Biomedical subjects

S Kanaya

Publications and source records attributed to S Kanaya.

At least 91 records · Page 5Linked to original sources

PubMed1997

An effective chemotherapy regimen for liver metastasis from retroperitoneal fibrosarcoma: report of a case.

Retroperitoneal fibrosarcoma is a rare disease that has proven difficult to treat due to its high incidence of postoperative local recurrence. We recently experienced a patient in whom retroperitoneal fibrosarcoma was followed by liver metastasis without local recurrence. A 34-year-old woman who initially presented with right upper quadrant pain was found to have a retroperitoneal tumor by diagnostic imaging techniques. Extirpation of the tumor was performed and the histopathological diagnosis was fibrosarcoma. A solitary metastasis was detected in the lateral segment 1 year after this operation and a lateral segmentectomy was carried out; however, a short time later, multiple liver metastases were found. Initially, ethanol injections were given with little effect, following which CYVADIC chemotherapy, consisting of cyclophosphamide, vincristine, farmorubicin, and dacarbazine was administered. An excellent responsiveness without severe toxicity was achieved after five cycles, with a significant reduction in tumor size, being estimated as a complete response. Thus, we consider that this chemotherapy regimen could be a promising mode of treatment for liver metastasis from retroperitoneal fibrosarcoma without local recurrence.

Adult↗

PubMed1996

Thermal stability of Escherichia coli ribonuclease HI and its active site mutants in the presence and absence of the Mg2+ ion. Proposal of a novel catalytic role for Glu48.

Escherichia coli ribonuclease HI, which requires divalent cations (Mg2+ or Mn2+) for activity, was thermostabilized by 2.6-3.0 kcal/mol in the presence of the Mg2+, Mn2+, or Ca2+ ion, probably because the negative charge repulsion around the active site was canceled upon the binding of these metal ions. The dissociation constants were determined to be 0.71 mM for Mg2+, 0.035 mM for Mn2+, and 0.16 mM for Ca2+. Likewise, various active site mutants at Asp10, Glu48, Asp70, or Asp134 were thermostabilized by 0.4-3.0 kcal/mol in the presence of the Mg2+ ion, suggesting that this ion binds to these mutant proteins as well. The dissociation constants of Mg2+ were determined to be 9.8 mM for D10N, 1.1 mM for E48Q, 18.8 mM for D70N, and 1.8 mM for D134N. Thus, the mutation of Asp10 or Asp70 to Asn considerably impairs the Mg2+ binding, whereas the mutation of Glu48 to Gln or Asp134 to Asn does not. Comparison of the thermal stability of the mutant proteins with that of the wild-type protein in the absence of the Mg2+ ion suggests that the negative charge repulsion between Asp10 and Asp70 is responsible for the binding of the metal cofactor. Glu48 may be required to anchor a water molecule, which functions as a general acid.

Anions↗

PubMed1996

Interaction of the basic protrusion of Escherichia coli ribonuclease HI with its substrate.

In order to determine the actual distance between the active site and the substrate binding site, termed the basic protrusion, of Escherichia coli ribonuclease HI, synthetic oligonucleotide duplexes with gradually extended overhangs were used, in which the enzymatic cleavage was restricted to a single site with 2'-O-methylnucleosides. The affinity of the enzyme for each substrate was determined by kinetic analysis. It was found that the affinity increased markedly when one nucleotide was attached to the 3' end of the DNA strand of the nine-base-pair hybrid duplex and then increased slightly as the DNA strand was extended further, whereas elongation of the strand in the other direction caused no change. When a mutant enzyme, in which three lysine residues in the basic protrusion were altered to alanine, was used, no increase in the kcat/K(m) value was observed. The results indicate that, for the productive binding, the axis from the 3' to the 5' end of the RNA strand of the substrate duplex must be oriented in agreement with the direction from the active site to the basic protrusion of the enzyme. The distance between the active site and the basic protrusion in the enzyme-substrate complex was shorter than that anticipated in modeling studies. A dynamic structure refinement, referred to as the normal mode analysis, was carried out in order to simulate the fluctuations of the basic protrusion.

Escherichia coli↗

PubMed1996

Unusual enzyme characteristics of aspartyl-tRNA synthetase from hyperthermophilic archaeon Pyrococcus sp. KOD1.

The aspA gene, encoding the aspartyl-tRNA synthetase (AspRS) from the hyperthermophilic archaeon Pyrococcus sp. KOD1, was expressed in Escherichia coli. The KOD1 AspRS, which was purified to homogeneity and was shown to be functional in dimeric form, aminoacylated tRNA from KOD1. The optimum temperature for this activity was 65 degrees C, which was lower than that for the cell growth of KOD1 (85 degrees C). However, it increased to 75 degrees C by the addition of polyamine molecules, such as putrescine, spermine, and spermidine. Analysis of the thermal denaturations of the enzyme and of KOD1-tRNA indicated that neither of them was denatured at temperatures below 70 degrees C. These results suggest polyamine is one of the factors which are required to stabilize the AspRS-tRNA complex in vivo. In order to determine whether the nucleotide triphosphate (NTP) is required for Asp-tRNA synthesis, the aminoacylation was examined in the presence of each of the four NTPs. AspRS most effectively aminoacylated tRNA in the presence of ATP. However, we also found that the enzyme aminoacylated it even in the presence of GTP and UTP as well. Archaeon synthetase may have an interesting system to utilize other NTPs than ATP. The extreme conditions of early life may have given rise to these unique characteristics which then disappeared from developed organisms through evolution.

Archaea↗

PubMed1996

Codon usage tabulated from the international DNA sequence databases.

Codon usage in 87 602 genes has been calculated using the nucleotide sequence data obtained from the GenBank Genetic Sequence Data Bank (Release 90.0; September 1995). The database is called the CUTG Database; the complete form of the database can be obtained by anonymous ftp from DDBJ and a part of the database, which lists the frequency of codon use in each organism, is made searchable through our World Wide Web server.

Base Sequence↗

PubMed1996

Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy.

The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). 1H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pKa values of the histidine residues obtained using 1H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D 1H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.

Amino Acid Sequence↗

PubMed1996

Synchronous gene expressions during embryogenesis of Oncorhynchus masou (Yamame).

In order to detect periods of synchronous gene expressions during embryogenesis of Oncorhynchus masou (Yamame), we analyzed the peptide constituents of the embryos by means of two-dimensional electrophoresis. New kinds of peptides were mainly detected in three periods, i.e. between the 12th and 13th day, between the 20th and 21st day, and between the 30th and 31st day after fertilization. Hatching occurred between the 34th and 37th day. These indicate that these three periods, which corespond to (1) the shifting stage from maternal to nuclear events in gene expression, (2) the development of external organs such as fins, and (3) the preparative stage for hatching, respectively, may be critical stages in gene expression.

Animals↗

PubMed1996

Detection of genes in Escherichia coli sequences determined by genome projects and prediction of protein production levels, based on multivariate diversity in codon usage.

We used principal component analysis to develop measures (called Z-parameters in this study) which reflect the diversity of codon usage in Escherichia coli genes. Protein production levels for 1500 CDSs (protein-coding sequences) identified by E.coli genome projects in Japan and the US were estimated from a correlation equation between Z1 and cellular protein content obtained through analysis of the genes experimentally characterized. Through the profile analysis of Z1 for E.coli sequences obtained by the Japanese Project, we predicted an additional 36 CDSs that had not been annotated in the International DNA Database. Thirty-one out of the 36 CDSs could be assigned to presumptive protein genes through a BLASTX search for recent protein databases in the Genome Net in Japan. Detailed examination of the Z1-parameter profile led us to assess sequencing errors which cause frame-shift.

Algorithms↗

PubMed1996

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI.

Three mutants of Escherichia coli ribonuclease HI, in which an invariant acidic residue Asp134 was replaced, were crystallized, and their three-dimensional structures were determined by X-ray crystallography. The D134A mutant is completely inactive, whereas the other two mutants, D134H and D134N, retain 59 and 90% activities relative to the wild-type, respectively. The overall structures of these three mutant proteins are identical with that of the wild-type enzyme, except for local conformational changes of the flexible loops. The ribonuclease H family has a common active site, which is composed of four invariant acidic residues (Asp10, Glu48, Asp70 and Asp134 in E.coli ribonuclease HI), and their relative positions in the mutants, even including the side-chain atoms, are almost the same as those in the wild-type. The positions of the delta-polar atoms at residue 134 in the wild-type, as well as D134H and D134N, coincide well with each other. They are located near the imidazole side chain of His124, which is assumed to participate in the catalytic reaction, in addition to the four invariant acidic residues. Combined with the pH profiles of the enzymatic activities of the two other mutants, H124A and H124A/D134N, the crystallographic results allow us to propose a new catalytic mechanism of ribonuclease H, which includes the roles for Asp134 and His124.

Aspartic Acid↗

PubMed1996

Reconstruction of the mitral annulus with porcine pericardium--report of a case with mitral annular disruption due to staphylococcal endocarditis.

A 60-year-old man was admitted to our hospital for investigation of dyspnea and disorientation with right hemiplegia. Echocardiography showed thickened mitral valve leaflets with vegetations and severe mitral regurgitation. Blood cultures grew Staphylococcus aureus. During the operation, perforation and destruction of the mitral valve leaflets and vegetations were confirmed. Debridement of the infected tissues resulted in segmental disruption of the posterior mitral fibrous annulus. Reconstruction of the mitral annulus with porcine pericardium treated with glutaraldehyde and mitral valve replacement were successful. The patient's postoperative course was complicated with metastatic cerebral and splenic abscesses. After splenectomy on the 8th postoperative day, he gradually recovered without major neurologic sequelae. We believe that reconstruction of the mitral valve annulus with pericardium, especially autologous pericardium, is reliable and useful for the treatment of patients with disruption of the mitral valve annulus.

Animals↗

PubMed1996

[Effect of reconstruction arc in myocardial SPECT imaging--comparison between 180 degrees and 360 degrees arc--].

A series of thallium-201 (201Tl) and technetium-99m (99mTc) myocardial perfusion images with or without perfusion defects were acquired by 360 degrees arc using a triple-head camera. The vertical long, horizontal long, and short axis images of 201Tl and 99mTc with 360 degrees, 180 degrees, and opposite 180 degrees were identically reconstructed and reoriented without attenuation correction. In order to detect the detailed differences between 360 degrees (30 degrees - 390 degrees) and 180 degrees (30 degrees - 210 degrees) images, the opposite 180 degrees (210 degrees - 30 degrees) images were utilized. Opposite 180 degrees images revealed the contribution of those to the 360 degrees images. In the normal perfusion images, the CV (coefficient of variation) of the counts in 360 degrees images with 201Tl by the segments was larger than 180 degrees images. On the contrary, the CV of the counts in 360 degrees images with 99mTc was smaller than 180 degrees images. This difference, caused by the difference of photon energy, were detected mainly as the extent of counts decreasing in the deep segments in the opposite 180 degrees images. Lesion contrasts of the opposite 180 degrees images were lower than those of 180 degrees images due to distance related collimator response (the detector response of the collimated gamma camera varies with source-to-detector distance) and scatter. Therefore, lesion contrasts were more decreased in the 360 degrees images compared with those of 180 degrees images, especially in the segments of lateral and anterior walls. The differences in lesion contrasts between 201Tl and 99mTc images were mainly in the segments of inferior and posterior walls (p < 0.05). Lesion contrast was dependent on the location of the perfusion defects, reconstruction arc, and photon energy.

Aged↗

PubMed1996

[Exposure to the technologists from radioactive patients during nuclear medicine studies].

In order to evaluate the exposure to the nuclear medicine technologists from patients who had been administrated with radiopharmaceuticals, we measured the exposure in 5 common diagnostic procedures (bone, lung, tumor scan, and brain, myocardial SPECT, n = 8 to 52) using a silicon semiconductor pocket dosimeter. We also measured the spatial dose rates at 5 cm, 50 cm, and 100 cm from skin surface of the patients (n = 10 to 21) using an ionization chamber, both 5 min after injection and right before the studies with the same procedures above. We further measured the spatial dose rate distributions around the patients in the 4 procedures (bone, renal, blood pool scan, and brain SPECT, n = 2 to 3). In results, the exposure to the technologists in each procedure was small (0.5, 0.5, 0.7, 1.6, and 0.3 muSv in each bone, lung, tumor scan, and brain, myocardial SPECT, respectively), compared with the dose limits of the medical workers. However, the dose-response relationships in cancer and hereditary effects, referred to as the stochastic effects, have been assumed linear and no threshold models; therefore, the exposure should be minimized. For this purpose, the measurements of spatial dose rates and spatial dose rate distributions were thought to be useful. The differences of these results among procedures were caused by the differences of dose distributions and physical and biological half lives of the radiopharmaceuticals. The results of the measurements in 7 consecutive weeks suggested that the direct measurement of the exposure using a high sensitive digital pocket dosimeter might result a reduced exposure to the technologists.

Adult↗

PubMed1996

[Factors predictive of early mortality after acute myocardial infarction].

OBJECTIVES: (1) To define the clinical and laboratory findings predictive of early mortality after myocardial infarction (AMI), 100 consecutive patients were studied. (2) To elucidate the serum potassium (K) concentration and its clinical significance after AMI, 38 patients were studied. RESULTS: (1) The 30-day mortality was greater for women than for men, for patients with diabetes mellitus than for those without, and for patients with previous MI than for those without. Clinical and laboratory findings predictive of early mortality were cardiogenic shock, congestive heart failure, perforation or rupture of the ventricle, and presence of bundle branch block and extensive anterior AMI on electrocardiogram. The presence of bradyarrhythmia, ventricular tachyarrhythmias, and high blood levels of peak creatinine phosphokinase (CPK) or myosin or atrial natriuretic peptide (ANP) were not independent predictors of early mortality. Patients complicated with congestive heart failure showed higher levels of peak CPK and ANP and patients with cardiogenic shock showed higher levels of ANP. (2) Serum K concentration was lower on admission than the following days, and it showed a negative correlation with plasma epinephrine. The lower serum K was accompanied with more severe ventricular arrhythmias on 24-hour electrocardiogram. CONCLUSION: (1) Early mortality after AMI is related to left ventricular function rather than to arrhythmias. (2) Serum K is lower in the early phase of AMI and is related with plasma catecholamines and severe ventricular arrhythmias.

Aged↗

PubMed1996

[Effects of exercise on mitral regurgitation in healthy subjects].

Transthoracic echocardiography including color Doppler echocardiography and graded exercise (supine bicycle ergometer) were performed to examine both the changes of mitral regurgitation (MR) flow during exercise and the effects of cathecholamine on the MR flow in 20 young males (mean age 19 years) and 9 male long-distance runners (mean age 20 years) with presumably normal hearts. MR flow was detected in 13 of the 20 young males at rest. The severity of MR was mild in 12 subjects, and moderate in 1. After exercise, MR flow vanished in 4 of the 13 subjects (group B). In the other nine subjects, MR flow lasted during and after exercise (group A). In the seven subjects without MR flow at rest, MR flow did not appear during and after exercise (group C). Group A had significantly lower left ventricular ejection fraction than group C at rest (A 64.9 +/- 4.25%, B 69.4 +/- 4.32%, C 73.9 +/- 3.46%; p < 0.05). However, no significant differences were observed in other echocardiographic parameters (including left ventricular end-diastolic dimension, mitral annulus diameter and left atrial diameter), heart rate and serum cathecholamine levels between the three groups before and after exercise. In the nine male long-distance runners, MR flow was detected in six of nine subjects at rest. The severity of MR was mild in all subjects. After exercise, MR flow vanished in two of these six subjects (group A). In the other four subjects, MR flow lasted during and after exercise (group B). In three subjects without MR flow at rest, MR flow did not appear during and after exercise (group C). There were no significant differences in echocardiographic parameters, heart rate and serum cathecholamine levels between the three groups before and after exercise. These data suggest that MR flow detected in healthy subjects correlates with tension and contractility of papillary muscle.

Adolescent↗

PubMed1995

Folding pathway of Escherichia coli ribonuclease HI: a circular dichroism, fluorescence, and NMR study.

The unfolding and refolding processes of Escherichia coli ribonuclease HI at 25 degrees C, induced by concentration jumps of either guanidine hydrochloride (GuHCl) or urea, were investigated using stopped-flow circular dichroism (CD), stopped-flow fluorescence, and NMR spectroscopies. Only a single exponential process was detected for the fast time scale unfolding (rate constants from 0.014 to 0.54 s-1, depending on the final denaturant concentration). For refolding, the far-UV CD value largely recovered within 50 ms of the stopped-flow mixing dead time (burst phase). This phase was followed by either one or two phases, with rate constants from 0.035 to 2.45 s-1 as detected by CD and fluorescence, respectively. Although this protein has a single cis-Pro residue, a very slow phase due to proline isomerization was not observed, for either unfolding or refolding. The difference in the amplitudes of the burst phases for refolding in the far- and near-UV CD spectra revealed that an intermediate state exists, with the characteristics of a molten globule. Because the one-phased fast exponential process detected by CD corresponds to the slower of the two phases detected by fluorescence, the intermediate detected by CD might be the most stable. GuHCl denaturation experiments revealed that this intermediate cooperatively unfolds, with a transition midpoint of 1.33 +/- 0.03 M. The Gibbs free energy difference (delta G) between the intermediate and the unfolded states, under physiological conditions (25 degrees C, pH 5.5, and 0 M GuHCl), was estimated to be 20.0 +/- 2.3 kJ mol-1. Therefore, it is reasonable to assume that the refolding intermediate, rather than the unfolded state, is the latent denatured state under physiological conditions. Approximately linear relationships between the GuHCl concentration and the logarithm of the microscopic rate constants determined by CD and fluorescence were also observed. By extrapolation to a GuHCl concentration of 0 M, activation Gibbs free energies of 98.5 +/- 1.1 kJ mol-1 for unfolding and 69.5 +/- 0.2 kJ mol-1 for refolding under physiological conditions were obtained. The hydrogen-exchange-refolding competition combined with two-dimensional NMR revealed that the amide protons of alpha-helix I are the most highly protected, suggesting that alpha-helix I is the initial site of protein folding. The CD and NMR data showed that the intermediate state has a structure similar to that of the acid-denatured molten globule.

Amino Acid Sequence↗

PubMed1995

Reconstitution of Escherichia coli RNase HI from the N-fragment with high helicity and the C-fragment with a disordered structure.

The Escherichia coli RNase HI variant with the Lys86-->Ala mutation is purified in two forms, as nicked and intact proteins. The nicked K86A protein, in which the N-fragment (Met1-Lys87) and the C-fragment (Arg88-Val155) remain associated, is enzymatically active. These N- and C-fragments were isolated and examined for reassociation. These peptides did not associate to form the nicked K86A protein at pH 3.0 in the absence of salt, but were associated, with a yield of 30-80%, when the pH was raised to 5.5 or when salt was added. Measurements of the CD spectra show that the alpha-helices are partially formed in the N-fragment at pH 3.0 in the absence of salt and are almost fully formed either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl. In contrast, the C-fragment remains almost fully disordered under these conditions. The N-fragment with this high (native-like) helicity shows the characteristics of a molten globule with respect to the content of the secondary and tertiary structures, the ability to bind a fluorescent probe (1-anilinonaphthalene-8-sulfonic acid), and the behavior on the thermal transition. These results suggest that the N-fragment contains an initial folding site, probably the alpha I-helix, and the completion of the folding in this site provides a surface that facilitates the folding of the C-fragment. This folding process may represent that of the intact RNase HI molecule.

Binding Sites↗

PubMed1995

Kinetic analysis of Escherichia coli ribonuclease HI using oligomeric DNA/RNA substrates suggests an alternative mechanism for the interaction between the enzyme and the substrate.

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-C10. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding.

Base Sequence↗

PubMed1995

High resistance of Escherichia coli ribonuclease HI variant with quintuple thermostabilizing mutations to thermal denaturation, acid denaturation, and proteolytic degradation.

To test whether the combination of multiple thermostabilizing mutations is a useful strategy to generate a hyperstable mutant protein, five mutations, Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His or Asn, were simultaneously introduced into Escherichia coli ribonuclease HI. The enzymatic activities of the resultant quintuple mutant proteins, 5H- and 5N-RNases HI, which have His and Asn at position 134, respectively, were 35 and 55% of that of the wild-type protein. The far-UV and near-UV CD spectra of these mutant proteins were similar to those of the wild-type protein, suggesting that the mutations did not seriously affect the tertiary structure of the protein. The differences in the free energy change of unfolding between the wild-type and mutant proteins, delta delta G, were estimated by analyzing the thermal denaturation of the proteins by CD. The 5H-RNase HI protein, which was slightly more stable than the 5N-RNase HI, was more stable than the wild-type protein by 20.2 degrees C in Tm and 5.6 kcal/mol in delta G at pH 5.5. In addition, the 5H-RNase HI was highly resistant to proteolysis and acid denaturation. The effects of each mutation on the thermal stability and the susceptibility to chymotryptic digestion were nearly cumulative, and the 5H-RNase HI undergoes chymotryptic digestion at a rate that is 41 times slower than that of the wild-type protein. Good correlation was observed between the thermal stability and the resistance to chymotryptic digestion for all proteins examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗