Search PubMed⌕ Search

Biomedical subjects

T Iwabuchi

Publications and source records attributed to T Iwabuchi.

At least 19 recordsLinked to original sources
PubMed2003

Expression of foreign proteins in Escherichia coli by fusing with an archaeal FK506 binding protein.

Improper protein-folding often results in inclusion-body formation in a protein expression system using Escherichia coli. To express such proteins in the soluble fraction of E. coli cytoplasm, we developed an expression system by fusing the target protein with an archaeal FK506 binding protein (FKBP). It has been reported that an archaeal FKBP from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TcFKBP18), possesses not only peptidyl-prolyl cis-trans isomerase activity, but also chaperone-like activity to enhance the refolding yield of an unfolded protein by suppressing irreversible protein aggregation. To study the effect of this fusion strategy with FKBP on the expression of foreign protein in E. coli, a putative rhodanese (thiosulfate sulfurtransferase) from a hyperthermophilic archaeon and two mouse antibody fragments were used as model target proteins. When they were expressed alone in E. coli, they formed insoluble aggregates. Their genes were designed to be expressed as a fusion protein by connecting them to the C-terminal end of TcFKBP18 with an oligopeptide containing a thrombin cleavage site. By fusing TcFKBP18, the expression of the target protein in the soluble fraction was significantly increased. The percentage of the soluble form in the expressed protein reached 10-28% of the host soluble proteins. After purification and protease digestion of the expressed antibody fragment-TcFKBP18 fusion protein, the cleaved antibody fragment (single-chain Fv) showed specific binding to the antigen in ELISA. This indicated that the expressed antibody fragment properly folded to the active form.

Archaeal Proteins↗
PubMed2002

Tubo-ovarian abscess after colonic vaginoplasty for high cloacal anomaly in a 13-year-old girl.

Recently a few articles have been published concerning the long-term follow-up of vaginoplasty of cloaca. However, no postoperative evaluation has been fully described and, in particular, the late complications are still unknown. We report a case of tuboovarian abscess after colonic vaginoplasty for high cloacal anomaly in a 13-year-old girl. She required a left salpingo-oophorectomy and postoperatively showed regular menstruation. Therefore we stress that tubo-ovarian abscess is one of the important late complications after colonic vaginoplasty for high cloacal anomaly.

Abscess↗
PubMed2000

FK506-binding protein-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum.

The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase). Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H. cutirubrum has an FKBP-type PPIase or not. In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H. cutirubrum and then sequenced. This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3kDa. Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs. Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp. KS-1. Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP. This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion. The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506. This FKBP suppressed aggregation of the unfolded protein.

Amino Acid Sequence↗
PubMed2000

A novel phenanthrene dioxygenase from Nocardioides sp. Strain KP7: expression in Escherichia coli.

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the alpha and beta subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8. 3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the alpha and beta subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.

Actinomycetales↗
PubMed1999

Functional conformation changes in the TF(1)-ATPase beta subunit probed by 12 tyrosine residues.

The effect of nucleotide binding on the structure of the F(1)-ATPase beta subunit from thermophilic bacillus PS-3 (TF(1)beta) was investigated by monitoring the NMR signals of the 12 tyrosine residues. The 3,5-proton resonances of 12 tyrosine residues could be observed for the specifically deuterated beta subunit. The assignment of 3,5-proton resonances of all of the tyrosine residues was accomplished using 14 mutant proteins, in each of which one or two tyrosine residues were replaced by phenylalanine. Binding of Mg. ATP induced an upfield shift of Tyr(341) resonance, suggesting that their aromatic rings are stacked to each other. Besides Tyr(341), the signal shift observed on Mg.ATP binding was restricted to the resonances of Tyr(148), Tyr(199), Tyr(238), and Tyr(307), suggesting that Mg.ATP induces a conformational change in the hinge region. This can be correlated to the change from the open to closed conformations as implicated in the crystal structure. Mg.ADP induced a similar but distinctly different conformational change. Therefore, the intrinsic conformational change in the beta subunit induced by the nucleotide binding is proposed to be one of the essential driving forces for the F(1) rotation. Reconstitution experiments showed that Tyr(277), one of the four conserved tyrosines, is essential to the formation of the alpha(3)beta(3)gamma complex.

Adenosine Diphosphate↗
PubMed1999

Characterization of genes for enzymes involved in the phenanthrene degradation in Nocardioides sp. KP7.

The nucleotide sequence of the gene cluster, phdEFABGHCD, encoding enzymes responsible for the transformation of phenanthrene to 1-hydroxy-2-naphthoate in Nocardioides sp. strain KP7 was determined. This gene cluster, which may constitute a single operon, resided at 6.1-kb downstream of the phdIJK gene cluster encoding the enzymes for the transformation of 1-hydroxy-2-naphthoate to o-phthalate. In general, the phd products exhibited moderate degrees of homology with isofunctional enzymes found in pathways for the degradation of other aromatic compounds. Remarkably, the phdC gene product had features of the [3Fe-4S] type ferredoxin, which has not been found so far as a component of the ring-hydroxylating dioxygenase. Escherichia coli carrying the genes for phenanthrene dioxygenase, phdABCD, was capable to oxidize phenanthrene.

Biodegradation, Environmental↗
PubMed1999

Effect of compression on the cochlear nerve: a short- and long-term electrophysiological and histological study.

The short- and long-term effects of static compression of the cochlear nerve were studied in dogs. The nerve was exposed in the cerebellopontine angle and a modified aneurysm clip was applied to reduce the diameter of the nerve trunk to 50%, 40%, 30% or 20% of normal (designated respectively as 50%, 60%, 70%, and 80% compression). Brainstem auditory evoked potentials (BAEPs) were monitored intraoperatively and post-operatively. The animals were sacrificed between 5 and 119 days after nerve compression and temporal bones were examined histologically. In the 50% compression group, all peaks except peak I disappeared immediately after nerve compression. After release of the clip, however, peak II and subsequent components recovered and prolonged interpeak latency (IPL) between peaks I and IV normalized within 7 days. In the 60% compression group, recovery was incomplete for as long as 49 days after compression. Significant histological changes were not always reflected in the electrophysiological recordings, as shown by the finding of multiple cavitations at the compressed portion of the cochlear nerve in cases in which conduction block of cochlear nerve impulses was reversible. In the 70% compression group, peak IV did not reappear for more than 1 week, and histological examination revealed severe damage to all cochlear nerve fibers except those from the apical turn, which lie in the center of the cochlear nerve trunk. Severe injury occurred to the cochlear nerve fibers that are situated more superficially in the nerve, which are tonotopically responsible for the perception of high-frequency sound and the generation of BAEPs. This means that the BAEP changes due to cochlear nerve compression would be detectable by BAEP monitoring, although changes in the apical region of the cochlea are not fully detectable by BAEP monitoring. In the 80% compression group, all peaks except peak I were lost permanently and the amplitude of peak I, which had been preserved in the acute phase, gradually decreased. Reversibility of impaired cochlear nerve impulse conduction was related to the severity of compression, and at some level of compression between 70% and 80% the nerve fibers generating BAEPs permanently lost the ability to conduct electrical impulses proximal to the site of compression. In the 70% and 80% compression groups, the amplitude of peak I gradually decreased over the first 30 days after compression and did not change significantly thereafter. Histologically, the branches of the internal auditory artery were resilient to compression, although they are easily avulsed due to stretch force. Furthermore, retrograde degeneration of cochlear neurons triggered by compression at the cisternal portion of the cochlear nerve was apparent. Such slowly progressive degeneration of nerve fibers may play a part in development of the delayed postoperative hearing disturbance.

Animals↗
PubMed1999

Structure of the ring cleavage product of 1-hydroxy-2-naphthoate, an intermediate of the phenanthrene-degradative pathway of Nocardioides sp. strain KP7.

1-Hydroxy-2-naphthoate (compound I) is a metabolite of the phenanthrene-degradative pathway in Nocardioides sp. strain KP7. This singly hydroxylated aromatic compound is cleaved by 1-hydroxy-2-naphthoate dioxygenase. In this study, the structure of the ring cleavage product generated by the action of homogeneous 1-hydroxy-2-naphthoate dioxygenase was determined upon separation by high-performance liquid chromatography at pH 2.5 by using nuclear magnetic resonance (NMR) and mass spectroscopic techniques. The ring cleavage product at this pH existed in equilibrium between two forms, 2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III) and 2,2-dihydroxy-3-(3-oxo-1, 3-dihydro-1-isobenzofuranyl)propanoate (compound IV). After the pH of the solution was raised to 7.5, the structure of the major species became (E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II; common name, trans-2'-carboxybenzalpyruvate), which was in equilibrium with compound III. Direct monitoring of the enzymatic formation of the ring cleavage product by 1H-NMR in a deuterated potassium phosphate buffer (pH 7.5) detected only compound II as a product, and the proton on carbon 3 of compound II was not exchanged with deuterium. Thus, compound II is likely to be the first stable product of dioxygenation of 1-hydroxy-2-naphthoate.

Actinomycetales↗
PubMed1998

Biochemical and molecular characterization of 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7.

1-Hydroxy-2-naphthoate dioxygenase, which cleaves the singly hydroxylated aromatic ring, was purified from phenanthrene-degrading Nocardioides sp. strain KP7. The purified enzyme had a molecular mass of 45 kDa by SDS-polyacrylamide gel electrophoresis and 270 kDa by gel filtration chromatography. The apparent Km and kcat values of this enzyme for 1-hydroxy-2-naphthoate were 10 microM and 114 s-1, respectively. One mole of molecular oxygen was consumed when 1 mol of 1-hydroxy-2-naphthoate was oxidized. This enzyme contained 1 mol of Fe(II)/mol of the subunit and was inactivated by o-phenanthroline. The enzyme that had been inactivated by o-phenanthroline was reactivated by incubating with FeSO4 and ascorbic acid. Thus, Fe(II) was required for the enzyme to exhibit activity. The structural gene for this enzyme was screened from a cosmid library and then sequenced, the length of the 1-hydroxy-2-naphthoate gene being 1161 base pairs. The deduced amino acid sequence of this enzyme was different from those of other ring-cleaving dioxygenases that cleave the doubly hydroxylated aromatic ring.

Amino Acid Sequence↗
PubMed1998

Biochemical and genetic characterization of trans-2'-carboxybenzalpyruvate hydratase-aldolase from a phenanthrene-degrading Nocardioides strain.

trans-2'-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The Km and kcat values of this enzyme for trans-2'-carboxybenzalpyruvate were 50 microM and 13 s(-1), respectively. trans-2'-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of trans-2'-hydroxybenzalpyruvate hydratase-aldolases from Pseudomonas putida PpG7 and Pseudomonas sp. strain C18.

Actinomycetales↗
PubMed1997

Gene for a cyclophilin-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum.

A gene encoding a cyclophilin (CyP)-type peptidyl prolyl cis-trans isomerase (PPIase) was cloned from a halophilic archaeum, Halobacterium cutirubrum DSM 669, and sequenced. Although amino-acid residues common to CyPs were conserved, an insertion that showed no homology to other CyPs was found in its N-terminal region. Sequence analysis revealed that the amino-acid sequence of this CyP was 40-45% identical to those of eukaryotes and Bacillus subtilis with high cyclosporin A sensitivity, but 27% identical to those of cyclosporin A-insensitive PPIases of Escherichia coli. The gene was also expressed in E. coli. The activity of purified recombinant CyP-type PPIase was stimulated by the addition of KCl, and was suppressed by cyclosporin A.

Amino Acid Sequence↗
PubMed1997

[Usefulness of 7 day therapy with cefluprenam in the management of respiratory tract infections].

Efficacy and safety of a newer injectable cephalosporin, cefluprenam (CFLP) on cases with bacterial pneumonia and chronic respiratory tract infections were evaluated at a dose of 1g (potency), d.i.d for 7 days. 1. Of 130 cases in total, 116 cases were enrolled for the clinical efficacy evaluation. The efficacy rate (excellent and good responses) was 94.8% (110/116). The efficacy rate was 93.8% (60/64) for cases with bacterial pneumonia, and 96.2% (50/52) for cases with chronic respiratory tract infections. The recurrence was noted in 1.2% (1/82). The bacteriological response rate was 100.0% (32/32) for gram positive cocci, 93.8% (15/16) for gram negative rods and 97.9% (47/48) in total. 2. Adverse drug reactions were noted in 3.9% (5/129), consisting of 2 cases with skin rash, 1 case with drug fever, 1 case with skin rash and skin itching and 1 case with drug fever and headache. The abnormal laboratory changes were noted in 23.6% (30/127), mainly containing the elevation in GPT and GOT, and eosinophylia. The safety rate (no problem evaluation) was 74.8% (95/127). 3. The usefulness rate (very useful and useful evaluations) was 93.1% (108/116). As suggested by the evaluation on the secondary endpoint in the phase III comparative studies with both bacterial pneumonia and chronic respiratory tract infections, it was confirmed that the 7 day therapy of CFLP was promising for treatment of moderate bacterial pneumonia and chronic respiratory tract infections, because the high clinical efficacy was obtained and also the incidence of allergic reactions with CFLP was almost the same as that of ceftazidime (CAZ) evaluated highly safe. Based on these results, it was concluded that CFLP was useful in the management of moderate respiratory tract infections and also the recommended therapeutic period with CFLP was within 7 days.

Adult↗
PubMed1997

Biochemical and genetic characterization of 2-carboxybenzaldehyde dehydrogenase, an enzyme involved in phenanthrene degradation by Nocardioides sp. strain KP7.

2-Carboxybenzaldehyde dehydrogenase from the phenanthrene-degrading bacterium Nocardioides sp. strain KP7 was purified and characterized. The purified enzyme had a molecular mass of 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 205 kDa by gel filtration chromatography. Thus, the homotetramer of the 53-kDa subunit constituted an active enzyme. The apparent Km and kcat values of this enzyme for 2-carboxybenzaldehyde were 100 microM and 39 s(-1), respectively, and those for NAD+ were 83 microM and 32 s(-1), respectively. The structural gene for this enzyme was cloned and sequenced. The length of the gene was 1,455 bp. The nucleotide sequence of the 10,279 bp of DNA around the gene for 2-carboxybenzaldehyde dehydrogenase was also determined, and seven open reading frames were found in this DNA region. These were the genes for 1-hydroxy-2-naphthoate dioxygenase (phdI) and trans-2'-carboxybenzalpyruvate aldolase (phdJ), orf1, the gene for 2-carboxybenzaldehyde dehydrogenase (phdK), orf2/orf3, and orf4. The amino acid sequence of the orf1 product was similar to that of the aromatic hydrocarbon transporter gene (pcaK) in Pseudomonas putida PRS2000. The amino acid sequence of the orf4 product revealed a similarity to cytochrome P-450 proteins. The region between phdK and orf4 encoded orf2 and orf3 on different strands. The amino acid sequences of the orf2 and orf3 products exhibited no significant similarity to the reported sequences in protein databases.

Actinomycetales↗
PubMed1996

Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products.

The method to internally label PCR products with multiple colored fluorescent dyes was developed and applied to multiple fluorescence-based PCR single-stranded conformational polymorphism (MF-PCR-SSCP) analysis. PCR-amplified fluorescent DNA fragments, which were internally labeled by adding fluorescent dUTPs ([F]dUTPs) to the PCR mixture, were heat-denatured and applied to a nondenaturing polyacrylamide gel (SSCP gel) set on an automated DNA sequencer with a gel temperature-controlling system. The image data were analyzed by GENESCAN 672 software. In spite of differences in species and amount of integrated [F]dUTPs, the SSCP profiles were not significantly affected, even by the different labeling methods used-internal labeling or post-labeling at the 3' ends-in regard to the three different [F]dUTPs examined. However, the salt concentration of the solution containing the DNA samples affected the SSCP profiles. The internally labeled [F]dUTP-containing DNA fragments beyond 1000 bp in length were successfully digested with restriction endonucleases and subjected to SSCP analysis. MF-PCR-SSCP analysis with internal fluorescence labeling affords a simple and sensitive method to detect alterations in DNA sequences.

DNA Mutational Analysis↗