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D Kobayashi

Publications and source records attributed to D Kobayashi.

At least 37 records · Page 2Linked to original sources
PubMed2001

Helicobacter pylori DNA in drinking water in Japan.

Helicobacter pylori has been detected in drinking water in Peru and Sweden, suggesting the possibility of water-borne transmission. To date there have been few reports of H. pylori being detected in water; one was of the ureA gene of H. pylori in wells and springs in rural Japan. We examined water sampled in or near urban areas of Japan for H. pylori DNA by three assays using the polymerase chain reaction (PCR). Near Tokyo, samples were obtained: 10 of tap water, 6 of well water, 10 of river water, and 10 of sea water. Samples were filtered with membranes with 0.05- or 0.22-microm pores, which bacterial cells are caught by. Bacterial nucleic acids were extracted and purified and the PCR was done to amplify adhesin specific for H. pylori and the ureA gene, if present. Real-time PCR that measured the yield in terms of fluorescence was done with primers for 16S rRNA. None of the samples of tap, river, or sea water contained adhesin, ureA or 16S rRNA. None of the 6 samples of well water contained adhesin or ureA, but 2 of the 6 samples contained 16S rRNA. Some of the users of the well had had H. pylori infection in the past. H. pylori DNA was detected in well water and the users had been infected, so water-borne transmission via well water may occur even in towns in Japan.

Adhesins, Bacterial↗
PubMed2001

Contribution of caspase-3 differs by p53 status in apoptosis induced by X-irradiation.

We investigated the effect of p53 status on involvement of caspase-3 activation in cell death induced by X-irradiation, using rat embryonic fibroblasts (REFs) transduced with a temperature-sensitive mutant (mt) p53 gene. Cells with wild-type (wt) p53 showed greater resistance to X-irradiation than cells with mt p53. In cells with wt p53, X-irradiation-induced apoptosis was not inhibited by the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspartyl-aldehyde (Ac-DMQD-CHO) and caspase-3 activity was not elevated following X-irradiation, although induction of p53 and p21 / WAF-1 protein was observed. In contrast, irradiated cells with mt p53 showed 89% inhibition of cell death with Ac-DMQD-CHO and 98% inhibition with the antioxidant N-acetyl-L-cysteine (NAC). In cells with mt p53, caspase-3 activity was increased approximately 5 times beyond baseline activity at 24 h after irradiation. This increase was almost completely inhibited by NAC. However, inhibition of caspase-3 by Ac-DMQD-CHO failed to decrease production of reactive oxygen species by cells with mt p53. Differential involvement of caspase-3 is a reason for differences in sensitivity to X-irradiation in cells with different p53 status. Caspase-3 activation appears to occur downstream from generation of reactive oxygen species occurring independently of wt p53 during X-irradiation-induced cell death.

Animals↗
PubMed2001

Augmented adriamycin sensitivity in cells transduced with an antisense tumor necrosis factor gene is mediated by caspase-3 downstream from reactive oxygen species.

While transduction of an antisense tumor necrosis factor (TNF) gene sequence can augment the cytotoxicity of adriamycin (ADM) in human cancer cells, the specific effect of introducing this sequence on the signal transduction pathway leading to cell death remains unclear. In ADM-resistant pancreatic carcinoma (PANC-1) cells, both the antioxidant N-acetyl-L-cysteine (NAC) and the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspartyl-aldehyde (Ac-DMQD-CHO) prevented ADM-induced cytotoxicity. NAC additionally inhibited caspase-3 activity induced by ADM treatment, while Ac-DMQD-CHO showed no suppressive effect on reactive oxygen species (ROS). Stable antisense-TNF transfectants showed higher ADM sensitivity and greater ADM-induced ROS production and caspase-3 activity than mock transfectant or parent cells. These results indicate that increased caspase-3 activity downstream from ROS production is among the mechanisms by which transduction of the antisense TNF sequence of augments ADM sensitivity of pancreatic carcinoma cells.

Acetylcysteine↗
PubMed2001

Tumor necrosis factor-alpha in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR.

BACKGROUND: The significance of serum concentrations of tumor necrosis factor-alpha (TNF-alpha) in the pathogenesis of inflammatory bowel disease (IBD) is uncertain. We measured TNF-alpha in serum from IBD patients by immuno-PCR to analyze the relationship between TNF-alpha and pathophysiologic state in IBD. METHODS: Serum samples were collected from 54 healthy blood donors, 29 patients with ulcerative colitis (UC; 46 samples), and 7 patients with Crohn disease (CD; 8 samples). DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to biotinylated third antibody. TNF-alpha sandwiched by antibodies was detected by PCR amplification of the DNA label. RESULTS: TNF-alpha could be measured in all samples. The median serum concentration in IBD patients overall was approximately 390-fold higher than in healthy donors (median increase, 380-fold for UC, 640-fold for CD). The median serum TNF-alpha concentration was 1.7-fold higher in the active stage of UC than in the inactive stage (P <0.05), and this difference could be detected in individual patients. CONCLUSIONS: Sensitive measurement of serum TNF-alpha could provide an important pathophysiologic marker for the presence and activity of IBD.

Adult↗
PubMed2000

Molecular and functional characterization of organic cation/carnitine transporter family in mice.

Carnitine is essential for beta-oxidation of fatty acids, and a defect of cell membrane transport of carnitine leads to fatal systemic carnitine deficiency. We have already shown that a defect of the organic cation/carnitine transporter OCTN2 is a primary cause of systemic carnitine deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na(+)-dependent, whereas that by OCTN3 was Na(+)-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for carnitine and significantly lower carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na(+)-independent carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of carnitine transport activity and may have a different role from other members of the OCTN family.

Amino Acid Sequence↗
PubMed2000

An immuno-polymerase chain reaction assay for human interleukin-18.

Conventional enzyme-linked immunosorbent assays (ELISA) are sufficient to measure normal and elevated serum interleukin (IL)-18 concentrations, but have limited sensitivity when measuring low concentrations of IL-18 such as in patients with the acquired immunodeficiency syndrome. We have developed a highly sensitive method for detecting human (h) IL-18 using an immuno-polymerase chain reaction (PCR). A mouse monoclonal anti-hIL-18 antibody and rabbit polyclonal anti-hIL-18 antibody was used for an indirect sandwich ELISA with a detection limit of 40 ng/l and a very low background. For immuno-PCR, biotinylated DNA was produced from the plasmid Bluescript by PCR amplification with biotinylated M13-20 primer and nonbiotinylated M13 reverse primer. Immuno-PCR for hIL-18 was performed for 40 cycles using 1 ng/l of biotinylated DNA. This immuno-PCR has a detection limit of 2.5 pg/l, 1.6x10(4) times lower than that of the ELISA. In addition, our system avoids sampling error caused by heat transfer from the ELISA plate to the PCR tube because all procedures from immobilization of the antibody to PCR amplification can be performed in the same tube. This immuno-PCR for hIL-18 is the most sensitive method for detecting hIL-18 reported to date.

Animals↗
PubMed2000

Identification and cDNA cloning of alveolin, an extracellular metalloproteinase, which induces chorion hardening of medaka (Oryzias latipes) eggs upon fertilization.

Chorion hardening is triggered by the contents of cortical alveoli that are released upon fertilization of medaka (Oryzias latipes) eggs. We purified the chorion hardening-inducing activity as a single protein from the exudate of cortical alveoli of medaka eggs. This activity was co-purified with proteolytic activity of the chorion protein ZI-1,2. Based on the amino acid sequence of purified protein, we cloned the cDNA of this protein from a medaka ovarian cDNA library. Sequence analyses revealed typical sequence features, a zinc-binding motif and a methionine turn motif, of the astacin metalloproteinase family. We termed this protein "alveolin." Alveolin has a molecular mass of 21.5 kDa deduced by the amino acid sequence and neutral optimal pH range. Alveolin hydrolyzes ZI-1,2. Alveolin activity was strongly inhibited by metal-chelating agents but not by various proteinase inhibitors. To our knowledge, this is the first description of the isolation and identification of the chorion hardening-inducing factor from cortical alveoli exudate of teleost eggs.

Amino Acid Sequence↗
PubMed2000

Establishment of quantitative reverse transcription--polymerase chain reaction assays for human telomerase-associated genes.

Telomerase is an enzyme that synthesizes and adds repetitive telomeric sequences of (TTAGGG)n to the ends of chromosomes. Recently, several telomerase-associated genes have been cloned, making it possible to study the expression of these genes. Quantitative comparisons of the expression of these genes and of telomerase activity might help clarify the regulation of telomerase activity. Therefore, we established the validity of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for the human telomerase catalytic subunit (hTERT) mRNA and telomerase associated protein (TEP1) mRNA using the TaqMan fluorogenic detection system. Using this assay, we quantitated hTERT mRNA and TEP1 mRNA expression in two human pancreatic cancer cell lines, AsPC-1 and PANC-1. Our results indicated that the levels of hTERT mRNA and TEP1 mRNA expression in AsPC-1 were 1.50 and 2.31 times higher than in PANC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the quantities of hTERT and TEP1 mRNAs in clinical specimens. Taken together, our results indicate that it is possible to measure the expression of the major telomerase genes subunits. Furthermore it is possible to apply this technique to determine the amount of other types of mRNA.

Carrier Proteins↗
PubMed2000

Helicobacter pylori infection: augmentation of telomerase activity in cancer and noncancerous tissues.

Telomerase adds hexameric repeats of 5'-TTAGGG-3' to the ends of chromosomal DNA (telomere) and has been implicated in cell immortalization and cellular senescence. The aim of this study was to measure quantitatively the telomerase activity and human telomerase RNA component (hTR) content in gastric cancer and to examine the relation between these values and histologic factors including Helicobacter pylori as a risk factor for gastric cancer. Telomerase activity was measured by a modified telomeric repeat amplification protocol in cancerous and noncancerous tissues (intestinal metaplasia, chronic gastritis, normal mucosa) from 27 gastric cancer patients; hTR expression was examined by the quantitative reverse transcriptase-polymerase chain reaction using fluorescent probes. Telomerase activity was higher in cancers (total product generated: 33.7) than in noncancerous tissues. Telomerase activity was higher in intestinal metaplasia (16.7) and chronic gastritis (10.6) than in normal mucosa (3.5). In patients with intestinal-type gastric cancer, telomerase activity was higher in intestinal metaplasia with H. pylori infection than in that without infection. hTR expression was not correlated with telomerase activity. H. pylori infection may influence telomerase activity in cancer and noncancerous tissues.

Aged↗
PubMed2000

Limitations of urinary telomerase activity measurement in urothelial cancer.

The reported frequency of detectable telomerase activity in spontaneously voided urine samples from patients with urothelial cancer varied from 0 to 85%. We examined stasis in the bladder and specimen storage as interfering conditions in this assay. Telomerase activity in exfoliated cells was measured by a polymerase-chain-reaction-based assay in spontaneously voided urine from urothelial cancer patients. Effects of retention in the bladder and specimen storage from voiding to measurement of telomerase activity were modeled by suspending 10(6) cells from the cancer-derived T24 line in normal urine (pH 6.5) at 37 degrees C and 25 degrees C, respectively. Hematuria was modeled by adding hemoglobin. In T24 cells suspended in urine at 37 degrees C, telomerase activity had decreased to approximately 20% of preincubation activity after 1 h, and had disappeared after 3 h. In urine at 25 degrees C, telomerase activity in T24 cells had decreased to approximately 40% of preincubation activity at 1 h and to <10% at 6 h. When we examined telomerase activity in exfoliated cells in spontaneously voided urine from urothelial cancer patients (excluding first-voided morning specimens), telomerase activity was detected in only 21% of samples (four of 19) despite measurement with 1 h of voiding and steps to avoid hemoglobin interference. Measurement of telomerase activity in spontaneously voided urine is insufficiently sensitive and reliable for the diagnosis of urothelial cancer.

Carcinoma, Transitional Cell↗
PubMed2000

A highly sensitive immuno-polymerase chain reaction assay for human angiotensinogen using the identical first and second polyclonal antibodies.

We describe an immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human angiotensinogen using identical first and second polyclonal antibodies. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated second antibody. Human recombinant angiotensinogen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. To reduce the effect of nonspecific amplification, the optimal concentrations of streptavidin and DNA label were determined to be 0.1 mg/l and 0.5 ng/l, respectively. The detection limit of the immuno-PCR assay was 0.1 ng/l, an approximately 2.5x10(5)-fold improvement compared with a conventional enzyme-linked immunosorbent assay. These results indicate that a highly sensitive immuno-PCR for human angiotensinogen can be developed even with identical first and second polyclonal antibodies.

Angiotensinogen↗
PubMed2000

Survivin as a radioresistance factor in pancreatic cancer.

We examined whether survivin acts as a constitutive and inducible radioresistance factor in pancreatic cancer cells. Using a quantitative TaqMan reverse transcription-polymerase chain reaction for survivin mRNA in five pancreatic cancer cell lines, we found an inverse relationship between survivin mRNA expression and radiosensitivity. PANC-1 cells, which had the highest survivin mRNA levels, were most resistant to X-irradiation; MIAPaCa-2 cells, which showed the least survivin mRNA expression, were the most sensitive to X-irradiation. Our results suggested that survivin could act as a constitutive radioresistance factor in pancreatic cancer cells. To determine whether radioresistance is enhanced by induction of survivin expression by irradiation, PANC-1 and MIAPaCa-2 cells were subjected to sublethal doses of X-irradiation followed by a lethal dose. Survivin mRNA expression was increased significantly in both PANC-1 and MIAPaCa-2 cell lines by pretreatment with a sublethal dose of X-irradiation, as was cell survival after exposure to the lethal dose. In this system, enzymatic caspase-3 activity was significantly suppressed in cells with acquired resistance. These results suggest that survivin also acts as an inducible radioresistance factor in pancreatic cancer cells. Survivin, then, appears to enhance radioresistance in pancreatic cancer cells; inhibition of survivin mRNA expression may improve the effectiveness of radiotherapy.

Caspase 3↗
PubMed2000

Role of human telomerase reverse transcriptase and telomeric-repeat binding factor proteins 1 and 2 in human hematopoietic cells.

Telomerase, an enzyme that adds hexameric repeats of 5'-TTAGGG-3', termed telomeres, to the ends of chromosomal DNA, has been implicated in cellular immortalization and cellular senescence. Recently several relevant genes have been cloned, including those encoding three major components of human telomerase: human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase-associated protein-1 (TEP1). Also important are genes encoding human telomeric-repeat binding factor proteins (TRF) 1 and 2. We compared 10 human malignant hematopoietic cell lines, 19 samples from patients with acute leukemia and normal granulocytes and monocytes to study telomerase activity and expression of these various genes using a reverse transcription-polymerase chain reaction (RT-PCR). In all 10 malignant cell lines with telomerase activity, hTR, hTERT mRNA, and TEP1 mRNA were expressed, while in normal monocytes and granulocytes without telomerase activity, expression of hTR, but not hTERT mRNA was detected. TEP1 mRNA was expressed in normal monocytes, but not granulocytes. Expression of TRF1 and TRF2 mRNAs was greater in the normal cells than in human malignant hematopoietic cell lines and in 16 samples of patients with acute leukemia. When differentiation of the malignant hematopoietic cell line HL-60 was induced using tumor-necrosis-factor 471 and all-trans retinoic acid (ATRA), telomerase activity decreased gradually during differentiation. Of the three telomerase components, only hTERT mRNA expression showed changes paralleling telomerase activity, becoming undetectable with differentiation. In contrast, initially low expression of TRF1 and TRF2 mRNAs increased during differentiation. Not only hTERT, but also TRF1 and TRF2 are important regulators of telomerase activity that represent potential targets for gene therapy against cancer.

Adolescent↗
PubMed2000

Synergistic effects of iontophoresis and jet injector pretreatment on the in-vitro skin permeation of diclofenac and angiotensin II.

A non-needle syringe (jet injector) was utilized to increase skin permeation of drugs by iontophoresis. Briefly, physiological saline was initially flushed by the injector to make a pore in the stratum corneum of excised hairless rat skin, and the iontophoretic skin permeation of two model compounds, sodium diclofenac and angiotensin II, was followed using a 2-chamber diffusion cell. Constant voltage and constant current iontophoresis treatments were evaluated. Pretreatment using the jet injector alone resulted in about 13- and 22-fold increases in the steady-state flux of diclofenac and angiotensin II, respectively, through the skin, compared with non-treated controls. Jet injector pretreatment with constant voltage iontophoresis further enhanced skin permeation of diclofenac and angiotensin II, and the enhancement was also greater than that by constant voltage iontophoresis alone. Thus, a synergistic effect was observed. The ratio of enhancement was greater compared with the control. Jet injector pretreatment with constant current iontophoresis, however, did not always yield higher skin permeation of the drugs than injector pretreatment alone, although the lag time was shortened. The difference in the enhancement between the constant voltage- and constant current iontophoresis can be explained by the electric current through the excised skin. Constant current iontophoresis after a short period of constant voltage iontophoresis with multiple jet injector pretreatments may be the best way to increase drug permeability while preventing severe skin damage.

Administration, Cutaneous↗