal fluids in patients with ovarian cancer.

Immunocytochemical methods were evaluated for their usefulness in the identification of malignant cells in cytologic preparations of peritoneal fluids of ovarian cancer. The antibodies used for this study were anti-cancer antigen-125 (CA-125) antibody, anti-carbohydrate antigen 19-9 (CA 19-9) antibody, anti-carcinoembryonic antigen (CEA) antibody and anti-epithelial membrane antigen (EMA) antibody. The immunohistochemical and cytochemical results using 4 antibodies for both the histologic specimens and the imprinted smears from tumor tissues of the same patients were compatible in 91.8% (both positive or both negative) of the cases. Reactivities of ascitic specimens taken from benign gynecological lesions were 39.4% with anti-CA-125, 12.1% with anti-CA 19-9, 6.1% with anti-EMA, while none of the 33 cases reacted with anti-CEA antibodies. Comparison of the immunoreactivity with the 4 antibodies in histologic specimens and peritoneal smears with Pap-positive cytology taken from the same patients showed 82.9% compatibility. However, 40% incompatibility was found in the anti-CA-125 staining. Some of the mesothelial cells contained in the peritoneal fluids reacted with the anti-CA-125 antibody. Except for the anti-CA-125 staining, compatibility was 90.3%. These results indicate that the anti-CA-125 antibody can not be used to distinguish benign from malignant cells, while anti-CEA antibody has high specificity for malignant cells. CA19-9, CEA and EMA stainings may be valuable criteria in the cytologic diagnosis of effusions. However, it is possible that individual use of these antibodies may lead to a false negative diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1990

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene.

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.

Animals↗
PubMed1987

The mechanism of inhibition of light-chain phosphorylation by purealin in chicken gizzard myosin.

The actin-activated Mg2+-ATPase activity of dephosphorylated chicken gizzard myosin reconstituted with actin, tropomyosin, myosin light-chain kinase (MLCK) and calmodulin was inhibited completely by purealin, 20 microM, whereas the activity of the phosphorylated and dephosphorylated myosin was not affected. Purealin inhibited the phosphorylation of myosin light chains caused by MLCK and calmodulin (IC50, 5 microM). On the other hand, purealin had no effect on myosin phosphorylation induced by Ca2+ -independent MLCK. The calmodulin-stimulated phosphodiesterase activity was inhibited by purealin (IC50, 7 microM) at concentrations very close to those that inhibit myosin phosphorylation. Kinetic analysis revealed a competitive mode of inhibition of calmodulin-stimulated phosphodiesterase activity by purealin. These results suggest that purealin acts as a calmodulin antagonist in reconstituted actomyosin from chicken gizzard, resulting in inhibition of light chain phosphorylation and the actin-activated ATPase activity of myosin.

Adenosine Triphosphatases↗
PubMed1986

Purealin, a novel stabilizer of smooth muscle myosin filaments that modulates ATPase activity of dephosphorylated myosin.

Effects of purealin isolated from a sea sponge, Psammaplysilla purea, on the enzymatic and physiochemical properties of chicken gizzard myosin were studied. At 0.15 M KCl, 40 microM purealin increased the Ca2+- and Mg2+-ATPase activity of dephosphorylated gizzard myosin to 2.5- and 3-fold, respectively, but decreased the K+-EDTA-ATPase activity of the myosin to 0.25-fold. In contrast, purealin had little effect on the ATPase activities of phosphorylated gizzard myosin. The ATP-induced decrease in light scattering of dephosphorylated gizzard myosin at 0.15 M KCl was lessened by 40 microM purealin. Electron microscopic observations indicated that thick filaments of dephosphorylated myosin were disassembled immediately by addition of 1 mM ATP at 0.15 M KCl, although they were preserved by purealin for a long time even after addition of ATP. Upon ultracentrifugation, dephosphorylated myosin sedimented as two components, the 10 S species and myosin filaments, in the solution containing 0.18 M KCl and 1 mM Mg X ATP in the presence of 60 microM purealin. These results suggest that purealin modulates the ATPase activities of dephosphorylated gizzard myosin by enhancing the stability of myosin filaments against the disassembling action of ATP.

Actin Cytoskeleton↗
PubMed1985

Purification of vitamin D-dependent 28,000-Mr calcium-binding protein from bovine cerebellum and kidney by calcium-dependent elution from DEAE-cellulose DE-52 column chromatography.

A vitamin D-dependent calcium-binding protein of 28,000 Mr was purified by a convenient method from the high-ammonium sulfate-saturated fraction (70 to 100% saturation) of bovine cerebellum and kidney. This method is based on the calcium-dependent DEAE-cellulose column chromatography that reflected the specific feature of this protein: different eluting salt concentrations in the presence or absence of Ca2+. The procedure of purification was easily performed by the detection of calcium-binding protein using 45Ca autoradiography. The purified calcium-binding protein showed the same molecular weight and calcium-dependent change of mobility in nondenaturing gel electrophoresis as vitamin D-dependent calcium-binding protein. The 28,000-Mr calcium-binding protein was induced in kidney by the injection of vitamin D in vitamin D-deficient rats.

Animals↗
PubMed1985

Enhancement of actin-activated myosin ATPase by an 84K Mr actin-binding protein in vertebrate smooth muscle.

A Ca2+-dependent actin-severing 84K Mr protein prepared from bovine aorta caused four-fold activation of smooth muscle actin-activated myosin ATPase at a 1/10(2) molar ratio to actin in the presence of tropomyosin and light chain kinase-calmodulin in a Ca2+-dependent manner, while it inhibited it markedly at a 1/5 molar ratio. Purified actin-tropomyosin filaments under the experimental ATPase conditions were distributed in a range of more than 10 micron in length and the addition of the 84K Mr protein changed the filament length to around 1 micron at a 1/10(2) molar ratio to actin or less than 50 nm at a 1/5 molar ratio in the presence of Ca2+. However, the apparent length of actin filaments alone does not appear to be responsible for the activation of ATPase activity, since in the absence of tropomyosin, the ATPase activation was much less in spite of actin filament length changes. These results indicate the possibility that the 84K Mr protein plays an important role with tropomyosin in at least in vitro smooth muscle actin-myosin interaction.

Actin Cytoskeleton↗