Biomedical subjects

T Kojima

Publications and source records attributed to T Kojima.

At least 217 records · Page 12Linked to original sources

PubMed1999

Purification and characterization of dibenzothiophene (DBT) sulfone monooxygenase, an enzyme involved in DBT desulfurization, from Rhodococcus erythropolis D-1.

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl by Rhodococcus erythropolis D-1. Three desulfurization (Dsz) enzymes--DszC, A, and B--and flavin reductase are involved in sulfur-specific DBT desulfurization. In this study, DszA was purified, characterized, and crystallized from R. erythropolis D-1. DszA, DBT sulfone monooxygenase, is the second enzyme in microbial DBT desulfurization metabolism and catalyzes the conversion of DBT sulfone to 2'-hydroxybiphenyl 2-sulfinic acid in the presence of flavin reductase with cleavage of the carbon-sulfur bond in the DBT skeleton. Using anion-exchange column chromatography, the four enzyme fractions responsible for DBT desulfurization were separated, and DszA was then purified to homogeneity. Polygonal crystals of DszA were observed within a week. DszA was found to have a molecular mass of 97 kDa and to consist of two subunits with identical masses of 50 kDa. The N-terminal amino acid sequence of the purified DszA completely coincided with the deduced amino acid sequence for dszA of R. erythropolis IGTS8 except for a methionine residue at the latter N-terminal. The optimal temperature and pH for DszA activity were 35 degrees C and about 7.5. The activity of the enzyme was inhibited by Mn2+, Ni2+, 2,2'-bipyridine, and 8-quinolinol, suggesting that a metal might be involved in its activity. DszA acted on not only DBT sulfone but also on dibenz[c,e][1,2]oxathiin 6-oxide and dibenz[c,e][1,2]oxathiin 6,6-dioxide. Dihydroxybiphenyl was formed from the latter two substrates.

Journal Article↗

PubMed1999

Probiotic preparations dose-dependently increase net production rates of organic acids and decrease that of ammonia by pig cecal bacteria in batch culture.

We tested probiotic preparations containing Bifidobacterium, Enterococcus, or Lactobacillus to see if they affect production of organic acids and ammonia by mixed cecal bacteria. Four preparations (Bibalance, Neorakuton, Yakult Seichoyaku, or Yakult Seichoyaku BL) were digested with HC and pancreatin before adding to batch cultures of 50% pig cecal contents. Concentrations of organic acids and ammonia in the culture were quantified. Two preparations stimulated the net production of short-chain fatty acids (SCFA) during the first 8 hr of incubation. From 8 to 24 hr of incubation, all preparations accelerated the net production of SCFA, succinic and lactic acids, and three preparations slowed the net production of isovaleric acid and ammonia, all dose-dependently. The above results indicate that the preparations tested accelerated the breakdown of hard-to-degrade carbohydrate(s) and, possibly thereby, decreased the breakdown of proteinous materials or increased bacterial cell body synthesis in a mixed culture of cecal bacteria.

Acetic Acid↗

PubMed1999

Histocompatibility antigens and alleles in Japanese haemophilia A patients with or without factor VIII antibodies.

We carried out human leukocyte antigen (HLA)-A, B, Cw, DR and DQ serological typing and HLA-DQA1, DQB1, DRB1 and DPB1 genetic typing for 46 Japanese haemophilia A patients, including 20 who had developed an antibody to factor VIII. It appears that anti FVIII inhibitor formation is associated with the major histocompatibility complex in Japanese haemophilia A patients. Absence of HLA-A24 is a principal risk factor for inhibitor formation in Japanese haemophilia A patients. As supplemental risk factors, HLA-DR4.1, DQ4 and DQA1*0301=2 are positively associated with patients exhibiting inhibitor compared with normal subjects. This and previous studies show that the association between HLA antigens and the formation of inhibitor depends on race. Data of HLA typing may be useful for the recognition of groups at high risk for the possible formation of inhibitor among Japanese haemophilia A patients.

Alleles↗

PubMed1999

Antibacterial activity of multiple antigen peptides homologous to a loop region in human lactoferrin.

An 11-residue peptide (FQWQRNMRKVR) homologous to just over half the loop region of human lactoferricin is thought to be responsible for antimicrobial properties of human lactoferricin. Multiple antigen peptides (MAP) of the 11-residue peptide exerted significant antibacterial effects against a broad spectrum of bacteria including MRSA. More than eight branching was favourable for increasing its antibacterial activity. Our report shows a novel possibility for MAP to increase the activity of antibiotic peptides other than simply to stimulate antibody production, as reported so far.

Amino Acid Sequence↗

PubMed1999

A signal sequence trap based on a constitutively active cytokine receptor.

Targeting of secreted and cell-surface proteins to the cell membrane is mediated by a short hydrophobic stretch of amino acids, termed the signal sequence. We have developed a method that detects signal sequences in cDNA fragments based on their ability to redirect a constitutively active mutant of a cytokine receptor to the cell surface, thereby permitting interleukin-3 (IL-3)-independent growth of Ba/F3 cells. Retrovirus-mediated expression of the fusions in IL-3-dependent cells was followed by selection of clones for growth in the absence of IL-3. Infection of cells with 5x10(6) viral particles in a pilot experiment led to the isolation of 150 known and 48 novel cDNA clones, and all the known cDNA clones were found to encode secreted and cell-surface proteins. In addition, we isolated type II membrane proteins, which have not been detected by existing signal sequence trap strategies.

Animals↗

PubMed1999

Identification of engrailed promoter elements essential for interactions with a stripe enhancer in Drosophila embryos.

BACKGROUND: The structures and functions of promoter sequences of most genes have been analysed using in vitro transcription and/or cultured cell systems, neither possessing tissue-specific enhancers. Promoter-enhancer interactions in vivo, in particular, during ontogeny, are still poorly understood. RESULTS: We have established a new method for the assessment of promoter activity in cells that participate in fly body formation, using the UAS/GAL4 system. A functional analysis was then conducted on the promoter sequence of the engrailed gene in Drosophila embryos. A 38-bp-long sequence, terminating with an initiator or RNA start site and a downstream promoter element, was found to be capable of receiving activation signals from the engrailed stripe enhancer. Transcriptional efficiency was improved significantly by the presence of upstream promoting elements, most functionally replaceable with synthetic GAGA factor binding sites. CONCLUSIONS: We identified the in vivo minimum promoter of engrailed and demonstrated that the GAGA factor binding sites serve primarily as quantitative elements which augment transcriptional efficiency. Evidence was also obtained that indicated that not only enhancer but also promoter sequences were involved in the determination of the tissue-specificity of gene expression.

Animals↗

PubMed1999

CagA and cytotoxicity of Helicobacter pylori are not markers of peptic ulcer in Japanese patients.

BACKGROUND: The infection with cagA-positive Helicobacter pylori strains is reported to be associated with peptic ulcer disease in developed countries, but it is controversial in Asia. To investigate the relationship between the virulence factors of H. pylori and peptic ulcer disease in Japan, we compared these between ulcer and nonulcer patients. MATERIALS AND METHODS: Seventy-four strains of clinically isolated H. pylori obtained from 22 gastric ulcer (GU), 23 duodenal ulcer (DU), and 29 chronic gastritis (CG) patients were studied. The presence of vacA and cagA gene was examined by polymerase chain reaction method using two different primer sets. We evaluated the proliferation-inhibiting and lethal cytotoxicity of culture supernatants using the alamarBlue assay. RESULTS: The vacA gene was identified in all strains by the original primers. S1 strains were found in 90.9% (20/22) from GU, 95.7% (22/23) from DU, and 96.6% (28/29) from CG patients. The prevalence of cagA gene determined by the first, and second primers was 90.9% (20/22), 90.9% (20/22) in strains from GU, 87.0% (20/23), 91.3% (21/23) from DU, and 86.2% (25/29), 89.7% (26/29) from CG patients, respectively. The supernatant showed cytolethal effect in 95.5% (21/22) of strains from GU, in 100% (23/23) from DU, and in 93.1% (27/29) from CG patients. There was no significant difference in the prevalence of the virulence factors between H. pylori strains isolated from patients with peptic ulcers and those with chronic gastritis. CONCLUSIONS: These results indicate that cagA gene status and the proliferation-inhibiting and lethal cytotoxicity of supernatant are not reliable markers of ulcerogenicity of H. pylori in Japanese patients.

Adolescent↗

PubMed1999

The fourth-compartment syndrome: its anatomical basis and clinical cases.

We propose a new term, the "fourth-compartment syndrome" to describe chronic dorsal wrist pain of the fourth compartment. Five main causes responsible for this syndrome are thought to be as follows: 1. Ganglion involvement, including an occult ganglion; 2. Extensor digitorum brevis manus muscle; 3. Abnormal extensor indicis muscle; 4. Tenosynovialitis; 5. Anomaly or deformity of carpal bones. Should the above mentioned conditions occur in the fourth compartment, pressure within the fourth compartment increases, ultimately compressing the posterior interosseous nerve directly or indirectly. Anatomical studies of the fourth compartment of the wrist and the posterior interosseous nerve are presented and the fourth-compartment syndrome is summarized with twelve clinical cases (six cases of occult ganglions, two cases of extensor digitorum brevis manus, two cases of tenosynovialitis, one case of abnormal extensor indicis muscle, and one case of carpal bossing).

Adolescent↗

PubMed1999

Tumor cell growth suppression by tannic acid.

Tannic acid was cultured together with tumor cells that had originated from human malignant tumors (HCT-15 & AGS). Significant suppression of tumor growth was observed. The 50% suppression was observed at the concentration between 50 micrograms/ml and 12.5 micrograms/ml. When tannic acid was injected into HCT-15 cells by electroporation at 1180 mu p and 200 ohm, the suppression rate was 34.3% at the concentration of 50 micrograms/ml in HBS solution. The suppression rate of cells only in contact with tannic acid solution for one hour under the same conditions as used for electroporation, was 26.1%. Histographic findings suggested that tannic acid almost completely blocked the S phase of the cell cycle.

Adenocarcinoma↗

PubMed1999

Tannic acid raises survival rate of mice bearing syngeneic tumors.

Tannic acid which was found earlier to have growth suppressive activity against cultured tumor cells, was given to Balb/c mice p.o. The mice had been inoculated with syngeneic tumor cells (Meth/A) i.p. When tannic acid was suspended in drinking water and given daily at doses of approximately 875 mg/kg/day and 1750 mg/kg/day, the respective survival rates were 59% and 48%, with that of the control mice being 29%. To study the cytotoxicity of tannic acid, we administered the tannic acid to mice at 875 and 1750 mg/kg/day for 35 days. The weight gain for each dose was lower than that of the control, although the difference was not significant. When tannic acid was given at 8750 mg/kg/day, the weight gain was significantly lower than the control. A histologic study did not show any pathological findings in the kidney, liver, or lungs in the mice given tannic acid at the above doses.

Administration, Oral↗

PubMed1999

Simple and sensitive analysis of nereistoxin and its metabolites in human serum using headspace solid-phase microextraction and gas chromatography-mass spectrometry.

A simple method for the analysis of nereistoxin and its metabolites in human serum using headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) is developed. A vial containing a serum sample, 5M sodium hydroxide, and benzylacetone (internal standard) is heated to 70 degrees C, and an SPME fiber is exposed for 30 min in the headspace of the vial. The compounds extracted by the fiber are desorbed by exposing the fiber in the injection port of the GC-MS. The calibration curves show linearity in the range of 0.05-5.0 micrograms/mL for nereistoxin and N-methyl-N-(2-methylthio-1-methylthiomethyl)ethylamine, 0.01-5.0 micrograms/mL for S,S'-dimethyl dihydronereistoxin, and 0.5-10 micrograms/mL for 2-methylthio-1-methylthiomethylethylamine in serum. No interferences are found, and the analysis time is 50 min for one sample. In addition, this proposed method is applied to a patient who attempted suicide by ingesting Padan 4R, a herbicide. Padan 4R contains 4% cartap hydrochloride, which is an analogue of nereistoxin. Nereistoxin and its metabolites are detected in the serum samples collected from the patient during hospitalization. The concentration ranges of nereistoxin in the serum are 0.09-2.69 micrograms/mL.

Adult↗

PubMed1999

Blinded, prospective, and serial evaluation by quantitative-EEG in interferon-alpha-treated hepatitis-C.

OBJECTIVES: To estimate the effect of brain function due to IFN-alpha in chronic hepatitis C patients by the quantitative EEG. METHODS: 56 chronic hepatitis C patients were administered IFN-alpha intramuscularly at 9x10(6) IU daily for the first 4 weeks and then 3 times/week for the next 20 weeks. Serial EEGs were obtained in each subject before, at 2 and 4 weeks, and after completing the treatment. Resting EEG without artifacts was selected for quantitative EEG analysis, which was performed blindly. The frequency range was divided into delta to beta. The absolute and relative powers of each frequency band in each subject were calculated and the differences of these powers at different stages were analyzed statistically. RESULTS: The absolute powers of slow waves (delta, theta 1, and theta 2) increased while alpha 2 and fast wave (beta) decreased significantly at all locations during IFN-alpha administration. The total power and alpha 1 values revealed no significant alterations. The relative power revealed the same alteration during treatment. These changes disappeared following the treatment. Such diffuse slowing in the EEG was revealed by the total change in the whole subjects. CONCLUSIONS: Diffuse slowing in the EEG was induced by IFN-alpha, was reversible, and was evident as the total change in the subjects. These findings suggested mild IFN-alpha-induced encephalopathy.

Analysis of Variance↗

PubMed1999

Polyphosphates in intraradical and extraradical hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita.

The amount of polyphosphate in the intraradical and extraradical hyphae of Gigaspora margarita was estimated from successive extractions with trichloroacetic acid (TCA), EDTA, and phenol-chloroform (PC). In the intraradical hyphae, most of the polyphosphate was present in TCA- and EDTA-soluble (short-chain and long-chain) fractions, whereas most of the polyphosphate in the extraradical hyphae was present in EDTA- and PC-soluble (long-chain and granular) fractions.

Biomass↗

PubMed1999

Epstein-Barr virus promotes epithelial cell growth in the absence of EBNA2 and LMP1 expression.

We attempted to infect primary gastric epithelia (PGE) with recombinant Epstein-Barr virus (EBV) carrying a selectable marker that made it possible to select EBV-infected cells. Cells dually positive for EBV-determined nuclear antigen (EBNA) and cytokeratin were detected in 3 of 21 primary cultures after 3 days of EBV inoculation. From one culture, EBV-infected cell clones were repeatedly obtained at a frequency of 3 to 5 cell clones per 10(6) cells. EBV-infected clones had enhanced population doubling and grew to attain a highly increased saturation density, together with acquisition of marked anchorage independence. The infected clones retained the ultrastructural morphology characteristic of gastric mucosal epithelium and have been growing stably for more than 18 months (corresponding to at least 300 generations) so far, in clear contrast to the parental PGE cells, which ceased growth after 60 generations. The p53 gene of the parental PGE cells was found to be overexpressed, perhaps thereby conferring the basal potential for long-term survival in vitro. Moreover, EBV infection accelerated, to a significant extent, the growth rate and agar clonability of NU-GC-3 cells, an established EBV-negative but EBV-susceptible human gastric carcinoma cell line. Both EBV-converted PGE and NU-GC-3 clones, like EBV-positive gastric carcinoma biopsy specimens, expressed a restricted set of EBV latent infection genes characterized by the absence of EBNA2 and latent membrane protein 1 (LMP1) expression. These results indicate that EBV infection causes a transformed phenotype on PGE in the setting of possible unregulated cell cycling and renders even established gastric carcinoma cells more malignant via a limited spectrum of viral latent-gene expression. This study may reflect an in vivo scenario illustrating multiphasic involvement of EBV in carcinogenesis of gastric or other epithelial cancers.

Cell Division↗

PubMed1999

Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants.

In Drosophila, two Frizzled proteins, Frizzled and Dfrizzled-2, have been reported to serve as receptors of Wingless. Here, we identified the third member of the Drosophila Frizzled family (Dfrizzled-3). In contrast to Dfrizzled-2, Dfrizzled-3 was transcriptionally upregulated by Wingless signaling. Although Dfrizzled-3 was capable of binding to Wingless in vitro, Wingless-dependent Armadillo/beta -catenin stabilization occurred much less effectively in Drosophila cells transfected with Dfrizzled-3 than in those with Dfrizzled-2. Flies lacking Dfrizzled-3 activity were viable and fertile, with few morphological defects. Genetic and immunochemical analysis indicated that the absence of Dfrizzled-3 activity suppresses the effects of hypomorphic wingless mutations such as failure of wing and antenna formation and restores target gene expression to the normal levels without change in wingless expression. Wingless signaling may thus be attenuated by Dfrizzled-3 at least in wingless hypomorphic mutants.

Amino Acid Sequence↗

PubMed1999

Bar homeobox genes are latitudinal prepattern genes in the developing Drosophila notum whose expression is regulated by the concerted functions of decapentaplegic and wingless.

In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. Here, we show that two homeobox genes at the Bar locus (BarH1 and BarH2) may belong to a different class of prepattern genes expressed latitudinally, and suggest that the developing notum consists of checker-square-like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar homeobox gene activity. Bar homeobox gene expression is restricted dorsally and posteriorly by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar homeobox genes is determined by wingless whose expression is under the control of Decapentaplegic signaling.

Animals↗

PubMed1999

Disruption of circumferential actin filament causes disappearance of occludin from the cell borders of rat hepatocytes in primary culture without distinct changes of tight junction strands.

We investigated the relationship of actin filament organization to occludin and tight junction strands in primary cultured rat hepatocytes using an actin depolymerizing agent, mycalolide B. In control cultures, well-developed circumferential actin filaments and occludin immunoreactivity were observed on the most subapical plasma membrane of the cells, and tight junction strands formed well-developed networks in freeze-fracture replicas. In hepatocytes treated with 3 microM mycalolide B for 6 h, circumferential actin filaments and occludin immunoreactivity disappeared from the cell borders. However, there were no marked abnormalities of tight junction strands in freeze fracture replicas. Similar results were obtained from cells cultured in medium with 0.05 mM Ca2+ for 6 h. The close association of occludin with actin and the existence of intact tight junction strands that are virtually free of both occludin and actin suggest a physiological role of occludin, but not the other proteins forming the tight junction strands, in the linkage between actin cytoskeleton and tight junction.

Actins↗

PubMed1999

TPA induced expression and function of human connexin 26 by post-translational mechanisms in stably transfected neuroblastoma cells.

Connexin 26 (Cx26) has been proposed to be a tumor suppressor gene and its expression may modulate development, cell growth and differentiation in various tissues, including the brain. 12-O-tetradecanoylphorbol-13-acetate (TPA) may serve as either tumor promoter (in mammary gland amd skin) or as a differentiating agent (in neuroblastoma and leukemic cells) and may also modulate expression, function and phosphorylation of gap junctions. In this study, to determine the effects of TPA on Cx26 expression and its function in neuroblastoma, we transfected N2A mouse neuroblastoma cells (which are gap junction deficient) with the coding region of human Cx26 gene (which lacks TPA response elements) and examined the changes of expression and function of Cx26 following 10 nM TPA treatment. Individual clones of transfectants stably expressed distinct levels of exogenous Cx26 as judged by Northern and Western blots, immunocytochemistry and electrophysiological recordings. Cx26 channels displayed unitary conductances of about 140-155 pS. Increase of Cx26 expression following TPA treatment was markedly observed using immunocytochemistry and Western blots of membrane fractions although it was not detected in Northern or Western blots of whole cells. This increase in Cx26 expression in the plasma membrane was accompanied by an increase of function as evidenced in measurements of junctional conductance. These results suggest that induction of exogenous Cx26 in neuroblastoma cells by TPA treatment is controlled by post-translational mechanisms.

Animals↗