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S Kanaya

Publications and source records attributed to S Kanaya.

At least 19 recordsLinked to original sources
PubMed2006

Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization.

Type I secretion system (TISS) of Gram-negative bacteria permits proteins to be secreted directly from the cytoplasm to the external medium by a single, energy-coupled step. To examine whether this system can be used as an extracellular production system of recombinant proteins, Escherichia coli alkaline phosphatase (AP) was fused to a C-terminal region of Pseudomonas sp. MIS38 lipase (PML) and examined for secretion using the E.coli cells carrying the heterologous TISS. PML is one of the passenger proteins of TISS and contains 12 repetitive sequences and a secretion signal at the C-terminal region. The fusion protein was efficiently secreted to the extracellular medium, while AP was not secreted at all, indicating that the secretion of AP is promoted by a secretion signal of PML. The repetitive sequences were not so important for secretion of the fusion protein, because the secretion level of the fusion protein containing entire repeats ( approximately 10 mg/l culture) was only 2-fold higher than that of the fusion protein without repeats. The fusion protein purified from the culture supernatant existed as a homodimer, like AP, and was indistinguishable from AP in enzymatic properties and stability.

Alkaline Phosphatase↗
PubMed2006

Family I.3 lipase: bacterial lipases secreted by the type I secretion system.

Based on the classification of bacterial lipolytic enzymes, family I.3 lipase is a member of the large group of Gram-negative bacterial true lipases. This lipase family is distinguished from other families not only by the amino acid sequence, but also by the secretion mechanism. Lipases of family I.3 are secreted via the well-known type I secretion system. Like most of proteins secreted via this system, family I.3 lipases are composed of two domains with distinct yet related functions. Recent years have seen an increasing amount of research on this lipase family, in terms of isolation, secretion mechanism, as well as biochemical and biophysical studies. This review describes our current knowledge on the structure-function relationships of family I.3 lipase, with an emphasis on its secretion mechanism.

Amino Acid Motifs↗
PubMed2005

Development of a simple method to evaluate medical staff radiation dose and its application to a software system supporting PET facility operation.

In recent years, positron-emitting labelled radiopharmaceuticals have come to be used in conjunction with positron emission tomography (PET) in various clinical diagnoses. Radiation exposure of the medical staff is a key issue in the design of PET facilities intended to handle large numbers of persons for PET diagnosis. As a first step, the radiation dose to individuals who received radiopharmaceuticals was calculated using a mathematical phantom model and the EGS4 electromagnetic cascade Monte Carlo code and MCNP Monte Carlo code. Dose rate behind a lead shield was also calculated for various lead thicknesses. The radiation dose distribution around a syringe containing a positron emitter was calculated. The calculated dose distributions were fitted to polynomial equations. These calculations were evaluated against measurements. The second step was to evaluate medical staff dose at a specified time by superimposing dose distribution from each person who received radioisotopes taking into account radioactive decay. In this way, we developed software to support PET facility operation, namely, planning, prediction, control of medical staff dose and facility operation. This system was also designed to schedule daily radiopharmaceuticals production and to manage radioactive wastes by taking decay time into account.

Body Burden↗
PubMed2005

Cleavage of PDGF receptor on periodontal ligament cells by elastase.

Human leukocyte elastase, a neutrophil serine protease, is considered to be a potential immunoregulatory protease. Since the PDGF receptor (PDGFR) on periodontal ligament (PDL) cells is a crucial element for various functions, such as wound healing in periodontal tissue, we investigated the effect of elastase on the expression of PDGFR on PDL cells by flow cytometry and Western blotting. We found that PDGFR-alpha disappeared with an increasing dose of elastase, and PDGFR-beta was degraded into several fragments. Elastase degraded both receptors on fixed cells, indicating that the degradation resulted from direct proteolysis on the cell surface. Elastase also then disturbed the phosphorylation of ERK1/2, JNK/SARK, and p38, triggered by PDGF-AA and PDGF-BB, suggesting that elastase inhibited PDGFR-dependent cell activation in PDL cells. These results suggest that elastase may modulate the PDGF-mediated activity of PDL cells during periodontal wound healing.

Adolescent↗
PubMed2001

Ca(2+)-induced folding of a family I.3 lipase with repetitive Ca(2+) binding motifs at the C-terminus.

In order to understand a role of the Ca(2+) ion on the structure and function of a Ca(2+)-dependent family I.3 lipase from Pseudomonas sp. MIS38, apo-PML, holo-PML, holo-PML*, and the N-terminal domain alone (N-fragment) were prepared and biochemically characterized. Apo-PML and holo-PML represent refolded proteins in the absence and presence of the Ca(2+) ion, respectively. Holo-PML* represents a holo-PML dialyzed against 20 mM Tris-HCl (pH 7.5). The results suggest that the C-terminal domain of PML is almost fully unfolded in the apo-form and its folding is induced by Ca(2+) binding. The folding of this C-terminal domain may be required to make a conformation of the N-terminal catalytic domain functional.

Amino Acid Motifs↗
PubMed2001

Intraplatelet tetrahydrobiopterin plays an important role in regulating canine coronary arterial thrombosis by modulating intraplatelet nitric oxide and superoxide generation.

BACKGROUND: Platelet-derived nitric oxide inhibits platelet aggregation via constitutive NO synthase (NOS). Tetrahydrobiopterin (BH(4)), a cofactor of NOS, augments NO formation, whereas its deficiency decreases NO bioactivity and increases superoxide generation by NOS. The roles of intraplatelet BH(4) in platelet aggregation and thrombus formation, however, are unknown. Accordingly, we investigated whether intraplatelet BH(4) is involved in regulating cyclic flow variations (CFVs) and platelet aggregation in a canine model with stenosed and endothelium-injured coronary arteries that mimics acute coronary syndromes in humans. METHODS AND RESULTS: After developing CFVs, dogs received saline or BH(4) (10 or 30 mg/kg) intravenously. Intraplatelet BH(4) and cGMP levels were decreased and intraplatelet nitrotyrosine production was increased during CFVs. ADP- and U46619-induced ex vivo platelet aggregation and platelet P-selectin expression were augmented during CFVs. BH(4) administration restored intraplatelet BH(4) and cGMP levels and decreased intraplatelet nitrotyrosine production, resulting in reduced CFVs and inhibited ex vivo platelet aggregation and platelet P-selectin expression. CFVs again developed after N(G)-monomethyl-L-arginine, an inhibitor of NOS, in BH(4)-treated dogs. Ex vivo platelet NOS activity at baseline, during CFVs, and after BH(4) administration did not differ. CONCLUSIONS: Intraplatelet BH(4) may play an important role in regulating thrombus formation by modulating platelet-derived nitric oxide and superoxide generation by platelet NOS.

Animals↗
PubMed2001

Analysis of codon usage diversity of bacterial genes with a self-organizing map (SOM): characterization of horizontally transferred genes with emphasis on the E. coli O157 genome.

With increases in the amounts of available DNA sequence data, it has become increasingly important to develop tools for comprehensive systematic analysis and comparison of species-specific characteristics of protein-coding sequences for a wide variety of genomes. In the present study, we used a novel neural-network algorithm, a self-organizing map (SOM), to efficiently and comprehensively analyze codon usage in approximately 60,000 genes from 29 bacterial species simultaneously. This SOM makes it possible to cluster and visualize genes of individual species separately at a much higher resolution than can be obtained with principal component analysis. The organization of the SOM can be explained by the genome G+C% and tRNA compositions of the individual species. We used SOM to examine codon usage heterogeneity in the E. coli O157 genome, which contains 'O157-unique segments' (O-islands), and showed that SOM is a powerful tool for characterization of horizontally transferred genes.

Algorithms↗
PubMed2001

Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site.

The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp(10) and Glu(48) are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011-11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu(48) is not responsible for Mg(2+) binding but is involved in the catalytic function.

Arginine↗
PubMed2001

Isolation and characterization of psychotrophic bacteria from oil-reservoir water and oil sands.

Four psychrotrophic strains, which grew at 4 degrees C but not at 37 degrees C, were isolated from Japanese oil-reservoir water (strains SIB1, SIC1, SIS1) and Canadian oil sands (strain CAB1). Strains SIB1, SIS1, and CAB1 had a maximum growth rate at 20 degrees C and grew to the highest cell densities at the cultivation temperature of 0-4 degrees C. Strain SIS1 was capable of growing even at -5 degrees C. The growth profile of strain SIC1 was rather similar to that of a mesophilic bacterium. Strains SIB1, SIC1, and SIS1 were identified as members of the genus Shewanella, and strain CAB1 was a member of the genus Arthrobacter. All these strains exhibited weak degradation ability against catechol, a hydroxylated aromatic hydrocarbon, and tributyrin. These strains are expected to be of potential use in the in situ bioremediation technology of hazardous hydrocarbons and esters under low-temperature conditions.

Aerobiosis↗
PubMed2001

Interaction of TIP26 from a hyperthermophilic archaeon with TFB/TBP/DNA ternary complex.

Interactions of TBP-interacting protein (TIP26), TBP, and TFB from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 with TATA-DNA were examined by electrophoretic mobility shift assay. Tk-TFB formed a ternary complex with Tk-TBP and TATA-DNA. Tk-TIP26 did not inhibit the formation of this ternary complex, but interacted with it to form a TIP26/TFB/TBP/DNA quaternary complex. This interaction is rather weak, and a large excess of Tk-TIP26 over Tk-TBP is required to fully convert the TFB/TBP/DNA ternary complex to the quaternary complex. However, determination of the concentration of Tk-TIP26 and Tk-TBP in KOD1 cells by Western blotting analysis indicated that the concentration of Tk-TIP26 is approximately ten times that of Tk-TBP, suggesting that the quaternary complex might also form in vivo.

Amino Acid Sequence↗
PubMed2001

Origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria is revealed by homology-hit analysis.

The origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria was proposed on the basis of the phylogenetic topologies of genes. However, it was not possible to conclude whether or not the genes involved were authentic representative genes. Furthermore, using the BLAST and FASTA programs, the similarity of open reading frame (ORF) groups between three domains (Eukarya, Archaea and Bacteria) was estimated at one threshold. Therefore, their similarities at other thresholds could not be clarified. Here we use our newly developed 'homology-hit analysis' method, which uses multiple thresholds, to determine the origin of the nucleus. We removed mitochondria-related ORFs from yeast ORFs, and determined the number of yeast orthologous ORFs in each functional category to the ORFs in six Archaea and nine Bacteria at several thresholds (E-values) using the BLAST. Our results indicate that yeast ORFs related to the nucleus may share their origins with archaeal ORFs, whereas ORFs that are related to the cytoplasm may share their origins with bacterial ORFs. Our results thus strongly support the idea of nucleus symbiosis.

Archaea↗
PubMed2001

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis.

The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA-RNA hybrid as a substrate revealed that the enzymatic properties of SIB1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was approximately 25 degrees C for SIB1 RNase HI and approximately 60 degrees C for E.coli RNase HI. The optimum temperature for the SIB1 RNase HI activity was also shifted downward by 20 degrees C compared with that of E.coli RNase HI. Nevertheless, SIB1 RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10 degrees C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the alphaI-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.

Alanine↗
PubMed2001

Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses.

The catalytic center of an archaeal Type 2 RNase H has been identified by a combination of X-ray crystallographic and mutational analyses. The crystal structure of the Type 2 RNase H from Thermococcus kodakaraensis KOD1 has revealed that the N-terminal major domain adopts the RNase H fold, despite the poor sequence similarity to the Type 1 RNase H. Mutational analyses showed that the catalytic reaction requires four acidic residues, which are well conserved in the Type 1 RNase H and the members of the polynucleotidyl transferase family. Thus, the Type 1 and Type 2 RNases H seem to share a common catalytic mechanism, except for the requirement of histidine as a general base in the former enzyme. Combined with the results from deletion mutant analyses, the structure suggests that the C-terminal domain of the Type 2 RNase H is involved in the interaction with the DNA/RNA hybrid.

Archaea↗
PubMed2001

Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence.

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.

Amino Acid Sequence↗
PubMed2001

Long-term smoking causes nitroglycerin resistance in platelets by depletion of intraplatelet glutathione.

We investigated whether platelet responsiveness to nitroglycerin (NTG) is maintained in long-term smokers and if not, the mechanism. In the absence or presence of NTG, intraplatelet reduced glutathione (GSH) levels and ADP-induced platelet aggregation and intraplatelet cGMP levels were measured in 10 long-term smokers and 10 age-matched nonsmokers. The intraplatelet GSH level was significantly lower in smokers than in nonsmokers (P<0.05). Platelet aggregation was dose-dependently inhibited by NTG in both groups; however, inhibition was significantly weaker in smokers. N-acetylcysteine (1 mmol/L), an exogenous thiol agent, significantly potentiated NTG-induced platelet inhibition in nonsmokers but not in smokers. The ADP-induced intraplatelet cGMP level was significantly greater in the presence of NTG in nonsmokers but not so in smokers. Because the effects of long-term smoking are multifactorial, a rabbit model was made by chronic administration of buthionine sulfoximine (BSO, n=6) to decrease intraplatelet GSH. The intraplatelet GSH level was significantly lower in BSO-treated rabbits than in saline-treated rabbits (P<0.001). The NTG-induced inhibition of platelet aggregation was significantly weaker in BSO rabbits. N-acetylcysteine-induced potentiation was not observed in BSO rabbits, whereas significant potentiation was found in saline rabbits. These findings were similar to those of long-term smokers. In contrast, the intraplatelet GSH-to-oxidized glutathione ratio, which represents the redox state of glutathione, was significantly lower in smokers than in nonsmokers, whereas no difference was found between saline rabbits and BSO rabbits. In conclusion, long-term smoking causes NTG resistance to aggregation in platelets, possibly through the depletion of intraplatelet GSH.

Animals↗
PubMed2001

Gene Cloning of an alcohol dehydrogenase from thermophilic alkane-degrading Bacillus thermoleovorans B23.

The gene encoding an alcohol dehydrogenase (Bt-ADH) was cloned from a newly isolated thermophilic alkane-degrading Bacillus thermoleovorans, strain B23. The gene conferred 1-tetradecanol dehydrogenase activity on Escherichia coli cells. Bt-ADH is composed of 249 amino acid residues and the calculated molecular mass is 27,196 Da. A tyrosine residue in the active site and a glycine-rich sequence (GGXXGI/LG) constituting probable nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+) binding site were completely conserved in the Bt-ADH sequence at positions 155 and 11, respectively. A phylogenetic analysis of Bt-ADH suggested that the enzyme belongs to the zinc-independent ADH Group II. Its highest similarity (48% identical) was to a hypothetical oxidoreductase from a hyperthermophile, Thermotoga maritima.

Journal Article↗