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Biomedical subjects

M Ohashi

Publications and source records attributed to M Ohashi.

At least 91 records · Page 5Linked to original sources
PubMed1998

Fidelity levels of DNA polymerases in tumorigenic state cells and serially transplantable tumor cells.

It is well known that point mutations exist in oncogenes and tumor suppressor genes of tumor cells, and one of the causes of these mutations may be misincorporation by error-prone DNA polymerases. This hypothesis is supported by the observation of decreased fidelity levels of DNA polymerases in mouse spleen containing tumorigenic cells after infection with Friend virus, and in aged animals that suffer high rates of tumorigenesis. However, this decrease in fidelity is disadvantageous for tumor cells maintained by serial transplantation. Therefore, we measured the fidelity levels of DNA polymerases in tumor cells transplanted through many passages. The fidelity levels of DNA polymerases from Yoshida ascites hepatoma, Rhodamine sarcoma, mouse ascites hepatoma-134, and Ehrlich ascites carcinoma cells derived from rats and mice are very high for in-vitro DNA synthesis on synthetic polynucleotides. These results suggest that many kinds of mutant cells arise during tumorigenesis. Among these mutant cells, cells showing decreased DNA polymerase(s) fidelities are present and these cells may undergo cell death. On the other hand, cells with mutations in various oncogenes and tumor suppressor genes and without mutations in DNA polymerase genes may survive as serially transplantable tumor cells.

Animals↗
PubMed1998

Age dependent decline in the 3'-->5' exonuclease activity involved in proofreading during DNA synthesis.

A 3'-->5' exonuclease found in rat liver excises mispaired nucleotides at the 3'-hydroxyl end of primer chains such as poly dA-d(T9-C). Consequently, the priming activity of the chain from which the mispaired base was cut is greatly increased during DNA synthesis. These results suggest that the 3'-->5' exonuclease acts as a proofreading enzyme during DNA synthesis. The activity of this 3'-->5' exonuclease in the liver of 24-month-old rats is approximately 30% lower than the activity found in 4-month-old rats. Furthermore, non-complementary nucleotide incorporations by DNA polymerases from aged rats are observed during DNA synthesis on poly dA-dT10. The number of misincorporations decreases in the presence of the 3'-->5' exonuclease, but not all errors are prevented even when DNA polymerase and 3'-->5' exonuclease are added at an activity ratio similar to that found in vivo. The data suggest that declines in both the fidelity of DNA polymerase and the 3'-->5' exonuclease activity related to proofreading during the aging process lead to a higher frequency of base misincorporations during DNA replication.

Aging↗
PubMed1998

Cloning and characterization of human MCM7 promoter.

MCM7 is a member of the MCM protein family which has been implicated in the regulatory machinery allowing DNA to replicate only once during S phase. In quiescent cells, human MCM7 (hMCM7) mRNA is almost undetectable. Stimulation of cells to enter the cell cycle results in induction of hMCM7 expression. Here, we report cloning and characterization of the hMCM7 promoter. We isolated and sequenced a 0.5 kb genomic fragment that contains putative transcription factor binding sites including three E2F sites, three GC boxes and an E box. Several transcription start sites, which were used upon growth stimulation, were identified. The minimal promoter region required for transcription of a luciferase reporter gene was delineated, and it contained an E box and one E2F site, which were important for promoter activity. Interestingly, the cloned sequence appears to act as a promoter for mu-adaptin-related protein 2 (mu-ARP2) gene in the opposite orientation.

Base Sequence↗
PubMed1998

Vasorelaxant effect of olprinone, an inhibitor of phosphodiesterase 3, on mesenteric small artery and vein of rabbits.

The effects of olprinone, a cardiotonic agent that inhibits cyclic GMP (cGMP)-inhibited phosphodiesterase, was studied on isolated rabbit mesenteric small artery and vein. In the presence of indomethacin and propranolol, olprinone at concentrations of 10 nM to 10 microM and 1 microM to 100 microM relaxed norepinephrine-stimulated mesenteric artery and vein in a concentration-dependent manner, respectively. The relaxation was not endothelium-dependent in the artery. Removal of the endothelium, however, increased marginally the response of the vein to olprinone. Olprinone-induced relaxation was less pronounced in arteries contracted with high KCl solution + norepinephrine than in those contracted with norepinephrine alone. Nicardipine inhibited this attenuating effect of high KCl solution on the olprinone-induced relaxation. Olprinone (1 microM) enhanced the relaxation of artery and vein in response to a cAMP-increasing agent, 6-(3-dimethylaminopropionyl) forskolin (NKH477), but not to a cGMP- increasing agent, glyceryl trinitrate. Norepinephrine (10 microM) and caffeine (5 mM) elicited a transient, phasic contraction of the artery in Ca2+-free solution. Both olprinone and NKH477 attenuated more potently the norepinephrine-induced contraction than the caffeine-induced contraction. When norepinephrine (10 microM) and caffeine (5 mM) were successively applied in Ca2+-free solution, the contractile effect of caffeine was diminished compared to that in artery which had not been pretreated with norepinephrine. When the contraction in response to norepinephrine was partially attenuated by 1 microM olprinone, the following contraction evoked by caffeine was enlarged. It is concluded that olprinone relaxes the small artery more strongly than the vein via its direct action on smooth muscles. It is suggested that olprinone attenuates norepinephrine-induced contraction through inhibition of receptor-operated transmembrane Ca2+ influx and Ca2+ release from intracellular storage sites.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Allelic loss analysis of gamma-ray-induced mouse thymic lymphomas: two candidate tumor suppressor gene loci on chromosomes 12 and 16.

A total of 429 gamma-ray-induced thymic lymphomas were obtained from F1 and backcross mice between BALB/c and MSM strains, about a half of which carried a p53-deficient allele. A genome-wide allelic loss analysis has revealed two loci exhibiting frequent allelic losses but no allelic preference, one is localized within a 2.9 cM region between D12Mit53 and D12Mit279 loci on chromosome 12, and the other is near the D16Mit122/D16Mit162 loci on chromosome 16. The frequency of allelic loss in the D12Mit279 region is 62% and does not differ in tumors between the presence and absence of the p53-deficient allele. In contrast, the loss frequency of D16Mit122 is raised by the existence of p53-deficient allele: 62% for p63(-/+) and 13% for p53(+/+), suggesting co-operative function of the two losses. The D12Mit279 and D16Mit122 regions probably harbor different types of tumor suppressor gene that play key roles in lymphoma development.

Animals↗
PubMed1998

Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentrations of 5-fluorouracil.

5-fluorouracil (5-FU), although a widely used chemotherapeutic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU. Escherichia coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate. The present study demonstrates that adenovirus-mediated transduction of E. coli UPRT gene results in marked sensitization of colon, gastric, liver, and pancreas cancer cell lines to low concentration of 5-FU in vitro. The in vitro bystander effect was observed when only 10% of the hepatoma Hep3B cells were infected with UPRT-expressing adenovirus. In addition, 5-FU treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with UPRT was demonstrated to result in significant in vivo antitumor effects. The adenovirus vector transduction of the UPRT gene followed by 5-FU administration is representative of a new chemosensitization strategy for cancer gene therapy.

Adenoviridae↗
PubMed1998

Effect of a 3'-->5' exonuclease with a proofreading function on the fidelity of error-prone DNA polymerase alpha from regenerating liver of aged rats.

A nuclease that releases noncomplementary nucleotides from the 3'-end of DNA was isolated and highly purified from rat liver extract. The d(T9-C) priming activities for DNA synthesis in vitro by DNA polymerases alpha and beta were recovered by the addition of this enzyme, which itself does not contain a DNA polymerase activity. This nuclease hydrolysed nucleotides from the 3'-end, but did not remove [32P]-labeled nucleotides from the 5'-terminus of specifically labeled DNA. Also, the reaction products released from the 3'-end of DNA were all mononucleotides. These results indicate that the exonuclease is a 3'-->5' exonuclease with properties the same as those of DNase VII from human placenta. Rat DNase VII requires 4 mM MgCl2 or 0.125 mM MnCl2 for maximum activity, and shows a pH optimum of 7.5. These optimal conditions are similar to those of DNA polymerases, and indicate that both rat DNase VII and DNA polymerases are able to act under same conditions. Non-complementary nucleotide incorporation by DNA polymerase alpha from aged rat has been observed during in vitro DNA synthesis on poly dA-dT10. The amount of this mis-incorporation is decreased by the coexistence of the 3'-->5' exonuclease, but not all errors are edited out. Thus, this rat DNase VII is suggested to play an important role in proofreading during DNA synthesis.

Aging↗
PubMed1998

Expression and function of a retinoic acid receptor in budding ascidians.

Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates.

Amino Acid Sequence↗
PubMed1998

Gene therapy for hepatic micrometastasis of murine colon carcinoma.

BACKGROUNDS/AIMS: Pit cells are located in the hepatic sinusoids and are organ-associated natural killer cells that contribute to immune surveillance in the liver. In the present study, the interleukin-2 gene was introduced into hepatocytes using an adenovirus vector to induce interleukin-2 production in an attempt to enhance the natural killer activity of pit cells, leading to inhibition of metastasis of colon carcinoma. METHODS: The recombinant adenovirus vector "Adex1CAmIL2" was constructed by inserting an expression unit which was composed of the CAG promotor (cytomegalovirus enhancer plus chicken beta-actin promotor), murine interleukin-2 cDNA, and a rabbit beta-globin polyadenylation signal. After administration of Adex1CAmIL2 to mice (4x10(7) pfu per animal), the expression of murine interleukin-2 in hepatocytes was examined by immunostaining and in situ hybridization, and the natural killer activity of hepatic mononuclear cells was measured. Inhibition of hepatic metastasis of colon carcinoma was examined after infusion of colon 38 tumor cells into the superior mesenteric vein. RESULTS: After administration of Adex1CAmIL2, interleukin-2 mRNA expression was demonstrated in hepatocytes until day 7, and the serum interleukin-2 level was increased. The natural killer activity of hepatic mononuclear cells was markedly enhanced for 7-10 days. Hepatic metastasis was inhibited by administration of Adex1CAmIL2 until day 7 after tumor cell inoculation. CONCLUSION: These results suggest that gene therapy using Adex1CAmIL2 could be potentially useful for inhibiting hepatic micrometastasis by enhancing the natural killer activity of pit cells.

Actins↗
PubMed1998

Correlation between magnetic resonance imaging and histopathology of an amputated forearm after an electrical injury.

A 53-year-old man sustained a 50 Hz, 60 kV electrical injury. The current flowed between his right hand and both feet. There was necrosis of the distal portion of the right forearm, and the fourth and fifth toes on the right foot. The skin on the amputated right upper extremity appeared normal except for an ulcer in the antecubital fossa and some blister scars. However, most of the muscles of the arm at the elbow were necrosed and partially replaced by fatty tissue or fibrosis. These necrotic areas corresponded to minimally increased signal intensities on T1-weighted MRI, and high signal intensities on T2-weighted images. MRI may be employed to predict amputation level after electrical injury.

Adult↗
PubMed1998

Effects of tibolone (Org OD14) treatment for 3 months on ovariectomy-induced osteopenia in 8-month-old rats on a low-calcium diet: preventive testing for 3 months.

Tibolone (Org OD14), (7alpha, 17alpha)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn++ +-3-one, is a synthetic steroid with weak estrogenic, progestational, and androgenic properties. We investigated the prophylactic effects of tibolone on bone loss, bone strength, and plasma and urinary parameters in 8-month-old ovariectomized rats on a low-Ca diet. Oral administration of tibolone (0.03-3 mg/kg/day) was started immediately after ovariectomy (ovx) and continued for 3 months. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. Oral administration of tibolone (1 or 3 mg/kg/day) significantly prevented a decrease in BMD and bone ash density (bone ash weight/volume) of the global femur, and BMDs in the femoral distal and proximal regions. Also in the lumbar vertebrae, the ovx-induced reduction in BMD was prevented by tibolone (1 and 3 mg/kg/ day) treatment, resulting in a significantly higher lumbar vertebral (L-2) bone compression strength compared to the ovx control group. Neither ovx alone nor supplemented with tibolone affected the BMD or bending strength of the femoral mid-diaphysial region. Tibolone (0.03-3 mg/kg/day) significantly reduced the ovx-induced increases in serum osteocalcin level. Furthermore, tibolone inhibited an increase in the urinary hydroxyproline/creatinine, pyridinoline/creatinine, and deoxypyridinoline/creatinine ratios induced by ovx. Tibolone also reduced body weight gain and serum cholesterol level, as has been reported for estrogen. These findings indicate that tibolone prevents reduction in bone mass associated with osteopenia by reducing increased trabecular bone resorption induced by a combination of ovx and a low-Ca diet.

Administration, Oral↗
PubMed1998

A monoarthritis model in rabbits induced by repeated intra-articular injections of lipopolysaccharide.

We attempted to develop a monoarthritis model using repeated intra-articular injections of high-dose Lipopolysaccharide (LPS). Furthermore, the effect of dexamethasone on this arthritis model by intra-articular administration was studied to examine whether the model can be used to screen anti-rheumatic drugs in a short time. Arthritis was induced by one, two or three intra-articular injections of LPS (5-50 micrograms/joint) at 4-day intervals into the knee joint. The rabbits were sacrificed at 7 days following the last injection of LPS. Three intra-articular injections of LPS at 50 micrograms/joint resulted in persistent joint swelling. Hyperplasia of synovium with some discolouration was macroscopically observed. Infiltration of mononuclear cells and lymphoid follicles were histologically observed as the synovial lesions. Concerning the articular bone/cartilage, trabecular destruction of gastrocnemius sesamoid bone and severe loss of safranin-O staining of articular cartilage were observed. Immunohistochemical analysis revealed that inflammatory cells and lymphoid follicles in the synovial lesions consisted predominantly of CD4+ T cells, with few CD8+ T cells. Treatment with dexamethasone markedly reduced the joint swelling and the articular destruction. The results suggest that this arthritis model in rabbits can be utilized to screen anti-rheumatic drugs as a model of rheumatoid arthritis.

Animals↗
PubMed1998

Endoscopic papillary balloon dilation for the management of common bile duct stones: experience of 226 cases.

BACKGROUND AND STUDY AIMS: Endoscopic sphincterotomy is a widely accepted technique for the treatment of patients with common bile duct stones. However, it is still associated with occasional complications. The recently developed technique of endoscopic papillary balloon dilation seems to be a safe and effective procedure, and to have great potential for replacing endoscopic sphincterotomy. However, few reports have been published on the use of this technique for bile duct stones. The present study was undertaken to evaluate its safety and efficacy. PATIENTS AND METHODS: Endoscopic papillary balloon dilation was used to remove common bile duct stones in 226 consecutive patients including 41 patients of ASA classification III/IV, 41 elderly patients (> 80 years) 24 with liver cirrhosis, and 86 with periampullary diverticulum. After dilation of the papilla with a balloon diameter of 8 mm, the stones were retrieved. RESULTS: In conjunction with the use of a mechanical or/and electrohydraulic lithotriptor in 79 patients (35%) with large stones (> 10 mm in diameter), clearance of the common bile duct was achieved in 225 of 226 patients (99%) without serious complications, such as hemorrhage or severe pancreatitis; mild (n = 13) or moderate (n = 2) pancreatitis occurred in 7% of cases. CONCLUSIONS: Endoscopic papillary balloon dilation is a safe and effective technique for the treatment of common bile duct stones, even in high-risk patients.

Adult↗
PubMed1998

In vivo adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing pancreatic cancer.

In gene therapy for malignancy, the herpes simplex virus thymidine kinase (HSVtk)-ganciclovir (GCV) system has been widely used. For pancreatic cancer targeting, we estimated the therapeutic efficacy of gene transduction by an adenovirus-carrying HSVtk gene under the control of a carcinoembryonic antigen (CEA) promoter (AdCEAtk) followed by systemic administration of GCV. Four cell lines, CEA-producing Su.86.86. BxPC-3 (pancreatic cancer cells), MKN45 (gastric cancer cells) and CEA-nonproducing HeLa, were used for analysis of GCV sensitivity induced by adenoviral gene transduction. To evaluate the therapeutic efficacy of AdCEAtk and GCV administration in human CEA-positive pancreatic cancer in vivo, a subcutaneously implanted tumor-bearing nude mouse model was used. When the HSVtk gene was transduced with a ubiquitous promoter into these cells, increase of the GCV sensitivity was independent of CEA-production. In contrast, when the cells were transduced with a CEA promoter, the cell-killing effect of GCV was increased in only CEA-producing cells. For in vivo analysis, AdCEAtk was delivered into subcutaneously established tumors of Su.86.86 cells. Immunohistochemical staining of the tumor showed that HSVtk protein was expressed only in tumor cells, and tumor growth was markedly suppressed by administration of GCV. These results suggest that the adenovirus-mediated transfer of HSVtk gene with CEA promoter specifically increases the GCV sensitivity of CEA-producing pancreatic cancer cells in vitro and in vivo. This strategy may provide a useful tool for treating pancreatic cancer, especially CEA-producing tumor cells.

Adenoviridae↗
PubMed1998

Typing of HLA-B*15 alleles using sequence-specific primers.

We have developed a DNA based typing method to detect 38 known B*15 alleles using sequence-specific primers (PCR-SSP). This method involves 38 primers and 39 PCR-SSP reactions with results that can be obtained in 3 hours. The method is easy, fast and suitable for clinical typing for bone marrow and organ transplantation. We have typed 106 HLA-B15 samples using this method. For homozygous HLA-B15 samples, some B*15 allele combinations need to be resolved by additional PCR reactions not included in this article. The method allows the detection of potential new alleles requiring sequencing for confirmation, and it is useful to resolve unusual serological reaction patterns for different HLA-B15 serological specificities. In addition, it could be used to resolve ambiguous PCR-SSOP typing results and for recognition of mismatches in serologically matched unrelated individuals.

Alleles↗
PubMed1998

Specific distribution patterns of hCDC47 expression in cutaneous diseases.

hCDC47 is a human member of the MCM family, which has been implicated to be concerned with the regulatory machinery causing DNA to replicate once per cell cycle. In a previous paper, we described hCDC47 expression as being localized in the proliferative component of normal tissues, and showed greater expression in squamous cell skin carcinomas than in seborrheic keratosis. In the present study, we compared its expression in another type of skin tumor and various non-neoplastic cutaneous proliferative diseases. Two patterns of distribution of hCDC47-positive cells were observed. Keratoacanthomas showed a peripheral pattern in which only the cells located the basal cell layers were positive. Psoriasis vulgaris also showed this peripheral type of location, with the cells in the suprabasal layers also occasionally expressing hCDC47. Verruca vulgaris demonstrated a diffuse pattern, with positive epithelial cells distributed throughout the layers. Basal cell carcinomas also showed a similar pattern. The keratoacanthomas showed the highest hCDC47 positive cell rate (43.1%), followed by verruca vulgaris (42.5%), psoriasis vulgaris (23.4%), and basal cell carcinoma (17.3%). Our results suggest participation of hCDC47 in these proliferative disorders involving keratinocytes.

Basal Cell Carcinoma↗
PubMed1998

A novel insulin sensitizer acts as a coligand for peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and PPAR-gamma: effect of PPAR-alpha activation on abnormal lipid metabolism in liver of Zucker fatty rats.

We investigated the biological activity of a novel thiazolidinedione (TZD) derivative, KRP-297, and the molecular basis of this activity. When administered to obese Zucker fatty rats (obese rats) at 10 mg/kg for 2 weeks, KRP-297, unlike BRL-49,653, restored reduced lipid oxidation, that is, CO2 and ketone body production from [14C]palmitic acid, in the liver by 39% (P < 0.05) and 57% (P < 0.01), respectively. KRP-297 was also significantly more effective than BRL-49,653 in the inhibition of enhanced lipogenesis and triglyceride accumulation in the liver. To understand the molecular basis of the biological effects of KRP-297, we examined the effect on peroxisome proliferator-activated receptor (PPAR) isoforms, which may play key roles in lipid metabolism. Unlike classical TZD derivatives, KRP-297 activated both PPAR-alpha and PPAR-gamma, with median effective concentrations of 1.0 and 0.8 micromol/l, respectively. Moreover, radiolabeled [3H]KRP-297 bound directly to PPAR-alpha and PPAR-gamma with dissociation constants of 228 and 326 nmol/l, respectively. Concomitantly, KRP-297, but not BRL-49,653, increased the mRNA and the activity (1.5-fold [P < 0.01] and 1.8-fold [P < 0.05], respectively) of acyl-CoA oxidase, which has been reported to be regulated by PPAR-alpha, in the liver. By contrast, KRP-297 (P < 0.05) was less potent than BRL-49,653 (P < 0.01) in inducing the PPAR-gamma-regulated aP2 gene mRNA expression in the adipose tissues. These results suggest that PPAR-alpha agonism has a protective effect against abnormal lipid metabolism in liver of obese rats.

Acyl-CoA Oxidase↗