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T Oshima

Publications and source records attributed to T Oshima.

At least 19 recordsLinked to original sources
PubMed1996

Electrostatic interaction between two domains of isocitrate dehydrogenase from Thermus thermophilus is important for the catalytic function and protein stability.

The role of electrostatic interaction between Lys96 and Glu147 of isocitrate dehydrogenase from Thermus thermophilus was investigated by site-directed mutagenesis. These two residues are located near the active site and involved in the interdomain interaction. Analyses of the catalytic properties and thermostability of the Glu147Gln mutant revealed that this interaction plays important roles in catalytic function and protein stability.

Amino Acid Sequence↗
PubMed1996

Eubacteria-type isocitrate dehydrogenase from an archaeon: cloning, sequencing, and expression of a gene encoding isocitrate dehydrogenase from a hyperthermophilic archaebacterium, Caldococcus noboribetus.

A gene coding for isocitrate dehydrogenase (ICDH) was cloned from a hyperthermophilic archaebacterium, Caldococcus noboribetus, and sequenced. The gene was preceded by a promoter-like sequence and was followed by a terminator-like sequence. The deduced amino acid sequence of C. noboribetus ICDH showed high similarities to eubacterial ICDH. In particular, extremely high identity scores were found for ICDHs from Vibrio sp. (48.2%) and Escherichia coli (47.9%). The gene was expressed in E. coli by connecting it with the T7 promoter. The molecular weight of the gene product was estimated to be 48,000, which is consistent with that calculated from the deduced amino acid sequence. The gene product showed NADP-dependent ICDH activity at 80 degrees C. While the host-derived ICDH was completely inactivated by treatment at 70 degrees C for 10 min, the ICDH from C. noboribetus showed much higher thermostability.

Amino Acid Sequence↗
PubMed1996

Cloning and sequencing of a gene encoding 16S ribosomal RNA from a novel hyperthermophilic archaebacterium NC12.

A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring. To characterize this organism, a gene coding for 16S rRNA was cloned and sequenced. The 16S rRNA sequence from NC12 shows the highest similarity with those from Pyrodictium occultum and Desulfurococcus mobilis among the sequences in the database, indicating that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain. However, since the highest identity score was only 91.2%, it is suggested that NC12 may constitute a new genus.

Archaea↗
PubMed1996

Redox-linked ionization of sulredoxin, an archaeal Rieske-type [2Fe-2S] protein from Sulfolobus sp. strain 7.

"Sulredoxin" of Sulfolobus sp. strain 7 is an archaeal soluble Rieske-type [2Fe-2S] protein and was initially characterized by several spectroscopic techniques (Iwasaki, T., Isogai, T., Iizuka, T. , and Oshima, T. (1995) J. Bacteriol. 177, 2576-2582). It appears to have tightly linked ionization affecting the redox properties of the protein, which is characteristic of the Rieske FeS proteins found as part of the respiratory chain. Sulredoxin had an Em(low pH) value of +188 +/- 9 mV, and the slope of pH dependence of the midpoint redox potential indicated two ionization equilibria in the oxidized form with pKa(ox1) of 6.23 +/- 0.22 and pKa(ox2) of 8.57 +/- 0.20. The absorption, CD, and resonance Raman spectra of oxidized sulredoxin are consistent with the proposed St2FeSb2Fe[N(His)]t2 core structure, and deprotonation of one of the two putative coordinated histidine imidazoles, having the pKa(ox2) of 8.57 +/- 0.20, causes a decrease in the midpoint redox potential, the change in the optical and CD spectra, and the appearance of a new Raman transition at 278 cm-1, without major structural rearrangement of the [2Fe-2S] cluster as well as the overall protein conformation. The redox-linked ionization of sulredoxin is also contributed by local changes involving another ionizable group having the pKa(ox1) of 6.23 +/- 0. 22, which is probably attributed to a certain positively charged amino acid residue that may not be a ligand by itself but located very close to the cluster. We suggest that sulredoxin provides a new tractable model of the membrane-bound homologue of the respiratory chain, the Rieske FeS proteins of the cytochrome bc1-b6f complexes.

Circular Dichroism↗
PubMed1996

Role of cytochrome b562 in the archaeal aerobic respiratory chain of Sulfolobus sp. strain 7.

The role of cytochrome b562, a fragile constituent of the respiratory terminal oxidase supercomplex of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, was investigated spectroscopically in the membrane-bound state. Cytochrome b562 did not react with CO or cyanide in the membrane-bound state, while it was irreversibly modified to a CO-reactive form (b59) upon solubilization in the presence of cholate and LiCl. Cyanide titration analyses with the succinate-reduced membrane suggested that cytochrome b562 was upstream of both the "gy = 1.89' Rieske FeS cluster and the a-type cytochromes. These results show that the b-type cytochrome functions as an intermediate electron transmitter in the terminal oxidase supercomplex.

Aerobiosis↗
PubMed1996

Molecular cloning, sequencing, and heterologous expression of a novel zinc-containing ferredoxin gene from a thermoacidophilic Archaeon Sulfolobus sp. strain 7.

The gene encoding a novel zinc-containing ferredoxin from a hyperthermophilic and acidophilic archaeon (archaebacterium) Sulfolobus sp. strain 7 was cloned and sequenced. The DNA sequence predicts a 103 residue protein after removal of N-terminal methionine, which is in good agreement with the results of the protein analysis. Surprisingly, the residues responsible for binding a zinc atom were conserved among three other thermoacidophilic archaea. A common sequence stretch VXGXHXGHX8-17PXXLGXHGTX38-56KXDPV is proposed as a new zinc-binding motif, where three histidines and an aspartic acid are ligated to a zinc atom. The ferredoxin gene was expressed in Eschericia coli. The recombinant ferredoxin was indistinguishable from the protein purified from Sulfolobus sp. strain 7 cells by several criteria so far investigated except that the methylation of the 29th lysine was suppressed.

Amino Acid Sequence↗
PubMed1996

Na+,K(+)-ATPase activity in the cochlear lateral wall of the gerbil.

We examined Na+,K(+)-ATPase activity in four regions of the gerbil cochlear lateral wall by measuring intracellular Na+ concentration ([Na+]i). The application of 1 mM ouabain and removal of external K+ caused a most striking increase in [Na+]i of the suprastrial fibrocytes, followed by the stria vascularis and the type II fibrocytes, but no detectable response in the type I fibrocytes. These results may imply the potency of the Na+,K(+)-ATPase activity. The high values of the resting [Na+]i in these cells under perilymph-like solution returned to the [Na+]i within physiological values after exposure to low-Na+ conditions similar to endolymph. Our results are interpreted in the light of recent morphological findings with regard to the roles in the secretion of K+ into endolymph of several cell types within the lateral wall.

Animals↗
PubMed1996

A 718-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 12.7-28.0 min region on the linkage map.

The 718,122 base pair sequence of the Escherichia coli K-12 genome corresponding to the region from 12.7 to 28.0 minutes on the genetic map is described. This region contains at least 681 potential open reading frames, of which 277 (41%) have been previously identified, 147 (22%) are homologous to other known genes, 139 (20%) are identical or similar to the hypothetical genes registered in databases, and the remaining 118 (17%) do not show a significant similarity to any other gene. In this region, we assigned a cluster of cit genes encoding multienzyme citrate lyase, two clusters of fimbrial genes and a set of lysogenic phage genes encoding integrase, excisionase and repressor in the e14 genetic element. In addition, a new valine tRNA gene, designated valZ, and a family of long directly repeated sequences, LDR-A, -B and -C, were found.

DNA, Bacterial↗
PubMed1996

Potentiation of the intracellular Ca2+ response to arginine vasopressin by increased cytosolic-free Mg2+ in rat vascular smooth muscle cells.

Although the inhibitory effects of extracellular Mg2+ on Ca2+ influx are well established, little is known about the effects of intracellular Mg2+ on Ca2+ handling. In the present study, the effects of cytosolic-free Mg2+ concentration in the physiological (submillimolar) range on Ca2+ handling were investigated after stimulation of rat vascular smooth muscle cells with arginine vasopressin. Cytosolic Mg2+ was manipulated by culturing cells in medium containing different Mg2+ concentrations. Peak cytosolic-free Ca2+ concentration responses to arginine vasopressin (1 mumol/1) were measured in the presence and absence of external Ca2+. The results suggest that an increase in cytosolic-free Mg2+ concentration increases both Ca2+ discharge from intracellular stores and Ca2+ influx, whereas a decrease in intracellular Mg2+ attenuates Ca2+ influx.

Animals↗
PubMed1996

The Na-K-Cl cotransporters in the rat cochlea: RT-PCR and partial sequence analysis.

Nonsensory epithelial cells of the mammalian cochlea contribute to the production of the endolymph. The presence as well as the function of the Na-K-Cl cotransporter has been suggested, but not yet been proved. To identify the Na-K-Cl cotransporter isoforms expressed in the cochlea, mRNA was extracted from the cochlear lateral wall. After reverse transcription, resulting cDNA was amplified by polymerase chain reaction (PCR) with primers specific for Na-K-Cl cotransporter isoforms. In the rat cochlear lateral wall, mRNA homologous to the mouse bumetanide-sensitive Na-K-Cl cotransporter (mBSC2) was detected. These results suggest that the basolateral localization of the cotransporter in the marginal cell is involved in the secretory process of the endolymph.

Animals↗
PubMed1996

A leader open reading frame is essential for the expression in Escherichia coli of GC-rich leuB gene of an extreme thermophile, Thermus thermophilus.

To improve expression efficiency of the leuB gene of an extreme thermophile. Thermus thermophilus, in Escherichia coli, the gene was placed under a potent promoter, tac. However, the expression was hardly improved, despite increased transcription. The expression under tac promoter was significantly improved by introducing a leader open reading frame in front of the gene. Similar improvement under a weak promoter, tet, with a leader open reading frame had been described previously. The present results provide evidence that the major limiting step in the expression of a GC-rich thermophile gene in E. coli is translation, and that the addition of a leader open reading frame is more crucial for high level expression of the gene than the use of a potent promoter.

Antigens, Differentiation↗
PubMed1996

A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.

3-Isopropylmalate Dehydrogenase↗
PubMed1996

Long-lasting potentiation in the secondary somatosensory cortex affects motor control: assessment by H-reflex.

We investigated descending projections from the secondary somatosensory cortex to the feline spinal cord and the effects of long-lasting potentiation in secondary somatosensory cortex on the activities of motoneurons of the cat. Electrophysiological examinations revealed that the low-intensity subthreshold secondary somatosensory cortex stimulation could change the H-Reflex induced by radial nerve stimulation. The H-wave amplitudes, recorded in wrist flexor muscles, were enhanced when the intervals from secondary somatosensory cortex to radial nerve stimuli were altered from 0 to 30 ms (initial excitation, 146 +/- 11% (mean +/- S.E.M.) of the control value). In contrast, the H-waves were suppressed with intervals longer than 30 ms (80 +/- 3%). The descending pathways from secondary somatosensory cortex to the spinal cord were assessed using an immunohistochemical technique. c-Fos and Zif268 proteins, induced by stimulation of the hand-represented secondary somatosensory cortex areas, could thus express in activated cervical neurons. The density of labeled cells was significantly higher in the seventh and eighth cervical segments than in other levels. The great majority of positive cells were distributed in the lateral part of the contralateral ventral horn and their somas ranged from 10 to 50 microns in size. Finally, we examined the effects of long-lasting potentiation, induced by high-frequency stimulation of the ventral posterolateral thalamic nucleus, on the activities of spinal motoneurons. Long-lasting potentiation altered the previously observed effects of secondary somatosensory cortex stimulation on the H-wave amplitude. The secondary somatosensory cortex-conditioned initial excitation of the H-reflex was enhanced (from 139 to 175%, P < 0.05), while late suppression was completely blocked (from 74 to 112%, P < 0.01). In conclusion, the descending pathways from secondary somatosensory cortex to the spinal cord modulated the H-reflex, and long-lasting potentiation in secondary somatosensory cortex affected this modulation. We have previously reported that corticocortical inputs from primary to secondary somatosensory cortex is required for induction of long-lasting potentiation in secondary somatosensory cortex. Taken together, the present study suggests that cortical plasticity in secondary somatosensory cortex amplifies somatic inputs from primary somatosensory cortex as a means of adaptive motor control by the sensory system.

Animals↗
PubMed1996

Subcellular distribution of protein kinase C in the living outer hair cell of the guinea pig cochlea.

Immunohistochemical staining using isoform-specific antibodies and intracellular localization using fluorescent probes for protein kinase C (PKC) were evaluated in the cochlear outer hair cell (OHC). Among three isoforms of classic PKC, PKC alpha was selectively stained in the fixed OHC as well as inner hair cells under a surface preparation method. Two types of fluorescent probes to detect subcellular localization of PKC were observed with a confocal laser scanning microscopy in the present study, fim-1 diacetate which binds to the ATP-competitive catalytic domain of PKC and Bodipy FL C12-phorbol acetate which binds to specific site localized to the first cysteine-rich loop of the C1 region in the regulatory domain. High fluorescence intensity of both dyes was observed in subcuticular and subsynaptic regions, infracuticular network, and along the lateral wall. The displacement experiments to evaluate binding specificity were performed by incubating Bodipy FL C12-phorbol acetate in the presence of 10 microM phorbol 12-myritate 13-acetate (PMA) and the fluorescence was totally disappeared. For the acute treatment of phorbol ester, cells were preincubated with 1 microM PMA 30 min before loading with fim-1 diacetate. The brightest area in the plasma membrane became much larger as compared with untreated cells, which suggests a dramatic translocation of PKC to the plasma membrane. The biological functions involving PKC in the OHC are discussed.

Adenosine Triphosphate↗
PubMed1996

Cardioscopic spectrum of the left ventricular endocardial surface and its relation to histologic changes in idiopathic myocarditis.

To examine feasibility of percutaneous cardioscopy for diagnosis of idiopathic myocarditis, cardioscopic appearance of the left ventricle and biopsy findings were compared in 21 such patients. The endocardial surface was milky white, red, pink, or reddish brown and edematous at the segments that exhibited histologic changes of acute myocarditis; purplish red in those that exhibited chronic active myocarditis; and yellow in those that exhibited chronic inactive or healed myocarditis. Follow-up study by repeated cardioscopy and biopsy in six patients revealed that the milky white surface disappeared and that the red, pink, and reddish brown surfaces changed to purplish red and then to yellow or white. The results indicate that the endocardial coloration of the left ventricle represents histologic changes and that cardioscopy is feasible for macroscopic pathologic diagnosis and for follow-up of idiopathic myocarditis.

Acute Disease↗
PubMed1996

An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The aspartate aminotransferase gene (AspAT, EC 2.6.1.1) of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced, and its gene product was overproduced. The purified T. thermophilus AspAT was stable up to about 80 degrees C at neutral pH. T. thermophilus AspAT was strictly specific for acidic amino acid substrates, such as aspartate, glutamate, and the respective keto acids. The gene coding for T. thermophilus AspAT showed that it comprised 1,155 bp with a high G+C content (70 mol%), and encoded a 385-residue protein with a molecular weight of 42,050. The amino acid sequence of T. thermophilus AspAT deduced from its gene showed about 15, 46, and 29% homology with those from Escherichia coli, Bacillus sp. YM-2, and Sulfolobus solfataricus, respectively. When the amino acid sequence of T. thermophilus AspAT was compared with that of E. coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13, all of which are known to be relatively labile at high temperatures. Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37. As shown by the E. coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule. This was quite different from that in the case of RecA protein, which shows an increased number of prolyl residues in the interior of its molecule. Different strategies of different proteins as to prolyl contribution to thermostability have been suggested. Despite the high degree of conservation of active-site residues, Arg292 in E. coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T. thermophilus AspAT. Arg89 may complement the function of Arg292.

Amino Acid Sequence↗