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Biomedical subjects

T Oshima

Publications and source records attributed to T Oshima.

At least 253 records · Page 14Linked to original sources
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

Cross-reactive idiotypes on rabbit anti-SEA antibodies stimulate anti-idiotype spleen and lymph node cell responses of mice infected with Schistosoma mansoni.

Antibodies to Schistosoma mansoni soluble egg antigens (SEA) generated in outbred rabbits from two different sources were used to study cross-reactive idiotype/anti-idiotype (Id/anti-Id) interactions in S. mansoni-infected mice in two different locations. Immunoaffinity purified rabbit polyclonal anti-SEA antibody preparations (RabId) were predominantly Ig by SDS-PAGE and demonstrated anti-SEA activity by ELISA and Western blot. RabId prepared from three separate rabbits and used at 40 micrograms/ml in vitro stimulated lymphocyte proliferative responses of spleen cells from mice with eight week infections (Mean +/- SEM [E-C]) of 3869 +/- 764, 18594 +/- 3046 and 8962 +/- 1734 cpm. Spleen cells from uninfected mice exposed to the same RabId preparations in vitro had respective [E-C] values of 206 +/- 144, 494 +/- 154 and 363 +/- 180. Lymph node cells from mice infected for 8 weeks demonstrated [E-C] of 123 +/- 400, 5073 +/- 828 and 2361 +/- 656 upon exposure to these 3 RabId preparations. Lymph node cells from uninfected mice had [E-C] of 220 +/- 76, 194 +/- 82 and 143 +/- 72 when exposed to these RabId. Maximum in vitro proliferative response to Ig from unimmunized rabbits was 761 +/- 400 by spleen cells from mice with eight week infections. These data indicate the presence of cross-reactive Id on rabbit anti-SEA antibodies from different rabbits that can stimulate in vitro lymphoproliferative responses of spleen or lymph node cells from S. mansoni-infected mice.

Animals↗
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↗
PubMed1996

Conversion of the coenzyme specificity of isocitrate dehydrogenase by module replacement.

The coenzyme specificity of isocitrate dehydrogenase from an extreme thermophilic bacterium was converted from NADP-dependent to NAD-dependent by replacing a "module" involved in the coenzyme binding site. The conversion was not possible with the replacement of a few residues that interact with the coenzyme. In addition, the module-replaced mutant dehydrogenase was as stable as the original, wild type enzyme. The results support a previous hypothesis that a module is a structural and functional unit of a protein.

Amino Acid Sequence↗
PubMed1996

2-oxoacid:ferredoxin oxidoreductase from the thermoacidophilic archaeon, Sulfolobus sp. strain 7.

The purified 2-oxoacid:ferredoxin oxidoreductase of a thermoacidophilic and aerobic crenarchaeote, Sulfolobus sp. strain 7, consists of 70-kDa alpha and 37-kDa beta subunits, and contains one thiamine pyrophosphate (TPP), one [4Fe-4S]2+.1+ cluster, and two magnesium atoms per alpha beta structure. It exhibits a broad substrate specificity toward 2-oxoacids such as 2-oxoglutarate, 2-oxobutyrate, and pyruvate. The gene encoding the archaeal oxidoreductase was cloned, and the two open reading frames encoding the alpha (632 amino acids) and beta subunits (305 amino acids), respectively, were sequenced. Careful sequence alignment revealed several consensus motifs of this enzyme family, as well as possible cofactor binding residues of the Sulfolobus enzyme. This new structural information also indicates that (i) several genetic fusions and reorganization of the early, possibly alpha beta gamma delta-type enzyme similar to those from hyperthermophiles have taken place during evolution of the 2-oxoacid:ferredoxin (flavodoxin) oxidoreductase superfamily, which might have occurred in different ways in early aerobic archaea and early anaerobic bacteria, and that (ii) enzymes with different subunit compositions should have an essentially similar catalytic mechanism with one TPP and at least one [4Fe-4S] cluster as the minimal set of redox centers.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1996

Bilateral sensorineural hearing loss associated with the point mutation in mitochondrial genome.

Mitochondrial DNA (mtDNA) mutation associated with sensorineural hearing loss (SNHL) has previously been described in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and in aminoglycoside-induced deafness. The authors of this study report three cases of SNHL associated with mtDNA mutation (3243A-->G). They examined the clinical features of this type of SNHL by audiologic studies and examined the mtDNA mutation by the polymerase chain reaction technique. In the three cases described, the SNHL had an adult onset and was bilateral and symmetrical. All patients had adult-onset diabetes mellitus. Audiologic studies revealed that the SNHL in all patients derived from the cochlea rather than from retrocochlear sites. It is presumed that mtDNA mutation results in mitochondrial dysfunction in cochlear tissues (i.e., hair cells and stria vascularis) and in neurons of the auditory pathway. Genetic analysis of mtDNA offers new insight into the diagnosis and treatment of SNHL.

Adolescent↗
PubMed1996

Pyrimidine biosynthesis genes (pyrE and pyrF) of an extreme thermophile, Thermus thermophilus.

We have isolated uracil auxotrophic mutants of an extreme thermophile, Thermus thermophilus. A part of the pyrimidine biosynthetic operon including genes for orotate phosphoribosyltransferase (pyrE) and for orotidine-5'-monophosphate decarboxylase (pyrF) was cloned and sequenced. The pyrE gene can be a bidirectional marker for the gene manipulation system of the thermophile.

Amino Acid Sequence↗
PubMed1996

Spontaneous tandem sequence duplications reverse the thermal stability of carboxyl-terminal modified 3-isopropylmalate dehydrogenase.

A mutant strain of Thermus thermophilus which contains deletions in the 3'-terminal region of its leuB gene showed a temperature-sensitive growth phenotype in the absence of leucine. Three phenotypically thermostable mutants were isolated from the temperature-sensitive strain by spontaneous evolution. Each pseudorevertant carried a tandem sequence duplication in the 3' region of its leuB gene. The mutated 3-isopropylmalate dehydrogenases encoded by the leuB genes from the pseudorevertants were more thermostable than the enzyme from the temperature-sensitive strain. Structural analyses suggested that the decreased thermostability of the enzyme from the temperature-sensitive strain was caused by reducing hydrophobic and electrostatic interactions in the carboxyl-terminal region and that the recovered stability of the enzymes from the pseudorevertants was due to the restoration of the hydrophobic interaction. Our results indicate that tandem sequence duplications are the general genetic way to alter protein characteristics in evolution.

3-Isopropylmalate Dehydrogenase↗
PubMed1996

Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method.

We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, by a suppressor mutation method. We previously constructed a chimeric IPMDH consisting of portions of thermophile and mesophile enzymes. The chimeric enzyme is less thermostable than the thermophile enzyme. The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T. thermophilus. The transformants were screened at 76 degrees C in minimum medium, and three independent stabilized mutants were obtained. The leuB genes from these three mutants were cloned and analyzed. The sequence analyses revealed Ala-172-->Val substitution in all of the mutants. The thermal stability of the thermophile IPMDH was improved by introducing the amino acid substitution.

3-Isopropylmalate Dehydrogenase↗
PubMed1996

Distribution of Ca2+ channels on cochlear outer hair cells revealed by fluorescent dihydropyridines.

Physiological evidence has shown that cochlear outer hair cells (OHC) possess L-type voltage-dependent Ca2+ channels through which Ca2+ enters the OHC during depolarization. Their subcellular distribution has, however, remained unclear. In this study, the distribution of L-type Ca2+ channels on the basolateral plasma membrane of OHC has been demonstrated by the use of a laser scanning confocal microscope (LSCM) and a fluorescent probe DMBODIPY-DHP. The fluorescent staining pattern on the basolateral wall is nonuniform, suggesting a heterogeneous distribution of the channels in the plasma membrane. Direct imaging of intracellular Ca2+ visualized in real time by means of the LSCM and the fluorescent Ca2+ probe fluo 3 revealed temporal and spatial integration of Ca2+ movements and Ca2+ channel distribution. Exposure to high-K+ solution induced heterogeneity in the subcellular increase in the intracellular Ca2+ concentration. These results suggest that the heterogeneous distribution of L-type Ca2+ channels on the basolateral membrane might induce heterogeneous intracellular Ca2+ distribution during electrical activity in the OHC.

Animals↗
PubMed1996

Culture and characterization of human nasal gland cells.

To study the regulation of ion transport by human nasal gland (HNG) cells, we developed a system for culturing HNG cells isolated by enzymatic digestion from human nasal polyps. When plated on flasks in media containing. Ultroser G serum substitute and a variety of growth factors, the HNG cells became confluent after 5-7 days. Cells were collected by trypsinization and replated at 10(6) cells/cm2 on porous filters. Confluent monolayers formed on days 3-5 after replating and were studied on days 5-7. Transepithelial resistance and short-circuit current (Isc) were 177 +/- 15 omega.cm2 and 12.2 +/- 1.3 microA/cm2 (means +/- SE, n = 35). Acetylcholine (ACh, 10(-5) M) induced a transient increase in Isc by 4.4 +/- 1.0 microA/cm2 when added to both apical and basolateral sides and returned to baseline levels within 5 min. Diphenylamine-2-carboxylate (10(-3) M) decreased baseline Isc by 30.2 +/- 1.7% (n = 6) and inhibited Isc responses induced by ACh. Amiloride also decreased baseline Isc by 12.3 +/- 3.4% (n = 6) but failed to inhibit ACh-induced Isc responses. ACh (10(-5) M) also induced an increase in intracellular Ca2+ concentration measured by fura 2-AM from 60.5 +/- 16.3 to 248.3 +/- 45.3 nM (n = 4). These findings suggest that gland cells from human nasal polyps can be grown in culture to produce epithelial cell sheets of high resistance, which secrete Cl- in response to cholinergic agent via an increase in intracellular Ca2+ concentration.

Adolescent↗
PubMed1996

Efficient oligonucleotide delivery using the HVJ-liposome method in the central nervous system.

We examined the efficiency and intracellular fate of oligodeoxy-nucleotides (ODN) in the central nervous system (CNS) after delivery with a hemagglutinating virus of Japan (HVJ)-liposome vector in vivo and in vitro. In primary cultured granular cells of the rat cerebellum, application of fluorescein isothiocyanate (FITC)-labeled ODN complexed with HVJ-liposomes in vitro resulted in strong fluorescence localized in nuclei that persisted for > or = 2 wk, in contrast to 3 days with ODN alone. In vivo ODN transfer was attempted by different approaches: infusions into the paraventricular nuclei of the hypothalamus and the lateral cerebroventricle. Injection of FITC-labeled ODN into the hypothalamus by the HVJ-liposome method produced a higher concentration and more persistent fluorescence than did injection of ODN alone. Administration of ODN into the lateral cerebroventricle with HVJ-liposomes yielded more conspicuous and prolonged fluorescence in the periventricular layer, predominantly in cell nuclei. Furthermore, the distribution of fluorescent cells was broader with the HVJ-liposome method. These results indicate that the HVJ-liposome method prolongs the half-life of ODN and concentrates them in cell nuclei. Thus it is an efficient method for ODN transfer and holds promise as a gene delivery method in the CNS.

Animals↗
PubMed1996

Prevalence of neurological disorders in a Japanese town.

The prevalence of all neurological disorders in a Japanese town was calculated, with a result of 91.1 per 1,000 population. The prevalence of cerebrovascular disease was 28.8; myelopathy and/or radiculopathy caused by deformity of the spine or disc herniation, 23.9; neuralgia, 11.5; dementia, 10.4; peripheral nerve disturbance, 5.5; epilepsy, 4.4; Parkinson's disease, 2.0; mental retardation, 2.9; brain/spinal tumor, 1.4; headache, 10.8, and vertigo/dizziness, 4.4. The prevalence of headache and vertigo/dizziness was also calculated from the results of the questionnaires sent to inhabitants: headache, 79.6, and vertigo/dizziness, 60.8. Neurological disorders are common in Japan and likely to continue to increase.

Adolescent↗