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Biomedical subjects

M Ohashi

Publications and source records attributed to M Ohashi.

At least 109 records · Page 6Linked to original sources
PubMed1997

Changes in fidelity levels of DNA polymerases alpha-1, alpha-2, and beta during ageing in rats.

DNA polymerases (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from the regenerating livers of rats of various ages. The extracts were separated into three DNA polymerase fractions (alpha-1, alpha-2, and beta) by phosphocellulose column chromatography, and their fidelity levels were then monitored with the synthetic template-primer, poly (dA-dT), poly dA-dT10, or poly dC-poly dG. The fidelity levels of the three DNA polymerases from regenerating liver of rats younger than 20 months were high, while those of DNA polymerases from rats older than 20 months were significantly lower with similar profiles on all three template-primers. On the other hand, the fidelity levels of enzymes from 23- and 26-month-old rats were similar. These results indicate that the levels of error-prone DNA polymerases increase rapidly in the regenerating liver of rats from ages 20 to 23 months. This may due to the amplification of DNA polymerase gene mutations by an error-prone enzyme itself. However, the cells in which mutations in the functional gene occur may undergo cell death because the fidelity levels of the DNA polymerases in the older animals did not increase.

Aging↗
PubMed1997

In vivo selective gene expression and therapy mediated by adenoviral vectors for human carcinoembryonic antigen-producing gastric carcinoma.

Previously, we reported that adenoviral vectors carrying the carcinoembryonic antigen (CEA) promoter sequences to direct the Echerichia coli beta-galactosidase gene (AdCEA-lacZ) or cytosine deaminase (CD) gene (AdCEA-CD) confer selective gene expression on a CEA-positive gastric cancer cell line (MKN45) in vitro. Here, adenovirus-mediated tumor-specific gene therapy for CEA-positive gastric carcinoma in vivo was investigated. Using an animal model with i.p. disseminated MKN45 tumors, adenovirus-mediated tumor-specific transgene expression and therapeutic efficacy were analyzed. After an i.p. injection of AdCEA-lacZ, beta-galactosidase activity was confined to tumor xenografts. Moreover, CD mRNA was expressed exclusively in MKN45 tumor xenografts after infection with AdCEA-CD, despite the fact that an adenovirus-mediated transfer of CD DNA was detected in all tissues tested. In contrast, CD mRNA was detected not only in tumor xenografts but also in other organs of mice infected with AdCA-CD, in which CD gene expression is governed by an ubiquitous promoter. Suppression of tumor growth and prolongation of survival were noted in tumor-bearing mice treated with AdCEA-CD and 5-fluorocytosine (5FC) without observable adverse effects. In contrast, significant hepatic toxicity was noted in animals treated with AdCA-CD. These results reveal that the CEA promoter restricts CD gene expression to CEA-positive tumor cells in the adenoviral context in vivo, along with the beneficial therapeutic effects of 5FC treatment, suggesting the i.p. AdCEA-CD/5FC system may provide a novel approach to treatment of i.p. disseminated gastric cancer.

Animals↗
PubMed1997

Residues within the polycationic region of cGMP phosphodiesterase gamma subunit crucial for the interaction with transducin alpha subunit. Identification by endogenous ADP-ribosylation and site-directed mutagenesis.

Interaction between the gamma subunit (Pgamma) of cGMP phosphodiesterase and the alpha subunit (Talpha) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an endogenous enzyme and site-directed mutagenesis of the Pgamma polycationic region to identify residues required for the interaction with Talpha. Pgamma, free or complexed with the alphabeta subunit (Palphabeta) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32P]NAD. Identification of ADP-ribose in the radiolabeled Pgamma and radiolabeling of arginine-replaced mutant forms of Pgamma indicate that both arginine 33 and arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is modified at a time. Pgamma complexed with Talpha (both GTP- and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with Talpha, was ADP-ribosylated in the presence of Talpha, suggesting that a Pgamma domain containing these arginines is masked by Talpha. A Pgamma mutant (R33,36K), as well as wild type Pgamma, inhibited both GTP hydrolysis of Talpha and GTP binding to Talpha. Moreover, GTP-bound Talpha activated Palphabeta that had been inhibited by R33,36K. However, another Pgamma mutant (R33,36L) could not inhibit these Talpha functions. In addition, GTP-bound Talpha could not activate Palphabeta inhibited by R33,36L. These results indicate that a Pgamma domain containing these arginines is required for its interaction with Talpha, but not with Palphabeta, and that positive charges in these arginines are crucial for the interaction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Expression cloning and intracellular localization of a human ZF5 homologue.

We isolated a cDNA encoding a human homologue of ZF5 (hZF5), which has five Kruppel-like C2H2 type zinc fingers at carboxyl terminus and the BTB/POZ (poxvirus and zinc finger) at the amino terminus, using autoimmune sera from a patient with overlap syndrome (dermatomyositis and scleroderma). Sequencing of the entire cDNA revealed an open reading frame (ORF) of 1349 bp with a deduced protein sequence of 449 amino acid residues and a calculated molecular weight of 51.3 kDa. The deduced amino acid sequence of hZF5 is highly homologous to mouse ZF5 (99.3% identity). Immunofluorescence studies revealed that HA-tagged hZF5 transiently expressed in COS-7 cells showed the nuclear dot pattern in the BTB/POZ domain-dependent manner.

Adult↗
PubMed1997

Immunolocalization of hCDC47 protein in normal and neoplastic human tissues and its relation to growth.

hCDC47 is a human member of the MCM family, which has been implicated in the regulatory machinery causing DNA to replicate once per cell cycle. We examined its protein expression and localization in normal human tissues, using immunostaining with polyclonal antibodies. Positive nuclei were found in the proliferative components of lymph nodes, bone marrow, epidermis and mucosa. Immunohistochemical analysis was also performed for 3 types of cutaneous keratinocytic tumor originating from same cell type but showing different grades of malignancy. In seborrheic keratosis, a benign condition, cells with hCDC47-positive nuclei were located in the outermost layers of the tumor lobules, while in Bowen's disease, carcinomas in situ and squamous-cell carcinomas, they were present throughout the lesions. The percentages of hCDC47-positive cells were 65.4% in squamous-cell carcinomas, 60.9% in Bowen's disease, 12.6% in seborrheic keratosis and 3.9% in normal epidermis (n = 5 in all cases). Further expansion of the analysis to include malignant tumors from several other organs revealed that all malignant lesions tested contained more nuclear hCDC47-positive cells than their normal counterparts. Our findings indicate that hCDC47 plays a role in normal and neoplastic cell growth in vivo and that hCDC47 immunolocalization could be used as an index of cell proliferation in tissue sections.

Carcinoma in Situ↗
PubMed1997

Adenovirus-mediated gene therapy of gastric carcinoma using cancer-specific gene expression in vivo.

The carcinoembryonic antigen (CEA) is a glycoprotein which overexpressed in the majority of human gastric cancers. We demonstrated that recombinant adenoviral vector (AdCEAtk), containing the CEA promoter, could transfer the herpes simplex virus thymidine kinase (HSVtk) gene into CEA-producing gastric cancer cells to confer sensitivity to ganciclovir (GCV) in vivo. In an ex vivo experiment, the tumor growth was inhibited after GCV treatment when the tumor contained more than 20% of AdCEAtk infected cells, indicating an efficient bystander killing effect. With intra-tumoral injection of AdCEAtk, the HSVtk were selectively expressed in approximately 30% of CEA producing cancer cells. By AdCEAtk injection and GCV administration, the growth of tumors was significantly inhibited by 20% as compared to untreated tumors. It is hoped that these results provide a strategy of tumor specific gene transfer for CEA producing gastric cancers.

Adenoviridae↗
PubMed1997

In vivo gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene.

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in human hepatocellular carcinoma (HCC). Here, we demonstrate that replication defective recombinant adenoviral vectors, containing the human AFP promoter/enhancer, can be used to express the Escherichia coli cytosine deaminase (CD) gene (AdAFPCD) and the beta-galactosidase gene (AdAF-PlacZ) in AFP-producing HCC cell lines. Expression of the CD gene by adenovirus from the AFP promoter/enhancer (AdAFPCD) induced cells sensitive to 5-fluorocytosine (5FC) in the AFP-producing cells but not in the AFP-nonproducing cells. Transduction by an adenoviral vector harboring an ubiquitous strong promoter and CD gene showed enzymatic activity and 5FC killing in all cell lines. When AdAFPlacZ was injected into the s.c. established hepatoma in vivo, expression of the beta-galactosidase gene was confined to AFP-producing HCC xenografts. Moreover, HCC xenografts regressed by transduction with AdAFPCD and subsequently with 5FC treatment in vivo. These findings suggest that utilization of the AFP promoter/enhancer in an adenoviral vector can confer selective expression of a heterologous suicide gene in hepatocellular carcinoma cells in vitro and in vivo.

Adenoviridae↗
PubMed1997

KB-R7785, a novel matrix metalloproteinase inhibitor, exerts its antidiabetic effect by inhibiting tumor necrosis factor-alpha production.

It has been suggested that tumor necrosis factor-alpha (TNF-alpha) is a key mediator of insulin resistance in non-insulin-dependent diabetes mellitus (NIDDM). TNF-alpha is synthesized as a membrane-bound precursor; this is proteolytically processed to an active form by a matrix metalloproteinase (MMP)-like enzyme. In this study, we have used KKAy mice which show insulin resistance like NIDDM to investigate the effects of KB-R7785, a novel MMP inhibitor, on blood glucose and insulin levels. Subcutaneous administration of KB-R7785 at 100 mg/kg twice daily (i.e., 200 mg/kg/day) for 4 weeks resulted in a significant decrease in plasma glucose levels which was observed after 3 weeks. Oral administration of pioglitazone (20 mg/kg twice daily or 40 mg/kg/day for 4 weeks), an agent known to ameliorate insulin sensitivity, significantly decreased plasma glucose levels during the treatment period. KB-R7785, but not pioglitazone, also significantly decreased plasma insulin levels. Lipopolysaccharide (LPS) increased plasma TNF-alpha levels to a significantly greater degree in KKAy mice than in normal C57BL mice; this was inhibitable in KKAy mice by KB-R7785. In contrast, pioglitazone did not affect the LPS-induced increase in plasma TNF-alpha levels in KKAy mice. These results suggest that KB-R7785 exerts its antidiabetic effect by ameliorating insulin sensitivity through the inhibition of TNF-alpha production.

Animals↗
PubMed1997

Possible mechanisms underlying the midazolam-induced relaxation of the noradrenaline-contraction in rabbit mesenteric resistance artery.

1. The mechanisms underlying the midazolam-induced relaxation of the noradrenaline (NA)-contraction were studied by measuring membrane potential, isometric force and intracellular concentration of Ca2+ ([Ca2+]i) in endothelium-denuded muscle strips from the rabbit mesenteric resistance artery. The actions of midazolam were compared with those of nicardipine, an L-type Ca2+-channel blocker. 2. Midazolam (30 and 100 microM) did not modify either the resting membrane potential or the membrane depolarization induced by 10 microM NA. 3. NA (10 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Midazolam (10-100 microM) did not modify the resting [Ca2+]i, but attenuated the NA-induced phasic and tonic increases in [Ca2+]i and force, in a concentration-dependent manner. In contrast, nicardipine (0.3 microM) attenuated the NA-induced tonic, but not phasic, increases in [Ca2+]i and force. 4. In Ca2+-free solution containing 2 mM EGTA, NA (10 microM) transiently increased [Ca2+]i and force. Midazolam (10-100 microM), but not nicardipine (0.3 microM), attenuated this NA-induced increase in [Ca2+]i and force, in a concentration-dependent manner. However, midazolam (10 and 30 microM), had no effect on the increases in [Ca2+]i and force induced by 10 mM caffeine. 5. In ryanodine-treated strips, which have functionally lost the NA-sensitive Ca2+ storage sites, NA slowly increased [Ca2+]i and force. Nicardipine (0.3 microM) did not modify the resting [Ca2+]i but partly attenuated the NA-induced increases in [Ca2+]i and force. In the presence of nicardipine, midazolam (100 microM) lowered the resting [Ca2+]i and further attenuated the remaining NA-induced increases in [Ca2+]i and force. 6. The [Ca2+]i-force relationship was obtained in ryanodine-treated strips by the application of ascending concentrations of Ca2+ (0.16-2.6 mM) in Ca2+-free solution containing 100 mM K+. NA (10 microM) shifted the [Ca2+]i-force relationship to the left and enhanced the maximum Ca2+-induced force. Under these conditions, whether in the presence or absence of 10 microM NA, midazolam (10 and 30 microM) attenuated the increases in [Ca2+]i and force induced by Ca2+ without changing the [Ca2+]i-force relationship. 7. It was concluded that, in smooth muscle of the rabbit mesenteric resistance artery, midazolam inhibits the NA-induced contraction through its inhibitory action on NA-induced Ca2+ mobilization. Midazolam attenuates NA-induced Ca2+ influx via its inhibition of both nicardipine-sensitive and -insensitive pathways. Furthermore, midazolam attenuates the NA-induced release of Ca2+ from the storage sites. This effect contributes to the midazolam-induced inhibition of the NA-induced phasic contraction.

Animals↗
PubMed1997

Progression of articular destruction and the production of tumour necrosis factor-alpha in antigen-induced arthritis in rabbits.

We examined the progression of articular destruction and the production of tumour necrosis factor-alpha (TNF-alpha) in antigen-induced arthritis (AIA) in rabbits, i.e. flare-ups of inflammation induced by repeated intra-articular injections (single, twice and three times) of antigen. A marked progression of articular destruction and an infiltration of inflammatory cells in the synovium were observed with the increase in the number of antigen injections. An immunohistochemical analysis of the synovial lesions following three injections of antigen revealed that the lymphoid follicles consisted mainly of CD4+ T cells and IgG/IgM+ B cells. There were marked infiltrations of IgG+ plasma cells around the lymphoid follicles. In contrast, the production of TNF-alpha in the synovial fluid and the erythrocyte sedimentation rate (ESR), which is a marker of systemic inflammatory activity in rheumatoid arthritis, peaked at 6 h 24 h, respectively, following the last injection of antigen. These values were also greater following the repeated injections of antigen compared with the single injection. The TNF-alpha was produced markedly in the joints at the onset of the flare-ups of arthritis following the repeated injections of antigen, and the elevation of the ESR and an acceleration of the inflammatory response in the synovium were observed with a concomitant progression of severe articular destruction, suggesting that the marked production of TNF-alpha at the time of flare-ups may be involved in the exacerbation of AIA in rabbits.

Animals↗
PubMed1997

Doppler echocardiographic evaluation of latent pulmonary hypertension by passive leg raising.

BACKGROUND: The prevalence of chronic lung disease increases with advancing age. The purpose of this study was to detect latent pulmonary hypertension noninvasively in elderly patients with chronic lung disease. METHODS: The changes of flow-velocity by passive leg raising were assessed using pulsed Doppler echocardiography in the right ventricular outflow tract in 19 patients with chronic lung disease and 13 normal healthy subjects. Pulmonary artery pressure was measured simultaneously using a thermodilution cardiac output catheter. RESULTS: In patients with chronic lung disease examined at rest, we found pulmonary hypertension in seven, and none in 12. The normal pattern observed at rest changed to pulmonary hypertension pattern after leg raising in six out of the 12 patients. Pulmonary arterial catheterization confirmed the data obtained by pulsed Doppler echocardiography. CONCLUSIONS: Some elderly patients with chronic lung disease have latent pulmonary hypertension which can be detected noninvasively by analyzing the changes of flow velocity profiles in the right ventricular outflow tract by passive leg raising.

Aged↗
PubMed1997

Apoptotic cell death of neutrophils in development of skin lesions of patients with anaphylactoid purpura.

The participation of apoptotic cell death of neutrophils in the development of skin lesions of patients with anaphylactoid purpura was examined by the in situ specific labeling of fragmented DNA. In the early stage of the skin lesions, there were few positively stained nuclei in infiltrating cells. The number of positive cells increased markedly in the fully developed stage of the lesions. A number of neutrophils were stained positively. Finally, a few fragmented nuclei were still positive in the late stage of the lesions. It was therefore suggested that fragmentation of neutrophils in the skin lesions from the patients might be due to apoptosis. Inducible nitric oxide synthase and nitrotyrosine were detected in infiltrates, and interleukin-8 was also detected in vascular endothelial cells in those skin lesions. The roles of nitric oxide and interleukin-8 in the apoptosis of neutrophils are discussed.

Adolescent↗
PubMed1997

Accurate typing of HLA-A antigens and analysis of serological deficiencies.

We are reporting the results of HLA-A typing by PCR-SSOP complemented by PCR-SSP of samples obtained from the National Marrow Donor Program (NMDP). These samples were a representative group from 2486 tested in duplicate by serology. A total of 390 samples gave HLA-A discrepant results. Comparing the molecular typing results of 238 samples (samples with available DNA) with the serological typing results, 54 homozygotes and 184 heterozygotes produced a total of 422 assignments by molecular methods. We found assignment discrepancies in 147/422 (35%) in laboratory 1 and 144/422 (34%) in laboratory 2 (a combined group of 4 NMDP laboratories; laboratory 1 is not included). The serological discrepancies found were of 3 categories: a) false negatives, b) incomplete typing (discrepancies due to the level of resolution within a cross-reactive or CREG group) and c) false positives. Major problems were identified using serology for typing HLA-A antigens: a) inability to identify all WHO-recognized specificities, more frequently in non-Caucasians or in HLA-A specificities known to be found more frequently in non-Caucasians for laboratory 1 and incorrect assignments of A19 specificities in laboratory 2, b) incorrect assignments in cells with poor viability and c) false-positive assignments in homozygotes. We propose a possible strategy to type HLA-A specificities with two steps: a) a minimum of serology for typing specificities for common CREG groups: A1, A2, A3, A11, A9, A10, A28, A19. However, a given laboratory can determine the level of serological assignments needed as a first step. And b) molecular methods to identify splits: A23, A24, A29, A30, A31, A32, A33, A34, A36, A66, A74 and A80. The technique described is useful for large-scale bone marrow donor typings for cells with poor viability, and for resolving ambiguous results including false-positive assignments of homozygous cells.

DNA↗
PubMed1997

Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods.

Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only.

DNA↗
PubMed1997

Synthesis and antitumor activity of 9-acyloxyellipticines.

Various kinds of water-soluble 9-acyloxyellipticine derivatives were synthesized in a search for compounds with potent antitumor activity. Antitumor activities against several tumors in mice (P388 leukemia, colon 26, Lewis lung carcinoma and B16 melanoma) were evaluated by using intravenous administration. Many compounds exhibited good antitumor activities; in particular, the glutarate derivative (5o) showed potent antitumor activity. This compound (5o) may be converted to 9-hydroxyellipticine (2) by enzyme-catalyzed hydrolysis in the body.

Animals↗
PubMed1997

Synthesis and antitumor activity of quaternary salts of 2-(2'-oxoalkoxy)-9-hydroxyellipticines.

Various kinds of water-soluble quaternary salts of 2-(2'-oxoalkoxy)-9-hydroxyellipticines were synthesized in a search for compounds with potent antitumor activity and low toxicity. Some compounds exhibited more potent antitumor activities than elliptinium (1) and SUN 4599 (3). In particular, 2-(3'-methoxy-2'-oxopropanoxy)-9- hydroxy-5,11-dimethyl-6H-pyrido[4,3-b]carbazolium bromide (4d) showed potent antitumor activities against P388 leukemia, colon 26, and Lewis lung carcinoma.

Animals↗
PubMed1997

Enhancement of interleukin-1 alpha mediated autocrine growth of cultured human keratinocytes by sho-saiko-to.

We investigated the effects of Sho-saiko-to, the most commonly used herbal medicine in Japan, on the production of interleukin (IL)-1 alpha by cultured human epidermal keratinocytes. IL-1 alpha production was significantly promoted by treatment with 100 or 500 micrograms/ml Sho-saiko-to for 24 or 48 hr. Expression of IL-1 alpha receptors was the most markedly upregulated after treatment with 500 micrograms/ml Sho-saiko-to for 24 hr and with 100 or 500 micrograms/ml for 48 hr; these cells showed the characteristics of multilayered differentiated keratinocytes. The presence of an anti-IL-1 alpha antibody during the treatment with 500 micrograms/ml of Sho-saiko-to for 24 or 48 hr or with 100 micrograms/ml for 48 hr significantly down-regulated the synthesis by the keratinocytes and induced damages in them. Keratinocytes treated with Sho-saiko-to might produce IL-1 alpha and express IL-1 alpha receptors. IL-1 alpha may regulate the proliferation and differentiation of keratinocytes after Sho-saiko-to treatment. These findings suggest that Sho-saiko-to enhances the autocrine growth mediated by IL-1 alpha.

Anti-Inflammatory Agents, Non-Steroidal↗