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T Oshima

Publications and source records attributed to T Oshima.

At least 19 recordsLinked to original sources
PubMed1997

Purification and characterization of isocitrate dehydrogenase from a hyperthermophilic archaebacterium, Caldococcus noboribetus.

Isocitrate dehydrogenase from a hyperthermophilic archaebacterium Caldococcus noboribetus produced in Escherichia coli was purified. The purification was performed by heat treatment at 80 degrees C followed by single column chromatography. N-terminal amino acid sequencing analysis revealed that the N-terminal methionine is removed from the purified enzyme. Gel filtration analysis suggests that the enzyme has a homodimeric structure with a molecular weight of 90,000. The isoelectric point of the enzyme was estimated to be 5.6 by isoelectric focusing electrophoresis. The circular dichroism spectrum suggests that the enzyme has a secondary structure consisting of 23% alpha-helix and 34% beta-sheet. Enzymatic activity was observed under neutral pH, and the highest specific activity was obtained using cacodylic acid-KOH (pH 7.0) buffer. MgCl2 or MnCl2 was essential for the activity, and KCl concentrations higher than 0.33 M had an inhibitory effect on it. Apparent Km values were 72 and 43 microM for D,L-isocitrate and NADP, respectively. The enzyme showed extremely high stability against heat treatment, and no activity loss was observed by the treatment at 80 degrees C. The specific activity of the enzyme increased as temperature rose. Nearly no activity was observed at 40 degrees C or lower.

Archaea↗
PubMed1997

Effect of polar side chains at position 172 on thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus.

To understand the role of the amino acid residue at position 172 in the conformational stability, four mutant enzymes of Thermus thermophilus 3-isopropylmalate dehydrogenase in which Ala172 was replaced with Asp, Glu, Asn, and Gln were prepared by site-directed mutagenesis. Three mutants were more stable than the wild-type enzyme. No significant change in catalytic properties was found in the mutant enzymes. The molecular modeling studies suggested that the enhanced thermostability of the mutant enzymes resulted from the formation of extra electrostatic interactions and/or improvement of hydrophobic packing of the interior core.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Extracellular Mg2+ inhibits capacitative Ca2+ entry in vascular smooth muscle cells.

BACKGROUND: Agonist-induced Ca2+ entry is thought to be mediated by capacitative Ca2+ entry other than L-type Ca2+ channels in vascular smooth muscle cells (VSMCs). The mechanism for capacitative Ca2+ entry has not been fully elucidated. Our objective was to examine the effect of external Mg2+ on capacitative Ca2+ entry in cultured rat aortic VSMCs. METHODS AND RESULTS: Three doses of external Mg2+ concentration (nominally 0, 1, and 5 mmol/L) were used. After exposure to 1 mumol/L, angiotensin II (Ang II) in Ca(2+)-free medium, addition of Ca2+ to the medium caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i), indicating Ang II-induced Ca2+ influx. This Ca2+ influx was attenuated in cells preincubated with high external Mg2+ concentrations or with 1 mumol/L nifedipine. After VSMCs in Ca(2+)-free medium were exposed to 1 mumol/L thapsigargin, which inhibits the sarcoplasmic reticulum Ca(2+)-ATPase and depletes Ca2+ stores, addition of Ca2+ to the medium induced an increase in [Ca2+]i, indicating capacitative Ca2+ entry. This entry pathway was found to be independent of dihydropyridine-sensitive Ca2+ channels and inhibited by increased external Mg2+ concentration. External Mg2+ concentration did not influence Ca2+ efflux across the plasma membrane after stimulation with Ang II plus thapsigargin. CONCLUSIONS: Results suggest that in VSMCs, capacitative Ca2+ entry is reduced by external Mg2+. This mechanism may explain in part the inhibitory effect of external Mg2+ on Ca2+ handling.

Angiotensin II↗
PubMed1997

Alternatively spliced isoforms of the Na+/Ca2+ exchanger in the guinea pig cochlea.

The cochlea has been suggested to express some Na+/ Ca2+ exchangers (NCX), since efficient acoustic transduction requires cytosolic calcium homeostasis. The present study revealed that several spliced isoforms of NCX are expressed in the guinea pig cochlea. Moreover, to determine their localization in the cochlea, microdissected RT-PCR was performed. The guinea pig cochlea was microdissected into three parts (lateral wall, the organ of Corti and modiolus). The cochlear lateral wall and the organ of Corti expressed only a single isoform of NCX1. On the other hand, five isoforms of NCX1 and four isoforms of NCX3 were detected in the cochlear modiolus. The alternative splicing may provide diverse functions for NCX in the cochlea.

Alternative Splicing↗
PubMed1997

Construction of a contiguous 874-kb sequence of the Escherichia coli -K12 genome corresponding to 50.0-68.8 min on the linkage map and analysis of its sequence features.

The contiguous 874.423 base pair sequence corresponding to the 50.0-68.8 min region on the genetic map of the Escherichia coli K-12 (W3110) was constructed by the determination of DNA sequences in the 50.0-57.9 min region (360 kb) and two large (100 kb in all) and five short gaps in the 57.9-68.8 min region whose sequences had been registered in the DNA databases. We analyzed its sequence features and found that this region contained at least 894 potential open reading frames (ORFs), of which 346 (38.7%) were previously reported, 158 (17.7%) were homologous to other known genes, 232 (26.0%) were identical or similar to hypothetical genes registered in databases, and the remaining 158 (17.7%) showed no significant similarity to any other genes. A homology search of the ORFs also identified several new gene clusters. Those include two clusters of fimbrial genes, a gene cluster of three genes encoding homologues of the human long chain fatty acid degradation enzyme complex in the mitochondrial membrane, a cluster of at least nine genes involved in the utilization of ethanolamine, a cluster of the secondary set of 11 hyc genes participating in the formate hydrogenlyase reaction and a cluster of five genes coding for the homologues of degradation enzymes for aromatic hydrocarbons in Pseudomonas putida. We also noted a variety of novel genes, including two ORFs, which were homologous to the putative genes encoding xanthine dehydrogenase in the fly and a protein responsible for axonal guidance and outgrowth of the rat, mouse and nematode. An isoleucine tRNA gene, designated ileY, was also newly identified at 60.0 min.

Base Sequence↗
PubMed1997

Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus.

The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability. The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme. The structure of the E. coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S. typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher thermostability of the T. thermophilus enzyme. A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues. The mutation in the hydrophobic core of T. thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

The crystal structure of zinc-containing ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7.

The crystal structure of ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 was determined by multiple isomorphous replacement supplemented with anomalous scattering effects of iron atoms in the Fe-S clusters, and refined at 2.0 A resolution to a crystallographic R value of 0.173. The structural model contains a polypeptide chain of 103 amino acid residues, 2 [3Fe-4S] clusters, and 31 water molecules; in this model, the cluster corresponding to cluster II in bacterial dicluster ferredoxins loses the fourth iron atom although it may originally be a [4Fe-4S] cluster. The structure of the archaeal ferredoxin consists of two parts: the core fold part (residues 37-103) and the N-terminal extension part (residues 1-36). The "core fold" part has an overall main-chain folding common to bacterial dicluster ferredoxins, containing two clusters as the active center, two alpha-helices near the clusters, and two sheets of two-stranded antiparallel beta-sheet (the terminal and central beta-sheets). The "N-terminal extension" part is mainly formed by a one-turn alpha-helix and a three-stranded antiparallel beta-sheet. The beta-sheet in the N-terminal extension is hydrogen-bonded with the terminal beta-sheet in the core fold to form a larger beta-sheet. The distinct structural feature of this archaeal ferredoxin lies in the zinc-binding center where the zinc ion is tetrahedrally ligated by four amino acid residues (His 16, His 19, and His 34 from the N-terminal extension, and Asp 76 from the core fold). The zinc ion in the zinc-binding center is located at the interface between the core fold and the N-terminal extension, and connects the beta-sheet in the N-terminal extension and the central beta-sheet in the core fold through the zinc ligation. Thus, the zinc ion plays an important role in stabilizing the structure of the present archaeal ferredoxin by connecting the N-terminal extension and the core fold, which may be common to thermoacidophilic archaeal ferredoxins.

Amino Acid Sequence↗
PubMed1997

Novel zinc-containing ferredoxin family in thermoacidophilic archaea.

The dicluster-type ferredoxins from the thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus sp. are known to contain an unusually long extension of unknown function in the N-terminal region. Recent x-ray structural analysis of the Sulfolobus ferredoxin has revealed the presence of a novel zinc center, which is coordinated by three histidine ligand residues in the N-terminal region and one aspartate in the ferredoxin core domain. We report here the quantitative metal analyses together with electron paramagnetic resonance and resonance Raman spectra of T. acidophilum ferredoxin, demonstrating the presence of a novel zinc center in addition to one [3Fe-4S] and one [4Fe-4S] cluster (Fe/Zn = 6.8 mol/mol). A phylogenetic tree constructed for several archaeal monocluster and dicluster type ferredoxins suggests that the zinc-containing ferredoxins of T. acidophilum and Sulfolobus sp. form an independent subgroup, which is more distantly related to the ferredoxins from the hyperthermophiles than those from the methanogenic archaea, indicating the existence of a novel group of ferredoxins, namely, a "zinc-containing ferredoxin family" in the thermoacidophilic archaea. Inspection of the N-terminal extension regions of the archaeal zinc-containing ferredoxins suggested strict conservation of three histidine and one aspartate residues as possible ligands to the novel zinc center.

Amino Acid Sequence↗
PubMed1997

Purification, catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli.

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg2+ or Mn2+, with Mn2+ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40 degrees C the K(m) of E. coli IPMDH was 105 microM for IPM and 321 microM for NAD, the kcat was 69 s-1. The half denaturation temperature was 64 degrees C, which was 20 degrees C lower than that of the thermophile enzyme.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Unilateral sinonasal disease without bone destruction. Differential diagnosis using diagnostic imaging and endonasal endoscopic biopsy.

OBJECTIVE: To evaluate the diagnostic algorithms leading to minimal-access surgery in unilateral sinonasal disease without evidence of bone destruction. DESIGN: In selected patients, we assigned qualitative preoperative diagnoses by performing computed tomography (CT), magnetic resonance imaging (MRI), and endonasal endoscopic biopsy and prospectively analyzed the results for 3 years, from 1992 to 1995. PATIENTS: Of 278 untreated patients who complained of nasal and sinus-related symptoms and underwent CT examinations, 130 were found to have unilateral sinonasal abnormalities without detectable changes in the bone structure. RESULTS: Accurate preoperative diagnoses were made using CT in 75% of the patients with chronic sinusitis with or without nasal polyps and 85% of the patients with mucoceles. In the remaining patients, preoperative diagnoses for both diseases were made using MRI. All fungal and vascular diseases were accurately diagnosed using MRI alone. Although neither CT nor MRI were useful in the qualitative diagnosis of neoplasm, the presence of neoplastic conditions, except papilloma, was indicated by MRI. Using endonasal endoscopy, biopsy was an accurate diagnostic tool, with minimal tissue damage, in 88% of the neoplasms. CONCLUSIONS: In differentiating untreated unilateral sinonasal disease without evidence of bone destruction, we conclude that CT is the first modality of diagnostic imaging and MRI is more sensitive than CT in identifying fungal disease and angiofibroma. Furthermore, MRI is helpful when neoplasm is indicated, but it is not an accurate diagnostic tool. The endonasal endoscopic approach for obtaining pathologic specimens is a qualitative diagnostic tool in the diagnosis of sinonasal neoplasm.

Adult↗
PubMed1997

Restoration of the mucociliary clearance of the maxillary sinus after endoscopic sinus surgery.

BACKGROUND: Whether endoscopic sinus surgery (ESS) restores the mucociliary clearance of the maxillary sinus needs further evaluation. METHODS: We evaluated the mucociliary clearance of the maxillary sinus by using a radionuclide technique in 12 patients with chronic sinusitis (sinusitis group) and in six patients who had undergone ESS 6 to 14 months after the surgery (post-ESS group). The mucosal cilia taken from the maxillary sinus in 12 patients with sinusitis before and after ESS (paired experiments) were examined by light and electron microscopy. RESULTS: The radionuclide placed endoscopically in the maxillary sinus in eight patients immediately after ESS maintained 81.2 +/- 16.3% of its radioactivity after 30 minutes. This result was consistent with results in four patients with untreated chronic sinusitis in whom the radionuclide had been instilled by antral puncture (86.9% +/- 3.5%). On the other hand, in six patients in the post-ESS group the radionucleotide maintained only 25.9% +/- 11.6% of its radioactivity, demonstrating statistically significant differences from those of both the sinusitis group without ESS (p < 0.005) and the group 4 days after ESS (p < 0.005). The absence of the cilia in the sinusitis condition was recognized in 35.5% +/- 12.1% (n = 12) of the epithelial cells. On the other hand, the absence of cilia was significantly (p < 0.01) reduced to 5.3% +/- 3.7% of the epithelial cells in the post-ESS condition. Electron microscopic observation also revealed abnormal cilia in the sinusitis condition, whereas the mucosal cilia were regularly arranged in the post-ESS condition. CONCLUSIONS: The mucociliary clearance of the maxillary sinus disturbed by chronic inflammation was restored by ESS, indicating the clinical effectiveness of ESS for the treatment of chronic sinusitis.

Adolescent↗
PubMed1997

Expression of voltage-dependent chloride channels in the rat cochlea.

Voltage-dependent chloride (ClC) channels have not yet been identified in the cochlea. In this study, an approach utilizing the reverse transcription-polymerase chain reaction (RT-PCR) was devised to clone the cDNA of ClC channels. PCR was performed using degenerate primers corresponding to two highly conserved regions of the ClC channels. By Southern hybridization and sequencing studies, the sequences corresponding to ClC-2 and ClC-3 were found in the cochlear lateral wall, while ClC-1 was not detected. These results suggest that ClC-2 and ClC-3 might be involved in Cl- transport in the cochlear lateral wall.

Animals↗
PubMed1997

Molecular and clinical implications of loop diuretic ototoxicity.

Recent advances in molecular biology have been applied to inner ear research. Loop diuretic ototoxicity has been suggested, but not proven, to share a common mechanism with diuretic effects on renal tubules. The discovery of the molecular nature of the Na-K-2Cl cotransporter in the cochlea provided a better understanding of loop diuretic ototoxicity. In this review, we describe clinical reports of loop diuretic ototoxicity and other information obtained by physiological, biochemical and morphological investigations related to the mechanism sensitive to loop diuretics. Based on recent evidence for the molecular nature of the Na-K-2Cl cotransporter expressed in the mammalian cochlea, the underlying mechanisms of ototoxicity induced by loop diuretics are described.

Animals↗
PubMed1997

Substrate recognition of isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8.

The substrate-binding sites of NADP-dependent isocitrate dehydrogenase and NAD-dependent 3-isopropylmalate dehydrogenase from Thermus thermophilus were analyzed by site-directed mutagenesis. Ser97 and Asn99 of isocitrate dehydrogenase were identified to be involved in the isocitrate recognition. In 3-isopropylmalate dehydrogenase, the corresponding residues, Leu90 and Leu91, appear to recognize the substrate by forming a hydrophobic pocket. Double mutation of Asp78 and Glu87 revealed that negative charge of these residues plays a crucial role in discriminating isopropylmalate from isocitrate.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

A mutation at the interface between domains causes rearrangement of domains in 3-isopropylmalate dehydrogenase.

The structure of a thermostable Ala172Leu mutant, designated A172L, of 3-isopropylmalate dehydrogenase from Thermus thermophilus was determined. The crystal belongs to space group P2(1), with cell parameters a = 55.5 A, b = 88.1 A, c = 72.0 A and beta = 100.9 degrees. There is one dimer in each asymmetric unit. The final R factor is 17.8% with 69 water molecules at 2.35 A resolution. The mutation is located at the interface between domains and the C alpha trace of the mutant structure deviates from that of the native structure by as much as 1.7 A, while the structure of each domain barely changes. The mutant enzyme has a more closed conformation compared with the wild-type enzyme as a result of the replacement of Ala with Leu at residue 172. These structural variations were found independent of the crystal packing, because the structure of wild type was the same in crystals obtained in different precipitants. The hinge regions for the movement of domains are located around the active cleft of the enzyme, an observation that implies that the mobility of domains around the hinge is indispensable for the activity of the enzyme. The larger side chain at the mutated site contributed to the thermostability of the mutant protein by enhancing the local packing of side chains, and also by shifting the backbone of the opposing domain.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Stabilization of Escherichia coli isopropylmalate dehydrogenase by single amino acid substitution.

To determine the key position for the unusual stability of isopropylmalate dehydrogenase from extreme thermophiles (Thermus thermophilus and T. aquaticus), sequence comparisons were carried out. As a result, a motif which is characteristic to the thermophilic dehydrogenases was found between two highly conserved stretches. The sequence motif was introduced into a mesophilic (Escherichia coli) isopropylmalate dehydrogenase, one by one. Contrary to our expectation, introduction of the whole motif led the mesophilic enzyme to be more unstable whereas substitution of only one amino acid residue in the motif thermostabilized the enzyme. From the 3D structure of the enzyme, a mechanism for the thermostabilization is speculated.

3-Isopropylmalate Dehydrogenase↗