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Biomedical subjects

S Kanaya

Publications and source records attributed to S Kanaya.

At least 109 records · Page 6Linked to original sources
PubMed1995

Characterization of the internal motions of Escherichia coli ribonuclease HI by a combination of 15N-NMR relaxation analysis and molecular dynamics simulation: examination of dynamic models.

The backbone dynamics of Escherichia coli ribonuclease HI (RNase HI) in the picosecond to nanosecond time scale were characterized by a combination of measurements of 15N-NMR relaxation (T1, T2, and NOE), analyzed by a model-free approach, and molecular dynamics (MD) simulation in water. The MD simulations in water were carried out with long-range Coulomb interactions to avoid the artificial fluctuation caused by the cutoff approximation. The model-free analysis of the 15N-NMR relaxation indicated that RNase HI has a rotational correlation time of 10.9 ns at 27 degrees C. The generalized order parameter (S2) for the internal motions varied from 0.15 to 1.0, with an average value of 0.85, which is much larger than that of the RNase H domain of HIV-1 reverse transcriptase (0.78). Large internal motions (small order parameters) were observed in the N-terminal region (Leu2-Lys3), the loop between beta-strands A and B (Cys13-Gly15), the turn between alpha-helix I and beta-strand D (Glu61, His62), the loop between beta-strand D and alpha-helix II (Asp70-Tyr71), the loop between alpha-helices III and IV (Ala93-Lys96), the loop between beta-strand E and alpha-helix V (Gly123-His127), and the C-terminal region (Gln152-Val155). The effective correlation time observed in these regions varied from 0.45 ns (Glu61, Lys96) to 2.2 ns (Leu14). The order parameters calculated from the MD agreed well with those from the NMR experiment, with a few exceptions. The distributions of most of the backbone N-H vectors obtained by MD are approximately consistent with the diffusion-in-a-cone model. These distributions, however, were elliptic, with a long axis perpendicular to the plane defined by the N-H and N-C alpha vectors. Distributions supporting the axial fluctuation model or the jump-between-two-cones model were also observed in the MD simulation.

Binding Sites↗
PubMed1995

Desk-top analysis of the structural stability of various point mutations introduced into ribonuclease H.

The structural stability of Escherichia coli ribonuclease HI mutants was analyzed by a pseudo-energy potential developed for evaluating structure-sequence compatibility. From the structure profile, the energy changes of the folding of mutant proteins relative to that of the wild-type, which correspond to the changes of free energy differences, were estimated. They are weakly but significantly correlated with the experimentally determined changes in the melting temperature between the mutant proteins and the wild-type. The correlation coefficient between the experimental data and the computation, estimated for all the known data (96 point mutations) and for the buried site data (32 point mutations), are -0.51 and -0.68, respectively. Experimentally known mechanisms to increase the structural stability are explained by the method: the main contributor to the stability in mutations of Val74 to either Ile or Leu is the side-chain packing energy, and that of Lys95 to Gly is the local conformational energy. This analysis is easy to do on a desk-top computer, and allows one to consider all the sites of possible candidates for point mutations. From the profile, new promising sites to increase the structural stability are suggested.

Computer Simulation↗
PubMed1995

Plasma sulpho-conjugated catecholamine dynamics up to 8 h after 60-min exercise at 50% and 70% maximal oxygen uptakes.

The prolonged effects of steady-state exercise and meals on plasma sulpho-conjugated catecholamines (CA) after exercise were examined. Seven male subjects exercised on 2 separate days for 60 min at 50% and 70% of maximal oxygen uptake (VO2max) on a cycle ergometer and then rested, for 8 h sitting in an armchair. A control trial without any exercise was also performed. At 2 h after the end of exercise the subjects were given a meal. The plasma free and sulphated CA, oxygen uptake (VO2) and heart rate (HR) were all measured before exercise, during exercise and hourly during the 8-h recovery period. The sulphated noradrenaline (NA-S) and adrenaline (A-S) concentrations increased after exercise, and, furthermore, only the NA-S concentrations remained elevated for 6 h after exercise at 50% VO2max and for 8 h at 70% VO2max trial, compared with the control trial. There were no changes in either the plasma NA-S or A-S concentrations after consuming a meal, whereas the dopamine sulphate concentration demonstrated a dynamic change. A significantly higher excess postexercise VO2 was observed at 2 h postexercise at 50% VO2max and at 6 h postexercise at 70% VO2max trials. The mean HR was still elevated at 6 and 8 h after exercise, which closely correlated with the duration of the elevated NA-S concentrations. These results suggest that sulphated CA, especially NA-S, could represent an additional index of sympathetic nerve activity after exercise, and that a meal containing small amounts of the amines would seem to have no effect on plasma NA-S and A-S concentrations.

Adult↗
PubMed1995

Long-term evaluation of distal splenorenal shunt with splenopancreatic and gastric disconnection.

BACKGROUND: This study was aimed at evaluating advantages of distal splenorenal shunt (DSRS) with splenopancreatic and gastric disconnection (DSRS-SPGD) over DSRS with splenopancreatic disconnection (DSRS-SPD) and standard DSRS (S-DSRS). METHODS: DSRS-SPGD, DSRS-SPD, and S-DSRS were performed on 62, 7, and 55 patients, respectively, from 1970 to 1992. Comparison was performed in the following aspects: (1) long-term results in ratio of rebleeding, survival rate, and quality of life and (2) portal hemodynamics evaluated by preoperative and postoperative angiography. Portal blood flow was assessed by the ratio of the diameter of portal vein (PV) to superior mesenteric vein (SMV), and shunt selectivity was evaluated by selectivity grade. RESULTS: Incidence of rebleeding was significantly lower in patients who underwent DSRS-SPGD than in those who underwent S-DSRS (p < 0.05). Grade 0 and I performance status was better in patients who underwent DSRS-SPGD. Accumulated survival ratio for 5 and 7 years was 78.3% and 70.5% in patients who underwent DSRS-SPGD, 59.7% and 44.1% in patients who underwent S-DSRS, and 75% and 75% in patients who underwent DSRS-SPD. Hemodynamic evaluation showed significantly lower PV/SMV ratio and degree of change in PV/SMV ratio of patients who underwent S-DSRS and DSRS-SPD. Many patients who underwent S-DSRS and DSRS-SPD exhibited loss of shunt selectivity at grades II and III. In contrast, patients who underwent DSRS-SPGD maintained satisfactory PV/SMV ratio and selectivity grade. CONCLUSIONS: DSRS-SPGD clearly showed advantages in decrease of rebleeding and improvement of quality of life resulting from maintenance of shunt selectivity and portal blood flow.

Cause of Death↗
PubMed1995

Characterization of vitelline membrane outer layer protein I, VMO-I: amino acid sequence and structural stability.

Vitelline membrane outer layer protein I (VMO-I) tightly bound to ovomucin fibrils of hen's egg yolk membrane was characterized in terms of its amino acid sequence and structural stability. The deduced sequence of VMO-I using the conventional sequencing method is: RTREYTSVITVPNGGHWGKWGIRQFCHSGYANGFALKVEPSQFGRDDTALNGIRLRCLD- GSVIESLVGKWGTWTSFLVCPTGYLVSFSLRSEKSQGGGDDTAANNIQFRCSDEAVLVGD- DLSWGRFGPWSKRCKICGLQTKVESPQGLRDDTALNNVRFFCCK. Thus, VMO-I is composed of 163 amino acid residues with a calculated molecular weight of 17,979. The sequence confirms the cDNA sequence of VMO-I we recently determined and does not show any significant similarity to proteins compiled in the NBRF database. Two of the four disulfide bonds found in VMO-I were estimated to lie between Cys26 and Cys57 and between Cys79 and Cys110. The sequence analyses show that VMO-I contains three 53-residue internal repeats that contain distinctive regions of turns flanked by beta-sheets consistent with the recent finding that the molecule contains a new beta-fold motif, the beta-prism. The molecular characteristics of VMO-I in solution were examined by CD spectroscopy in the far and near ultraviolet regions, NMR spectroscopy, and high sensitive differential scanning calorimetry (DSC). CD spectra in the far UV region at room temperature were similar to that assigned to a random coil, while in the near UV region, small positive peaks were observed. The ellipticity in both regions decreased on raising the temperature. Proton NMR experiments showed the native structure unfolds to unordered conformations at 70 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Effects of ajmaline on non-sodium ionic currents in guinea pig ventricular myocytes.

The lack of currently available data stimulated us to investigate the electrophysiological effects of ajmaline, a classical class Ia antiarrhythmic agent, on various currents responsible for the action potential plateau and repolarization phases. The whole cell patch clamp recording technique was applied to guinea pig ventricular myocytes. Ajmaline suppressed the Ca2+ current (Ica) in a dose-dependent manner (Kd = 1.2 x 10(-5) M) without affecting the steady-state inactivation kinetics and the voltage dependency of the current-voltage relationship. Ajmaline inhibited the inward portion of the inward rectifying K+ current (IKl). Ajmaline decreased the delayed rectifier K+ current (IK) without altering the activation or deactivation time courses. All these inhibitory effects of ajmaline prolonged the action potential duration in a dose dependent manner. The inhibitory actions of ajmaline on the action potential upstroke and various currents responsible for the plateau and repolarization may contribute to the observed suppression of depolarization-induced abnormal automaticities by this agent.

Action Potentials↗
PubMed1995

Coronary dilating effects of intracoronary nicorandil. Comparison with isosorbide dinitrate.

Although nicorandil, N-(2-hydroxyethyl) nicotinamide dinitrate, is a nitrate ester, its cardiovascular action differs from that of nitrate compounds in several aspects. In this quantitative angiographic study, the acute coronary dilating effect of intracoronary nicorandil (0.25, 0.50, 1.0 mg) was compared with that of isosorbide dinitrate (ISDN; 1.0 mg) in 46 patients with or without ischemic heart disease (IHD). Dose-dependent right coronary dilating action was observed by intracoronary administration of nicorandil without any adverse effects. The same degree of right coronary dilation was achieved by the intracoronary application of equivalent doses of ISDN. We conclude that intracoronary administration of nicorandil is beneficial for the supportive treatment of IHD during coronary artery investigation and intervention without the risk of severe systemic hypotension.

Angina Pectoris↗
PubMed1995

[Transendoscopic microvascular Doppler sonography for the assessment of hemodynamics of esophageal and gastric varices].

The importance of unravel hemodynamics at the stomach and esophagus in patients with portal hypertension has been described for understanding pathophysiology and treatment of varices associated with portal hypertension. Microvascular Doppler sonogrlaphy (MF20) is a pulse Doppler method with 20 MHz which is able to measure velocity and direction of blood flow in microvessel. We invented transendoscopic microvascular Doppler sonography (EMDS) by connecting a tiny probe of one mm in diameter with an endoscopy. We evaluated direction of blood flow in the esophageal and gastric varices by EMDS and angiography in 21 untreated and 26 shunt-operated patients with portal hypertension. In all of untreated patients direction of blood flow in the varices was toward the probe (forward). Results obtained with EMDS were consistent with angiographic results available in 13 patients. Direction of blood flow after shunt operation was away from the probe in 21 out of 26 patients. The comparison with angiography revealed that post-operative patients with "away" blood flow in varices had maintained satisfactory shunt selectivity without hepatofugal collateral and it proved that the direction of blood flow was toward the shunt at lower pressure. In conclusion EMDS is useful as an evaluation method for hemodynamics in varices of the esophagus and stomach.

Adult↗
PubMed1994

Kinetic analyses of DNA-linked ribonucleases H with different sizes of DNA.

A series of DNA-linked ribonucleases H with DNA adducts varying in size and sequence, ranging from heptamer to nonamer, were constructed and examined for their ability to cleave the 12-base RNA (5'-CGGAGAUGACGG-3') site-specifically. The DNA-linked RNase H with the 9-base DNA (5'-GTCATCTCC-3') cleaved the 12-base RNA specifically at A6-U7. Kinetic studies revealed that the DNA-linked RNase H with the 8-base DNA (5'-TCATCTCC-3') cleaved it slightly more effectively than that with the 9-base DNA. Factors that may affect the specificity and catalytic efficiency of a DNA-linked RNase H are described.

Base Sequence↗
PubMed1994

A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions.

A strategy to genetically select Escherichia coli ribonuclease HI mutants with enhanced thermostability is described. E. coli strain MIC3001, which shows an RNase H-dependent, temperature-sensitive growth phenotype, was used for this purpose. Introduction of the rnhA gene permits the growth of this temperature-sensitive strain, whereas the gene for the truncated protein, 142-RNase HI, which lacks the carboxyl-terminal 13 residues, cannot. Analyses of the production levels and the stability of a series of mutant proteins with COOH-terminal truncations suggested that 142-RNase HI is nonfunctional in vivo because of a dramatic decrease in the protein stability. Polymerase chain reaction-mediated random mutagenesis of the rnhA142 gene, encoding 142-RNase HI, followed by selection of revertants, allowed us to isolate 11 single amino acid substitutions that render 142-RNase HI functional in vivo. Of them, eight substitutions were shown to enhance the thermal stability of the wild-type RNase HI protein, and of these, six were novel. The genetic selection strategy employed in this experiment was thus shown to be effective for identifying amino acid substitutions that enhance the thermal stability of E. coli RNase HI. Such a strategy would be versatile if a protein of interest could be destabilized by a deletion or a truncation and a conditional-lethal strain were available.

Amino Acid Sequence↗
PubMed1994

Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR.

All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.

Amino Acid Sequence↗
PubMed1994

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis.

The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the alpha V helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. coli strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins [D134H]RNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp134 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the alpha V helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its delta 1 position impacts catalysis.

Amino Acid Sequence↗
PubMed1994

Sequence-specific cleavage of RNA by a hybrid ribonuclease H.

Site-specific cleavage of the 22-, 132- and 534-base RNAs by the DNA/protein hybrid RNase H were examined. The 22-base RNA was chemically synthesized, and 132- and 534-base RNAs were prepared by run-off transcription. The hybrid enzyme cleaves these RNAs, which contain a single target sequence, primarily at the unique phosphodiester bond within the target sequence. The hybrid enzyme performs multiple turnovers, and at a substrate/enzyme ratio of 10:1 the RNAs are almost completely cleaved by the hybrid enzyme at 37 degrees C within 1 h. We propose that hybrid RNase H molecules with various oligodeoxyribonucleotides function as RNA restriction enzymes and are useful for structural and functional studies of RNA.

Base Sequence↗
PubMed1994

Overproduction, purification, and characterization of ferrochelatase from Escherichia coli.

To establish a system for overproduction of the ferrochelatase [EC 4.99.1.1] from Escherichia coli, a plasmid designated pFC3 was constructed. The 35-kDa protein was accumulated in E. coli DH5 alpha cells that harbored pFC3 to a level equal to approximately 9% of the total protein (roughly 50 mg/liter) upon thermal induction. This 35-kDa protein was identified as the ferrochelatase of E. coli by Western blotting and amino-terminal amino acid sequence analysis. The protein with ferrochelatase activity was purified from the cells by three simple steps with a yield of 17%. The optimum pH of the purified enzyme was around 8.0. The molecular weight of the enzyme was estimated to be 35-kDa from column chromatography on Sephacryl S-300, a value consistent with that estimated from SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a monomer. The isoelectric point of the enzyme was approximately 4.7. Determination of the far-ultraviolet circular dichroism spectrum allowed us to calculate the alpha-helix and beta-sheet contents of the enzyme as 10 +/- 0.2 and 39 +/- 0.2%, respectively. High-level production of the ferrochelatase from E. coli will greatly facilitate detailed structural analysis of this protein.

Amino Acid Sequence↗
PubMed1994

Effects of hydrocortisone on transmembrane currents in guinea pig ventricular myocytes--possible evidence for positive inotropism.

Steroid hormones are thought to have a positive inotropic action, but the precise electrophysiological effects which underlie this positive inotropism remain unknown. The effects of hydrocortisone on the L-type calcium current (ICa) of the isolated guinea pig ventricular cells were investigated under the whole-cell voltage-clamp recording. Hydrocortisone (10(-6) to 10(-5) M) increased the amplitude of ICa without any changes in the voltage-dependency in ICa. This agent retarded the fast inactivation component of ICa, but not the slow component. On the other hand, hydrocortisone slightly reduced the slope conductance of inward rectifying potassium current (IK1) at potentials less than -50mV. This effect on IK1 is favorable for the activation of ICa. Thus, these results indicate that the positive inotropism of hydrocortisone is mediated, at least in part, by a direct potentiating effect on ICa.

Animals↗
PubMed1994

Thrombotic obstruction of the right coronary artery in a postoperative patient with Bland-White-Garland syndrome.

A 53-year-old female presented with symptoms of severe chest and back pain associated with oliguria. The patient had a history of exertional dyspnea since the age of 20, and easy fatigability since the age of 27. At the age of 41, she noted marked exacerbation of these symptoms after suffering from a cold and was ultimately diagnosed as having Bland-White-Garland (BWG) syndrome with mitral valve regurgitation. The patient then underwent re-implantation of an anomalous left coronary artery from the pulmonary artery to the posterolateral wall of the aorta. Eleven years later, she re-presented with symptoms of angina and congestive heart failure. Coronary angiography was subsequently performed and a total occlusion of the right coronary artery with probable thrombus was revealed. The right coronary artery was filled via collaterals from the implanted left coronary artery. Mitral regurgitation was noted during angiography. The patient underwent aorto-coronary artery bypass grafting of the right coronary artery and concomitant mitral valve replacement. Her postoperative condition remained excellent.

Coronary Angiography↗