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Biomedical subjects

D Kobayashi

Publications and source records attributed to D Kobayashi.

At least 19 recordsLinked to original sources
PubMed2006

Human beta-defensin-3 induction in H. pylori-infected gastric mucosal tissues.

AIM: To examine human beta-defensin-3 (hBD-3) expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H. pylori infection for better understanding the innate immune response to H. pylori. METHODS: We used reverse transcription-polymerase chain reactions and immunohistochemistry to examine hBD-3 expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H. pylori. Effects of hBD-3 against H. pylori were also evaluated. RESULTS: The mean mRNA expression of hBD-3 in H. pylori-positive specimens was significantly higher than that in H pylori-negative specimens (P = 0.0002, Mann-Whitney). In addition, unlike uninfected samples, 8 of 15 (53.33%) infected mucosal samples expressed hBD-3 protein. H. pylori dose-dependently induced mRNA expression of hBD-3 in MKN45 cells, an effect inhibited by adding anti-toll-like receptor (TLR)-4 antibody. HBD-3 protein completely inhibited H. pylori growth. CONCLUSION: Our results suggest that like hBD-2, hBD-3 may be involved in the pathophysiology of H. pylori-induced gastritis.

Antibodies, Anti-Idiotypic↗
PubMed2004

Large-scale isolation of ESTs from medaka embryos and its application to medaka developmental genetics.

The medaka is becoming an attractive model organism for the study of vertebrate early development and organogenesis and large-scale mutagenesis projects that are aimed at creating developmentally defective mutants are now being conducted by several groups in Japan. To strengthen the study of medaka developmental genetics, we have conducted a large-scale isolation of ESTs from medaka embryos and developed tools that facilitate mutant analysis. In this study, we have characterized a total of 132,082 sequences from both ends of cloned insert cDNAs from libraries generated at different stages of medaka embryo development. Clustering analysis with 3-prime sequences finally identified a total of 12,429 clusters. As a pilot analysis, 924 clusters were subjected to in situ hybridization to determine the spatial localization of their transcripts. Using EST sequence data generated in the present study, a 60-mer oligonucleotide microarray with 8,091 unigenes (Medaka Microarray 8K) was constructed and tested for its usefulness in expression profiling. Furthermore, we have developed a rapid and reliable mutant mapping system using a set of mapped EST markers (M-marker 2003) that covers the entire medaka genome. These resources will accelerate medaka mutant analyses and make an important contribution to the medaka genome project.

Animals↗
PubMed2003

Cavernous lymphangioma of the leg in children treated by the injection of OK-432 after resection.

We describe the treatment of three boys with cavernous lymphangioma of the legs. The suggested guidelines for treatment are extensive surgical resection, although complete resection is usually impossible, and approximately two weeks after the operation aspiration of serous fluid which has accumulated at the operation site, followed by injection of OK-432 (Picibanil). There was pyrexia and local inflammation for several days but the accumulation of serous fluid disappeared after the injection. There were no complications with no recurrence, joint contracture, or pain during a mean follow-up of 48 months (24 to 72). We conclude that an injection of OK-432 after surgical resection of cavernous lymphangioma of the legs in children is an effective treatment.

Antineoplastic Agents↗
PubMed2002

Successful treatment of advanced peripheral T-cell lymphoma with an angiocentric growth pattern complicated with hemophagocytic syndrome by high-dose chemotherapy and autologous peripheral blood stem cell transplantation.

Peripheral T-cell lymphomas (PTCL) account for about 10% of all lymphomas in Western countries, respond poorly to therapy, and have short survival with no sustained remission. Furthermore, the complication of hemophagocytic syndrome (HPS) sometimes makes the prognosis of this disease extremely worse. We report here a case of PTCL with an angiocentric growth pattern complicated with HPS successfully treated by high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Our case suggests this approach is an excellent candidate for the treatment of this disease.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2002

Pyrazinamide resistance associated with pncA gene mutation in Mycobacterium tuberculosis in Japan.

Thirty Japanese clinical isolates of Mycobacterium tuberculosis were analysed by pyrazinamide susceptibility testing and pyrazinamidase assay, as well as polymerase chain reaction for single-strand conformational polymorphism and direct sequencing of the gene encoding pyrazinamidase (pncA). All sensitive isolates showed pyrazinamidase activity and a wild-type pncA gene, but three resistant isolates had pncA gene mutations and lacked pyrazinamidase activity. The latter isolates showed a minimum inhibitory concentration of at least 100 mg/l by the 7H10 agar proportion method and 400 mg/l by the 7H9 liquid medium method. Isolate 28 showed T-to-C change at position 11, leading to Leu4 --> Ser substitution; isolate 29 had an 8-bp deletion from position 382; and isolate 30 had A-to-C change at position 29, leading to Gln10 --> Pro substitution. The deletion has not been described previously. This is the first demonstration of pncA gene mutations in PZA-resistant M. tuberculosis strains isolated from Japanese patients.

Amidohydrolases↗
PubMed2002

Comparison of arbitrarily primed-polymerase chain reaction and pulse-field gel electrophoresis for characterizing methicillin-resistant Staphylococcus aureus.

AIMS: The aim of this study was to analyse genotypes for clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including hetero-vancomycin-resistant Staph. aureus (VRSA), at a Japanese university hospital. METHODS AND RESULTS: Seventy-eight clinical isolates of MRSA were analysed by arbitrarily primed-polymerase chain reaction (AP-PCR) using ERIC2 primer and by pulse-field gel electrophoresis (PFGE) following SmaI digestion. Analyses of the nine genotypes and 28 subtypes defined by PFGE, and of the three genotypes and 22 subtypes defined by AP-PCR, both facilitated epidemiological tracing. Used in combination, AP-PCR and PFGE provided more precise classification than the use of a single genotyping method. The six hetero-VRSA isolates were classified into four genotypes defined by the combination of both methods, but these genotypes contained non-VRSA isolates. CONCLUSIONS: The results suggest that both PFGE and AP-PCR are useful in discriminating MRSA, but not hetero-VRSA, isolates for epidemiological analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining the results of PFGE with the results of AP-PCR can provide more detail differentiation of MRSA and hetero-MRSA isolates than either method alone.

DNA, Bacterial↗
PubMed2002

Gastric mucosal density of Helicobacter pylori estimated by real-time PCR compared with results of urea breath test and histological grading.

The accuracy of the urea breath test (UBT) and histological grading for estimation of the density of Helicobacter pylori in gastric mucosa is not known. Real-time (TaqMan) PCR was used to estimate the total number of H. pylori genomes in biopsy samples. These values were compared with those obtained by the UBT and the histological grade obtained by the Sydney system. The UBT and endoscopy with antral and corporal biopsies were performed in 88 consecutive untreated patients with dyspepsia. Bacterial culture and the rapid urease test were done with fresh biopsy materials. TaqMan PCR and histological examination were done on serial paraffin sections of the biopsy samples. Of the five methods tested, TaqMan PCR had the highest sensitivity and specificity (both 100%) in the diagnosis of H. pylori infection. The mean density of H. pylori genomes for pairs of biopsy samples from individual patients was compared with the individual values obtained by the UBT; correlation between the results was significant. The density of H. pylori genomes was higher in histological grades 1, 2 and 3 than in grade 0, without significant differences between adjacent grades from 1 to 3. These results suggest that the severity of H. pylori infection of the stomach can be estimated by the UBT and that histopathologists might state whether the organism is present or absent, rather than making a quantitative statement as recommended in the Sydney system.

Biopsy↗
PubMed2001

Effect of poly-L-arginine on the nasal absorption of FITC-dextran of different molecular weights and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats.

The effect of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of FITC-dextrans with a mean molecular weight ranging from 4.3 to 167 kDa and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats were studied. When FITC-dextrans were co-administered intranasally with 1.0 w/v% poly-L-Args of different molecular weight (MW, ca. 45.5 and 92 kDa, poly-L-Arg (50) and poly-L-Arg (100)), the bioavailability (F(infinity)) increased markedly compared with that after administration of FITC-dextran alone. However, the F(infinity) decreased exponentially with the increasing molecular weight of FITC-dextrans. There was no significant difference between the enhanced nasal absorption of FITC-dextrans achieved by the co-administration of poly-L-Arg (50) and poly-L-Arg (100). Moreover, the relationship between the F(infinity) and the molecular weight of FITC-dextrans indicated that the molecular weight of protein drugs, which exhibited efficient absorption with poly-L-Arg, was about 20 kDa, when the lower limit of bioavailability for developing a potent transnasal delivery system was assumed to be about 10%. Indeed, the nasal absorption of rhG-CSF, which has a molecular weight of 18.8 kDa, was also increased after co-administration of 1.0 w/v% poly-L-Arg (50) and the F(infinity) was about 11%. It seems likely that poly-L-Arg can be used to provide adequate nasal absorption of various protein drugs which have a molecular weight of about 20 kDa, thereby allowing the successful development of a variety of transnasal drug delivery systems.

Absorption↗
PubMed2001

Telomerase reverse transcriptase and telomeric-repeat binding factor protein 1 as regulators of telomerase activity in pancreatic cancer cells.

Telomerase adds hexameric repeats of 5'-TTAGGG-3' termed telomeres to ends of chromosomal DNA. This enzyme has been implicated in cellular immortalization and cellular senescence. Recently, a number of relevant genes have been cloned, including these 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 protein (TRF) 1 and 2. To clarify mechanisms regulating telomerase activity, we studied telomerase activity, the telomeric restriction fragment (TRF) length and gene expression of these telomerase components and the telomeric-repeat binding factor proteins in sequential observation following X-irradiation of cultured pancreatic cancer cells. We previously reported that PANC-1 cells are better able to tolerate thermal stress, antineoplastic drugs, and exposure to tumour necrosis factor than MIAPaCa-2 cells. MIAPaCa-2 and PANC-1 cells were exposed to X-irradiation, their telomerase activity was increased at 2 days and then decreased gradually. Of the three telomerase components, only hTERT mRNA expression showed parallel changes. TRF length was stable just before and after X-irradiation. Among binding factor proteins, TRF1 mRNA showed reciprocal changes possibly directed toward maintaining a stable telomere length. In this study, our results demonstrate that not only hTERT but also TRF1 are important regulator of telomerase activity.

Cell Count↗
PubMed2001

Na(+)-coupled transport of L-carnitine via high-affinity carnitine transporter OCTN2 and its subcellular localization in kidney.

The mechanism of Na(+)-dependent transport of L-carnitine via the carnitine/organic cation transporter OCTN2 and the subcellular localization of OCTN2 in kidney were studied. Using plasma membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN2, transport of L-carnitine via human OCTN2 was characterized. Uptake of L-[(3)H]carnitine by the OCTN2-expressing membrane vesicles was significantly increased in the presence of an inwardly directed Na(+) gradient, with an overshoot, while such transient uphill transport was not observed in membrane vesicles from cells that were mock transfected with expression vector pcDNA3 alone. The uptake of L-[(3)H]carnitine was specifically dependent on Na(+) and the osmolarity effect showed that Na(+) significantly influenced the transport rather than the binding. Changes of inorganic anions in the extravesicular medium and of membrane potential by valinomycin altered the initial uptake activity of L-carnitine by OCTN2. In addition, the fluxes of L-carnitine and Na(+) were coupled with 1:1 stoichiometry. Accordingly, it was clarified that Na(+) is coupled with flux of L-carnitine and the flux is an electrogenic process. Furthermore, OCTN2 was localized on the apical membrane of renal tubular epithelial cells. These results clarified that OCTN2 is important for the concentrative reabsorption of L-carnitine after glomerular filtration in the kidney.

Animals↗
PubMed2001

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate.

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange-red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3' region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20-25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Animals↗
PubMed2001

Expression of telomerase-associated genes: reflection of telomerase activity in gastric cancer?

Telomerase activation is a characteristic of immortalized tumor cells but not of normal cells. Telomerase activity has been detected in approximately 85% of malignant tumors, and assaying for telomerase activity is thought to be useful for diagnosing cancer. Three telomerase-associated molecules [human telomerase RNA component (hTR), telomerase-associated protein (TEP1), and human telomerase reverse transcriptase (hTERT)] have been cloned. We semiquantitatively measured telomerase activity and the expression of these genes in cancerous and noncancerous regions of gastric cancer patients. We also investigated whether the expression of these genes correlated with telomerase activity. Telomerase activity in cancerous regions was significantly higher than in noncancerous regions, but there was no correlation between telomerase activity and the expression of these genes. Furthermore, no clear difference was observed between cancerous and noncancerous regions. These data indicate that the level of three telomerase-associated genes (i.e., hTR, TEP1 mRNA, hTERT mRNA), do not reflect telomerase activity, and the RNA levels of these genes are not useful markers for diagnosing gastric cancer.

Carrier Proteins↗
PubMed2001

Improved nasal absorption of drugs using poly-L-arginine: effects of concentration and molecular weight of poly-L-arginine on the nasal absorption of fluorescein isothiocyanate-dextran in rats.

The effects of the concentration and molecular weight of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of fluorescein isothiocyanate-labeled dextran (MW, 4 kDa, FD-4) in rats were studied. When poly-L-Arg with a range of different molecular weights (MW, 8.9, 45.5 and 92.0 kDa) was applied intranasally at various concentrations, the bioavailability (F(0-9 h)) of FD-4 increased with the increasing concentration of poly-L-Arg. The enhanced absorption was also dependent on the molar concentration, in that the poly-L-Arg with a higher molecular weight increased F(0-9 h) at a lower molar concentration. In addition, for each applied concentration, the poly-L-Arg exhibited a molecular weight-dependence as far as the enhancement of FD-4 absorption was concerned. On the other hand, the maximum absorption rate (MAR) of FD-4, calculated by means of a deconvolution method, tended to reach a maximum plateau level at a lower applied concentration for the poly-L-Arg with the highest molecular weight, but this plateau level was almost the same for poly-L-Arg with molecular weights of 45.5 and 92.0 kDa. Moreover, the simulated absorption profiles of FD-4 indicate that the degree of enhancement (the level of MAR and the subsequent reduction in the absorption rate) was dependent on the molecular weight of poly-L-Arg, while the effect of poly-L-Arg was maintained for a longer period, depending on the applied concentration, although the MAR was relatively similar. These results indicate that the molecular weight of poly-L-Arg appears to affect both the enhancing efficiency (absorption rate) and the time-frame of this enhancing effect, whereas the concentrations of each poly-L-Arg system applied only have an effect on the time-frame. These effects may also be associated with the charge density of a poly-L-Arg molecule.

Absorption↗
PubMed2001

Neonatal alloimmune thrombocytopenia due to anti-human leukocyte antigen antibody: a case report.

Anti-HLA antibodies reportedly exist in 31% of pregnant women. However, few ocurrences of neonatal alloimmune thrombocytopenia (NAIT) caused by anti-HLA antibody have been reported. In this study, maternal anti-HLA B60 and B61 antibodies were identified in patient serum at birth, but no anti-platelet antibodies were present. No maternal anti-HLA A2, A24, B51, or B52 antibodies were detected in patient serum. Platelet transfusion from the third donor was effective because these platelets expressed HLA A24 and B52 but not B60 or B61. Cross-matching tests between patient leukocytes or platelets and maternal serum were strongly positive, indicating that maternal anti-HLA antibodies were responsible for NAIT. This report is the first to demonstrate NAIT probably caused by maternal anti-HLA A24 and B52.

Female↗
PubMed2001

Enzyme immunoassay for conjugated 7alpha-hydroxy-3-oxo-4-cholenoic acid in human urine.

A microplate enzyme immunoassay (EIA) was developed for the measurement of glycine- and taurine-conjugated 7alpha-hydroxy-3-oxo-4-cholenoic acids (CDCA-delta4-3-one) in human urine. The antiserum was prepared by immunizing rabbits with N-(7alpha-hydroxy-3-oxo-4-cholen-24-oyl)-3-aminopropionic acid--bovine serum albumin conjugate. A colorimetric EIA was established using horseradish peroxidase-labeled antigen having a shorter bridge length than that of the immunogen, and 3, 3', 5, 5'-tetramethylbenzidine /hydrogen peroxide for the measurement of the enzyme activity. The reactivities of the antiserum for glycine and taurine conjugates of CDCA-delta4-3-one was almost the same. The specificity of the antiserum was investigated by determining the cross-reactivities of various bile acids and related compounds. An appropriate dose-response curve for conjugated CDCA-delta4-3-one was obtained in the range of 0.05-10 ng/well. This method was used for direct analysis of conjugated CDCA-delta4-3-one in urine of healthy infants and patients with liver diseases.

Chenodeoxycholic Acid↗
PubMed2001

Caspase-3 activation downstream from reactive oxygen species in heat-induced apoptosis of pancreatic carcinoma cells carrying a mutant p53 gene.

In the present study we investigated the intracellular signaling pathway leading to p53-independent activation of caspase-3 during heat-induced apoptosis of pancreatic carcinoma cells. Induction of mutant p53 protein, but not p21/WAF-1, was observed after heat treatment of both heat-resistant (PANC-1) and heat-sensitive (MIAPaCa-2) cells. A specific inhibitor of caspase-3 (Ac-DMQD-CHO) caused 84% and 92% inhibition of apoptosis in MIAPaCa-2 and PANC-1 cells, respectively. Caspase-3 mRNA expression was increased in both cell lines after heat treatment. Further, heat-induced caspase-3 activity detected by fluorogenic assay in MIAPaCa-2 cells was almost completely inhibited by addition of the antioxidant N-acetyl-L-cysteine. In contrast, Ac-DMQD-CHO had no inhibitory effect on amounts of reactive oxygen species in heat-treated MIAPaCa-2 cells. These results suggest a possible pathway by which reactive oxygen species lead to caspase-3 activation to cause heat-induced death of pancreatic carcinoma cells carrying mutant p53.

Acetylcysteine↗