Biomedical subjects

T Ohshima

Publications and source records attributed to T Ohshima.

At least 91 records · Page 5Linked to original sources

PubMed2000

Simultaneous chiral analysis of methamphetamine and related compounds by capillary electrophoresis.

A capillary electrophoretic method for the simultaneous chiral analysis of nine cationic drugs (18 enantiomers) has been developed. These drugs are methamphetamine (MA), amphetamine, dimethylamphetamine, ephedrine (EP), norephedrine, methylephedrine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxy-N-ethylamphetamine. The chiral selector, which was added to the electrolyte, was a mixture of beta-cyclodextrin and heptakis(2,6-di-O-methyl)-beta-cyclodextrin. The detection limits of all enantiomers were 0.1 microg/ml, and the intermediate precisions of migration time and peak area of within-run assays (n=6) were under 0.3% and 1.4%, respectively. The calibration curves of the peak area of (1R,2S)-(-)-EP and S-(+)-MA were linear in the range 0.2-500 microg/ml. This method was applicable to urine analysis.

Calibration↗

PubMed2000

Forensic wound examination.

Wound examination is of prime importance in forensic pathology, and it is desirable to establish a wound examination system in order to evaluate and record the nature of wound more accurately and objectively. Modern diagnostic techniques and devices as well as advanced cell-biological methods should be introduced as the means for this aim. For example, radiological, endoscopic or magnetic resonance imaging (MRI) examination have been used in addition to examination with the naked eye. In our department, a binocular surgical operating microscope is routinely employed at forensic autopsy, which is useful for elucidating the nature of wound in more detail. It is also necessary to determine whether a wound has vitality, and, if antemortem, how long before death the wound has been sustained. For the determination of wound age including vitality, various biological factors such as cytokines and extracellular matrix components involved in wound healing have been examined by histopathological methods. Our studies have shown that interleukin (IL)-1alpha, IL-1 b, IL-6, IL-10 and tumor necrosis factor-alpha are possibly useful markers for wound age determination as well as cell-biological indicators of vitality. Furthermore, molecular biological techniques have been intended to be applied to wound examination; our experimental study has shown that even mRNA of cytokines mentioned above can be histologically detected by reverse transcriptase-polymerase chain reaction or in situ hybridization. A trial of forensic wound examination from macroscopic to molecular level is discussed.

Attitude of Health Personnel↗

PubMed2000

Genetic variation and relationships at five STR loci in five distinct ethnic groups in China.

Five short tandem repeat (STR) systems of TH01, vWA, LPL, F13B and FES/FPS were investigated in five ethnic groups living in China (Tujia, Miao, Bai, Chaoxian and Han). All five loci did not deviate from the Hardy-Weinberg equilibrium (P>0.05). At the five loci of each ethnic group, the observed heterozygosity, the mean exclusion chance (MEC), and the power of discrimination (PD) ranged from 0.42 to 0.86, from 0.20 to 0.66 and from 0.61 to 0.95, respectively. For the five ethnic groups, the combined MEC and combined PD were >0. 9360 and >0.9998, respectively, suggesting that combinations of these five systems are feasible for DNA typing in forensic investigations such as personal identification or paternity testing. Furthermore, the allelic frequencies at the five loci suggested that these five ethnic groups were distinctly different communities. Judging from the phylogenetic tree constructed based on the genetic distance among the five ethnic groups, Han, Chaoxian and Tujia were involved in an identical cluster, and Miao and Bai in another. These findings indicate that each of the five groups examined is not only a distinct community, but also has a relationship with each of the others.

Alleles↗

PubMed2000

Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells.

Fabry disease is a compelling target for gene therapy as a treatment strategy. A deficiency in the lysosomal hydrolase alpha-galactosidase A (alpha-gal A; EC ) leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop vascular occlusions that cause cardiovascular, cerebrovascular, and renal disease. Unlike for some lysosomal storage disorders, there is limited primary nervous system involvement in Fabry disease. The enzyme defect can be corrected by gene transfer. Overexpression of alpha-gal A by transduced cells results in secretion of this enzyme. Secreted enzyme is available for uptake by nontransduced cells presumably by receptor-mediated endocytosis. Correction of bystander cells may occur locally or systemically after circulation of the enzyme in the blood. In this paper we report studies on long-term genetic correction in an alpha-gal A-deficient mouse model of Fabry disease. alpha-gal A-deficient bone marrow mononuclear cells (BMMCs) were transduced with a retrovirus encoding alpha-gal A and transplanted into sublethally and lethally irradiated alpha-gal A-deficient mice. alpha-gal A activity and Gb3 levels were analyzed in plasma, peripheral blood mononuclear cells, BMMCs, liver, spleen, heart, lung, kidney, and brain. Primary recipient animals were followed for up to 26 weeks. BMMCs were then transplanted into secondary recipients. Increased alpha-gal A activity and decreased Gb3 storage were observed in all recipient groups in all organs and tissues except the brain. These effects occurred even with a low percentage of transduced cells. The findings indicate that genetic correction of bone marrow cells derived from patients with Fabry disease may have utility for phenotypic correction of patients with this disorder.

Animals↗

PubMed2000

Releasing profiles of gene products from recombinant Escherichia coli in a high-voltage pulsed electric field.

Release of recombinant proteins from gene-engineered Escherichia coli by applying a pulsed electric field (PEF) to a cell suspension was studied. When E. coli/pNC1, which produces beta-glucosidase and accumulates it in cytoplasm, was exposed to PEF, the most effective release of this enzyme was achieved in the cell suspension of 5% glycine and 15% PEG solution under 10kV/cm and 280J/ml of a PEF in a needle-plate electrode chamber. However, the amount of released beta-glucosidase by PEF treatment was only 26% of that by ultrasonic treatment. On the other hand, alpha-amylase produced by E. coli/pHI301A and accumulated in the periplasmic space could be easily released by PEF treatment. When this recombinant E. coli was suspended in 0.9% NaCl and 10% PEG solution and exposed to 10kV/cm and 200J/ml of a PEF in a plate-plate electrode chamber, 89% of intracellular alpha-amylase with nine-times higher specific activity compared with that by ultrasonic treatment was released. The release tendency of cellobiohydrolase, produced by E. coli/pNB6 and accumulated in both the cytoplasm and periplasmic space, was intermediate between those of beta-glucosidase and alpha-amylase. In this case, 70% of cellobiohydrolase with 1.9-times higher specific activity compared with that by ultrasonic treatment could be released when E. coli/pNB6 was suspended in 15% PEG and 10kV/cm and 200J/ml of a PEF was applied in a needle-plate electrode chamber. These results indicated that PEF treatment could easily disrupt the outer membrane, but it was difficult to disrupt the cytoplasmic membrane simultaneously. Therefore, PEF treatment is useful for easy release of periplasmic protein with selectivity.

Journal Article↗

PubMed2000

CBP: A target molecule of HTLV-1 Tax in synoviocyte activation.

Previous studies have shown that human T-cell leukemia virus type 1 (HTLV-1) Tax is a key molecule of synoviocyte activation in HTLV-1 associated arthropathy (HAAP). To clarify the molecular mechanism of HTLV-1 Tax-induced transcriptional activation in synoviocytes from HAAP, we investigated the role of cyclicAMP (cAMP)-regulated enhancer (CRE) binding protein (CREB)-binding protein (CBP), as a target molecule of HTLV-1 Tax. Activation of cyclic AMP (cAMP)/protein kinase-A (PK-A) pathway resulted in a significantly high response of CRE promoter in synoviocytes from patients with HAAP as well as in Tax-transiently transfected synoviocytes from patients with rheumatoid arthritis (RA). Mammalian two-hybrid analysis showed that the recruitment of CBP was responsible for CREB activation. Furthermore, PK-A activation induced CBP-Tax complex in synoviocytes from HAAP and the complex contained CREB. These findings demonstrated that complex formation of CBP and Tax is critical for enhanced CREB activity in synoviocytes from HAAP.

CREB-Binding Protein↗

PubMed2000

Vergence disorders in patients with spinocerebellar ataxia 3/Machado-Joseph disease: a synoptophore study.

Diplopia, a common symptom in spinocerebellar ataxia 3/Machado-Joseph disease (SCA3/MJD) cases, is not always due to asymmetric ophthalmoplegia. We found a Japanese SCA3/MJD family, in which three patients clearly had an impairment of divergence eye movement. We thus quantitatively examined the vergence ranges in eight Japanese SCA3/MJD cases using the synoptophore test. An impairment of the vergence eye movements was found in all patients, and the vergence impairment pattern, but not the ophthalmoplegia pattern, was found to be compatible with the diplopia pattern. The diplopia in SCA3/MJD cases is, therefore, attributed, at least in part, to the impairment of the vergence eye movements.

Abducens Nerve↗

PubMed2000

Catalytic asymmetric synthesis of 19,20-dihydroakuammicine.

An enantiocontrolled total synthesis of 19,20-dihydroakuammicine using a catalytic asymmetric Michael addition of dimethyl malonate to cyclohexenone as the key step is described. The above catalytic asymmetric Michael addition proceeds quite efficiently in the presence of a heterobimetalic asymmetric catalyst (ALB-KO-t-Bu-MS 4A, 0.3 mol%), giving the corresponding Michael adduct in 94% yield and 99% ee.

Journal Article↗

PubMed2000

The expression of mRNA of proinflammatory cytokines during skin wound healing in mice: a preliminary study for forensic wound age estimation (II).

This study examined the temporal expression of mRNA for the proinflammatory cytokines, interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor-alpha (TNFalpha) in incised wounds in mice using the reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) techniques. After incision, an increase in each cytokine mRNA level was observed by RT-PCR. The local IL-6 mRNA level peaked at 6 h, while the peak levels of mRNA for IL-1alpha, IL-1beta and TNFalpha occurred between 48 and 72 h. All cytokine mRNA levels were almost normalized after 240 h. In the early phase of wound healing, infiltrating polymorphonuclear cells were labeled with antisense probes for IL-1alpha, IL-1beta and TNFalpha mRNA by the ISH technique. Thereafter, infiltrating mononuclear cells and spindle-shaped mesenchymal cells showed positive signals for all the cytokines examined. Regenerating epidermal cells were also labeled with the antisense probes for IL-1alpha, IL-6 and TNFalpha mRNA, indicating that IL-1, IL-6, and TNFalpha are involved in skin wound healing and their local production by various cells involved in the healing process is suggested. From the viewpoint of forensic pathology, the temporal characteristics of the cytokine mRNA expression may have a potential to indicate wound age or wound vitality.

Animals↗

PubMed2000

Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications.

NADP-dependent glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95 degrees C for 30 min, and the maximum activity in L-glutamate deamination was obtained around 100 degrees C. The enzyme showed a strict specificity for L-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The Km values for NADP, L-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively. On the basis of the N-terminal amino acid sequence, the encoding gene was identified in the A. pernix K1 genome, cloned, and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46170. Phylogenetic analysis revealed that the glutamate dehydrogenase from A. pernix K1 clustered with those from aerobic Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota. The branching pattern of the enzymes from A. pernix K1, S. solfataricus, S. shibatae, and Pb. islandicum in the phylogenetic tree coincided with that of 16S rDNAs obtained from the same organisms.

Amino Acid Sequence↗

PubMed2000

Determination of triazolam by GC-MS in two autopsy cases: distribution in body fluids and organs.

A detailed procedure for analysis of triazolam by GC-MS was constructed in our laboratory. At the concentration of 100 ng/ml, recoveries of triazolam in plasma and urine were 84.9 and 91.0%, respectively. The coefficients of variation in terms of its recovery were 11.5 (plasma) and 10.2% (urine). The detection limit for quantitation by the method was approximately 5 ng/g. This method was applied to two autopsy cases, giving triazolam distribution in body fluids and organs. In one case (33-year-old woman), concentrations of triazolam in the heart blood, urine, brain, lung, liver, kidney, skeletal muscles and stomach contents were 83.9, 741, 106, 165, 507, 293, 125 and 343 ng/g, respectively. From these toxicological data together with autopsy findings, her cause of death was diagnosed as triazolam poisoning. In the other case (45-year-old man), triazolam concentrations in the urine and stomach contents were 7.81 and 41.1 ng/g, respectively, but it could not be detected in the pleural blood; his cause of death was judged to be a traumatic shock, based on autopsy findings.

Journal Article↗

PubMed2000

Immunohistochemical study on Fas and Fas ligand in skin wound healing.

An immunohistochemical study on the expression of Fas and Fas ligand (Fas L) was performed in order to examine the role of apoptosis through Fas-Fas L in mouse skin wound healing. After a 1-cm-long incision in the central dorsum skin, mice were sacrificed at intervals ranging from 0.5 to 240 h, followed by the sampling of wound margin. The expression of Fas and Fas L in the wound margins and in uninjured skin controls was studied using frozen sections. In uninjured skin controls, a very weak expression of Fas and Fas L was detected immunohistochemically in hair follicles, sebaceous glands and epidermal cells. In wounded specimens, polymorphonuclear cells and inflammatory mononuclear ones (round-shaped and spindle-shaped types) were evident. A single immunostaining showed that Fas or Fas L was detectable in inflammatory mononuclear cells involved in the skin wound healing process. Double immunostaining for Fas and Fas L revealed that inflammatory mononuclear cells co-expressed both antigens. In situ TUNEL combined with immunostaining showed that the inflammatory mononuclear cells expressing Fas or Fas L and the polymorphonuclear cells were TUNEL-stained, although neither Fas nor Fas L was detected in the polymorphonuclear cells. The number of TUNEL-positive, inflammatory mononuclear cells expressing Fas or Fas L per 0.01 x 0.01 cm2 was counted. The average number of 10 randomly selected microscope fields reached a peak at the fibro-proliferative phase of wound healing. These results indicate that apoptosis through Fas and Fas L may play an important role for reducing the cellularity during skin wound healing in mice.

Animals↗

PubMed2000

Immunohistochemical study on the expression of c-Fos and c-Jun in human skin wounds.

An immunohistochemical study on the temporal expression of c-Fos and c-Jun, both of which designate proto-oncogene products, was performed on 60 human skin wounds with different post-infliction intervals. In unwounded skin, c-Fos or c-Jun was immunolocalized at the nuclei of the epidermal cells in the basal layer, hair follicle cells and sweat gland cells. During the early inflammatory phase of wound healing, the nuclei of polymorphonuclear cells (probably neutrophils), mainly infiltrating at the wound site, were labeled with anti-c-Fos or -c-Jun antibody. As the wound age increased, the neutrophils had disappeared at the wound site, and both mononuclear cells (probably macrophages) and spindle-shaped fibroblastic cells, which expressed a c-Fos or c-Jun positive reaction in the nuclei, were mainly observed. Morphometrically, the distribution of the c-Fos-positive ratio was very similar to that of the c-Jun-positive ratio; the positive ratio was considerably increased in wound specimens with a post-infliction interval of > or = 1 day, thus indicating the late inflammatory or proliferative phase. This study showed that c-Fos and c-Jun were closely involved in the inflammatory phase as well as the proliferative phase of the wound healing process.

Humans↗

PubMed2000

Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin.

A family of basic-helix-loop-helix (bHLH) nuclear factors play important roles in controlling cell growth and differentiation as critical regulatory components in transcription. Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin. Transactivation property of POD-1/Capsulin was analyzed by the Gal4 fusion system in six mammalian cell lines. The results indicated that an activation property was shown in HT1080 and HeLa cells, but a repression activity in HepG2 cells. Mapping analysis for the transactivation and repression activities revealed that the C-terminal domain of POD-1/Capsulin is essential for the transactivation and both the N-terminal and C-terminal domains are contributed to the repression activities. Furthermore, in order to identify possible interactants of the POD-1/Capsulin, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified a class A bHLH protein, ITF-2 as potential heterodimeric partner of the bHLH protein.

3T3 Cells↗

PubMed2000

The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 degrees C.

The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 degrees C. It also showed high stability of 18.3 kcal.mol-1 (76.5 kJ.mol-1) at 25 degrees C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA-like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 degrees C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 degrees C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 degrees C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 degrees C being the critical threshold temperature.

Adenosine Triphosphatases↗

PubMed2000

Biochemical characterization, cloning, and sequencing of ADP-dependent (AMP-forming) glucokinase from two hyperthermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis.

The ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51,265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53,621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed.

Amino Acid Sequence↗

PubMed2000

The effect on intracuff pressure of various nitrous oxide concentrations used for inflating an endotracheal tube cuff.

UNLABELLED: We sought to determine the optimal concentration of nitrous oxide (N(2)O) for inflating endotracheal tube cuffs, to avoid overinflation and air leaks. Female patients undergoing endotracheal intubation (inner diameter 7.5 mm) during anesthesia with 67% N(2)O were randomly assigned to five groups of 25 subjects each, in which cuffs were inflated with 0% (Air), 30% (N30), 40% (N40), 50% (N50), or 67% (N67) N(2)O. The cuff pressure and the N(2)O concentration in the cuff were measured. In an additional 15 patients (N40-a group), pilot balloons were replaced with metal tubes, and the mouths and noses of the patients were wrapped with tape, to minimize N(2)O efflux into the air. Postoperative sore throats were evaluated in double-blinded interviews. Cuff pressures increased significantly in the Air and N30 groups but decreased in the N67 group. Cuff pressures were <22 mm Hg in the N40 and N50 groups, but the N50 group had air leaks. The N(2)O concentration in the cuff in the N40 group was significantly smaller than that in the N40-a group, suggesting N(2)O rediffusion. The incidence of sore throats (40% in the Air group) was reduced significantly in the N40 and N50 groups. Therefore, 40% N(2)O is optimal for filling the cuff during anesthesia with 67% N(2)O. IMPLICATIONS: Nitrous oxide (N(2)O) diffuses into the cuff, equilibrating at a smaller concentration than the gas mixture with which patients are ventilated. Our data indicate that inflation of the cuff with 40% N(2)O is recommended to prevent both excessive endotracheal cuff pressure and air leaks during anesthesia with 67% N(2)O, reducing postoperative sore throats.

Adolescent↗

PubMed2000

Automated specific capture of hepatitis C virus RNA with probes and paramagnetic particle separation.

We developed and evaluated a prototype automated specimen preparation instrument for the specific capture of hepatitis C virus (HCV) RNA with probes and magnetic bead-fluid separation. HCV RNA was isolated from serum by lysis of virus particles with a chaotropic agent, followed by hybridization of the RNA with biotinylated probes and capture of the hybridized RNA with streptavidin-coated paramagnetic particles. After washing of the hybrid-particle complexes to remove nonspecifically bound materials, the particles were resuspended in a specimen diluent and were then ready for amplification and detection with a fully automated PCR system (COBAS AMPLICOR; Roche Diagnostic Systems). The analytical sensitivity in the dilution series was 33 copies per ml or greater. Comparison of the test results with those obtained by a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R; Sanko Junyaku Co., Ltd.) showed 93% (49 of 53 samples) sensitivity and 100% (12 of 12 samples) specificity. There was 94% overall agreement between results. When RNA was extracted by the manual method from serum containing 10(3) or 10(5) copies of HCV per ml in the presence of heparin, there was an inhibitory effect on detection of both HCV RNA and the internal control. In contrast, when RNA was extracted from the serum by the automated method, there was no inhibitory effect. This inhibitory effect of heparin on the manual method was also observed for a series of serum specimens from a hemodialysis patient, but the inhibitory effect was eliminated by the automated specimen preparation method. In summary, a fully automated RNA extraction system for PCR detection of HCV RNA by use of specific capture with probes and magnetic bead-fluid separation was shown to have performance similar to that of the conventional manual method. In addition, it successfully eliminated the inhibitory effect of the heparin in the serum and permitted the detection of HCV RNA in serum samples from a hemodialysis patient. The prototype automated RNA extraction system is suitable as a totally automated system, starting with RNA extraction to detection of HCV, if it was combined with the fully automated COBAS AMPLICOR PCR system.

Automation↗