Biomedical subjects

S Kanaya

Publications and source records attributed to S Kanaya.

At least 55 records · Page 3Linked to original sources

PubMed1999

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.

Amino Acid Sequence↗

PubMed1999

A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon.

A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

Adenosine Triphosphatases↗

PubMed1999

Identification of the genes encoding Mn2+-dependent RNase HII and Mg2+-dependent RNase HIII from Bacillus subtilis: classification of RNases H into three families.

Database searches indicated that the genome of Bacillus subtilis contains three different genes encoding RNase H homologues. The ypdQ gene encodes an RNase HI homologue with 132 amino acid residues, whereas the rnh and ysgB genes encode RNase HII homologues with 255 and 313 amino acid residues, respectively. RNases HI and HII show no significant sequence similarity. These genes were individually expressed in Escherichia coli; the recombinant proteins were purified, and their enzymatic properties were compared with those of E. coli RNases HI and HII. We found that the ypdQ gene product showed no RNase H activity. The 2.2 kb pair genomic DNA containing this gene did not suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that this gene product shows no RNase H activity in vivo as well. In contrast, the rnh (rnhB) gene product (RNase HII) showed a preference for Mn2+, as did E. coli RNase HII, whereas the ysgB (rnhC) gene product (RNase HIII) exhibited a Mg2+-dependent RNase H activity. Oligomeric substrates digested with these enzymes indicate similar recognition of these substrates by B. subtilis and E. coli RNases HII. Likewise, B. subtilis RNase HIII and E. coli RNase HI have generated similar products. These results suggest that B. subtilis RNases HII and HIII may be functionally similar to E. coli RNases HII and HI, respectively. We propose that Mn2+-dependent RNase HII is universally present in various organisms and Mg2+-dependent RNase HIII, which may have evolved from RNase HII, functions as a substitute for RNase HI.

Amino Acid Sequence↗

PubMed1999

Na+-K+ pump inhibition caused by chronic amiodarone in guinea pig myocardium.

Although it is known that amiodarone inhibits myocardial Na+-K+ pump activity, the potency and the time course of this inhibition are unknown. The aim of this study was to investigate these aspects with reference to digoxin, using guinea pigs treated with either intraperitoneal amiodarone (20mg/kg per week, up to 12 weeks, n = 26) or the same amount of vehicle as a control (n = 24). ECG recording and microelectrode experiments were conducted every 2 weeks. QT interval corrected by heart rate and action potential duration were prolonged as a function of the time of exposure to amiodarone. Hyperpolarization observed immediately after the overdrive (1.0Hz) termination or K+-replenishment following K+-depletion in the presence of 0.1mM Ba2+ was compared in the amiodarone-treated and untreated groups, as an index of the Na+-K+ pump activity. The resting membrane potential recovery from overdrive-induced depolarization was slower and the amplitude of K+-induced hyperpolarization was smaller in the amiodarone-treated group than in the untreated group. These changes were evident as the chronic amiodarone treatment progressed, although the changes in these parameters were greater in the case of acute application of 50 microM digoxin. In conclusion, this study indicates that treatment with amiodarone for longer than several weeks moderately inhibits the myocardial Na+-K+ pump.

Amiodarone↗

PubMed1999

Effects of oral repetitive loading of disopyramide on acute-onset atrial fibrillation with concurrent monitoring of serum drug concentration.

We investigated the efficacy and safety of oral repetitive loading of disopyramide, for the termination of acute-onset (i.e., therapy started within 2 days after the onset of palpitations) atrial fibrillation (AF) in 96 consecutive patients, with concurrent monitoring of the serum concentration of this agent in fifteen of the patients. Outpatients with AF verified by standard electrocardiogram (ECG) were hospitalized and received disopyramide (200 mg) every 4-6 h, with a maximal dose of 800 mg daily, until the termination of AF under ECG monitoring was obtained. Conversion to sinus rhythm occurred within the first day of treatment in 88 patients (92%), on the second day of treatment in six patients (6%), and on the third and fifth days of treatment in the remaining two patients. No major adverse effects, such as hypotension, congestive heart failure, proarrythmic events or systemic embolism were noted. The serum levels of disopyramide evaluated in fifteen of the enrolled patients were found to be maintained within the therapeutic range throughout the treatment period. In spite of the absence of any placebo-controlled group in this study, these findings suggest that repetitive oral loading of disopyramide (200 mg) with an interval of 4-6 h is effective and safe for the termination of acute-onset AF under a stable therapeutic serum drug concentration, hence offering the possibility of self-medication for patients with episodic AF.

Acute Disease↗

PubMed1999

Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium, strain HD-1.

The hd-ald gene encoding aldehyde dehydrogenase (hd-ALDH) from an mixotrophic petroleum-degrading bacterium, strain HD-1 was cloned and sequenced. hd-ALDH (506 amino acids) is a member of the NAD+-dependent aldehyde dehydrogenase group. The hd-ald gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically and enzymatically. The molecular weight of the enzyme was estimated to be 55,000 by SDS-PAGE, and 224,000 by gel filtration chromatography, suggesting that it acts as a tetramer. The CD spectrum suggests that the helical content of the enzyme is 10%. hd-ALDH was active on various aliphatic aldehyde substrates. The K(m) values of the enzyme were 6.4 microM for acetaldehyde, 4.2 microM for hexanal, 2.8 microM for octanal, and 0.84 microM for decanal, whereas the kcat values for these substrates were nearly equal (51-64 min(-1)). These results indicate that hd-ALDH acts preferentially on long-chain aliphatic aldehydes.

Journal Article↗

PubMed1999

Molecular diversities of RNases H.

RNase H is an enzyme that specifically cleaves RNA hybridized to DNA. The enzyme is ubiquitously present in various organisms. Single bacterial and eucaryotic cells often contain two RNases H, whereas single archaeal cells contain only one. To determine whether there is a physiological significance in the ubiquity and multiplicity of the enzyme, and whether all enzymes are evolutionarily diverged from a common ancestor, we carried out phylogenetic analyses of the RNase H sequences. In this report, we demonstrated that RNases H are classified into two major families, Type 1 and Type 2 RNases H, of which only the Type 2 enzymes are present in all living organisms, including bacteria, archaea, and eucaryotes, suggesting that they represent an ancestral form of RNases H. Based on this information, we discuss the evolutionary relationships and possible cellular functions of RNases H.

Journal Article↗

PubMed1999

Characterization of petroleum-degrading bacteria from oil-contaminated sites in Vietnam.

Four petroleum-degrading bacterial strains, 2TN-NB, 6TBX-CL, MVK2-5, and XCK, were isolated from various oil-contaminated sites in Vietnam. Determination of the nucleotide sequence of the gene encoding 16S rRNA allowed 2TN-NB to be identified as Acinetobacter sp. and the other three stains as Pseudomonas sp. Among the four isolates, 2TN-NB was most effective in degrading crude oil: in 1 d, it degraded 95% of the crude oil in the culture medium (5%, v/v). The isolated strains could also degrade a sulfur-containing aromatic hydrocarbon, dibenzothiophene (DBT), with low efficiency. Except for MVK2-5, which degraded crude oil least efficiently, the isolates produced biosurfactants in amounts sufficient for structural analysis. FT-IR measurement suggested that strains 6TBX-CL and XCK produced glycolipid-type biosurfactants while that produced by 2TN-NB was of the polysaccharide type.

Journal Article↗

PubMed1999

The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages.

phi CTX is a cytotoxin-converting phage isolated from Pseudomonas aeruginosa. In this study, we determined the complete nucleotide sequence of the phi CTX phage genome. The precise genome size was 35,538 bp with 21 base 5'-extruding cohesive ends. Forty-seven open reading frames (ORFs) were identified on the phi CTX genome, including two previously identified genes, ctx and int. Among them, 15 gene products were identified in the phage particle by protein microsequencing. The most striking feature of the phi CTX genome was an extensive homology with the coliphage P2 and P2-related phages; more than half of the ORFs (25 ORFs) had marked homology to P2 genes with 28.9-65.8% identity. The gene arrangement on the genome was also highly conserved for the two phages, although the G + C content and codon usage of most phi CTX genes were similar to those of the host P. aeruginosa chromosome. In addition, phi CTX was found to share several common features with P2, including the morphology, non-inducibility, use of lipopolysaccharide core oligosaccharide as receptor and Ca(2+)-dependent receptor binding. These findings indicate that phi CTX is a P2-like phage well adapted to P. aeruginosa, and provide clear evidence of the intergeneric spread and evolution of bacteriophages. Furthermore, comparative analysis of genome structures of phi CTX, P2 and other P2 relatives revealed the presence of several hot-spots where foreign DNAs, including the cytotoxin gene, were inserted. They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection.

Amino Acid Sequence↗

PubMed1999

Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1.

The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus. The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.

Amino Acid Sequence↗

PubMed1999

Video-assisted transsternal radical esophagectomy: three-field lymphadenectomy without thoracotomy for esophageal cancer.

To reduce the invasiveness of radical esophagectomy, we developed a new approach: video-assisted transsternal radical esophagectomy (VATRE). This article presents the operative techniques and our initial results. In our new procedure, cervical U-shaped and longitudinal sternoabdominal incisions are made, and a complete midline sternotomy is carried out. Lymph node clearance from the neck to the upper mediastinum and from the lower mediastinum to the upper abdomen is performed under direct vision. In the middle mediastinum, a video-assisted technique is used to dissect the lymph nodes. After esophageal resection and three-field lymphadenectomy, reconstruction is performed. One-lung ventilation is unnecessary. We have performed this procedure in two cases. These patients had no major complications and recovered more rapidly than patients undergoing conventional transthoracic esophagectomy. Our initial experience shows that VATRE is a technically feasible and less invasive procedure for cancer surgery, and it enables us to easily perform three-field lymphadenectomy.

Aged↗

PubMed1999

Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme.

The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.

Adenosine Triphosphatases↗

PubMed1999

Isolation of RNase H genes that are essential for growth of Bacillus subtilis 168.

Two genes encoding functional RNase H (EC 3.1.26.4) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilis RNase HIII. The B. subtilis genome has an rnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB and rnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both the rnhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single rnhC homologue in the small genome of Mycoplasma species.

Bacillus subtilis↗

PubMed1999

Heart rate variability analysis of patients with idiopathic left ventricular outflow tract tachycardia: role of triggered activity.

There have been several reports with respect to idiopathic ventricular tachycardias (VTs) originating from the left ventricular outflow tract (LVOT). A previous report suggested that triggered activity plays a partial role in idiopathic LVOT tachycardia from the electrophysiological as well as the electropharmacological viewpoint. However, the exact role of triggered activity in this type of VT remains unknown. In the present study the relationship of the frequency of premature ventricular contractions (PVCs) and heart rate was examined and heart rate variability (HRV) was analyzed in 2 cases of LVOT tachycardia using 24-h Holter electrocardiographic (ECG) monitoring. The relation between the PVCs frequency and heart rate showed a persistently positive correlation, indicating frequent PVCs as heart rate increased. In HRV analysis, NN50(%), a time-domain variable of parasympathetic activity, showed no change prior to ventricular arrhythmias. In frequency-domain analysis of HRV, the high frequency (HF) component tended to fall prior to repetitive PVCs and VTs. The ratio of the low frequency to high frequency (LF/HF) components increased prior to single PVCs, repetitive PVCs and VTs. Sympathetic predominance predisposes the genesis of these kinds of arrhythmias originating from the LVOT and it is suggested that triggered activity plays an important role in LVOT tachycardia, at least in its initiation.

Electrocardiography↗

PubMed1999

Experimental ablation study using a new long linear probe in isolated porcine hearts.

We studied a new technique for creating long linear lesions in hearts using a custom-made linear probe. Radiofrequency (RF) energy applications using a 25-mm long stainless steel linear probe and a corresponding 500-kHz energy generator were tested, creating 90 lesions in isolated porcine hearts. The RF current was applied between the linear probe and a large patch electrode attached to the back of the specimen. Three parameters, comprising the power of the delivered energy, the pressure of contact between the probe and the specimen, and the duration of energy delivery were changed independently and the size of the resulting lesions was measured. All 90 lesions were transmural, well demarcated and created by a single stationary RF application. Lesion length and width increased with: 1) increasing power, when the other two parameters were maintained at constant levels, 2) increasing contact pressure, when the other two parameters were maintained at constant levels, and 3) increasing duration of energy delivery when the other two parameters were maintained at constant levels. The maximum width of the lesions was 3.7 mm. No overheating of any of the specimens was observed. In conclusion, the new original long linear probe used in this study was effective for creating transmural linear lesions, presenting the possibility of a worthwhile contribution to the maze surgical procedure applied to atrial fibrillation.

Animals↗

PubMed1999

Possible evidence for transmembrane K(+)-H+ exchange system in guinea pig myocardium.

The aim of this study was to obtain evidence for a transmembrane K(+)-H+ exchange system in Langendorff-perfused whole hearts and isolated ventricular myocytes of guinea pig. Effluent relation between K+ and pH in the whole hearts perfused with HEPES-buffered Tyrode's solution indicated a significant (p < 0.05) functional coupling of K+ uptake and H+ extrusion that was energy-dependent and omeprazole (OPZ)-sensitive. Administration of OPZ (0.3 mM) or dimethylamiloride (0.1 mM), an inhibitor of Na(+)-H+ antiport, to whole hearts subjected to the repetitive NH4Cl applications implied that both Na(+)-H+ and putative K(+)-H+ countertransports contribute to the regulation of intracellular pH. In isolated myocytes, voltage-dependent L-type Ca current (ICa) was inhibited by OPZ (0.3 mM) under K(-)- and Na(+)-free condition by 11 to 14%, and was inhibited to a greater extent (i.e., by 36 to 40%) by this agent in the presence of K+. OPZ-induced inhibition of the putative K(+)-H+ exchanger likely resulted in subsarcolemmal acidification which was responsible for the rate-independent suppression of ICa. In conclusion, these data provide functional evidence for a myocardial transmembrane K(+)-H+ exchanger.

Animals↗

PubMed1999

The effects of tea catechins on fecal conditions of elderly residents in a long-term care facility.

This study was carried out to evaluate the effects of tea catechins on fecal contents and metabolites of elderly people who were on a diet of solid food. The subjects were 35 residents in a long-term care facility who were all on the same diet, consisting of rice gruel and minced food. Tea catechins (300 mg), which were divided into 3 doses a day, were a meal supplement every day for 6 weeks. Fecal specimens were collected by the nursing staff, and their moisture content, pH, ammonia, sulfide, and oxidation-reduction potential (ORP) were determined before, during, and after the administration of tea catechins. In a comparison of values before the administration, all these fecal parameters decreased significantly during the tea catechin administration. After termination of the administration, these data tended to return toward the levels before administration. The reduction of such fecal parameters as moisture, pH, ammonia, sulfide, and ORP by tea catechin administration indicated very favorable improvements of the subjects' bowel conditions.

Aged↗

PubMed1998

Comparison of synonymous codon distribution patterns of bacteriophage and host genomes.

Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available.

Bacillus subtilis↗