Biomedical subjects

M Namba

Publications and source records attributed to M Namba.

At least 109 records · Page 6Linked to original sources

PubMed1997

Pyruvate alleviates toxic effects of ethanol on cells in culture.

Cytotoxic effects of ethanol on cultured human hepatocytes and fibroblasts differed with the type of culture medium used. When cytotoxic effects of ethanol were compared between culture systems using either RPMI-1640 or Dulbecco's modified Eagle's medium (DMEM), the cytotoxicity was more prominent with the former medium than with the latter. A reduction of the cytotoxic effects appeared to be due to the pyruvate contained in DMEM. The protective effect of pyruvate against ethanol toxicity may be related to tricarboxylic acid (TCA) cycle activity because addition of malate to the medium also reduced the cytotoxic effects. Our results suggest that drug cytotoxicity testing in vitro must be done using various types of culture medium.

Cell Division↗

PubMed1997

Immortalization of mutant p53-transfected human fibroblasts by treatment with either 4-nitroquinoline 1-oxide or X-rays.

The study of in vitro cell transformation is valuable for understanding the multistep carcinogenesis of human cells. The difficulty in inducing neoplastic transformation of human cells by treatment with chemical or physical agents alone is due to the difficulty in immortalizing normal human cells. Thus, the immortalization step is critical for in vitro neoplastic transformation of human cells. We transfected a mutant p53 gene (mp53: codon 273Arg-His) into normal human fibroblasts and obtained two G418-resistant mp53-containing clones. These clones showed an extended life span but ultimately senesced. However, when they were treated with either 4-nitroquinoline 1-oxide or X-rays, they were immortalized. The immortalized cells showed both numerical and structural chromosome abnormalities, but they were not tumorigenic. The expression of mutant but not wild type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in increased cdk2 and cdc2 kinase activity. However, there was no significant difference between the normal and immortalized human cells in expression of another tumor suppressor gene, p16. These findings indicate that the p53-p21 cascade may play an important role in the immortalization of human cells.

4-Nitroquinoline-1-oxide↗

PubMed1997

The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit.

According to the telomere hypothesis of senescence, the progressive shortening of telomeres that occurs upon division of normal somatic cells eventually leads to cellular senescence. The immortalisation of human cells is associated with the acquisition of a telomere maintenance mechanism which is usually dependent upon expression of the enzyme telomerase. About one third of in vitro immortalised human cell lines, however, have no detectable telomerase but contain telomeres that are abnormally long. The nature of the alternative telomere maintenance mechanism (referred to as ALT, for Alternative Lengthening of Telomeres) that must exist in these telomerase-negative cells has not been elucidated. It has previously been shown that abnormal lengthening of yeast telomeres may occur due to mutations in the yeast telomerase RNA gene. That this is not the mechanism of the abnormally long telomeres in ALT cell lines was demonstrated by the finding that seven of seven ALT lines have wild-type human telomerase RNA (hTR) sequence, including a novel polymorphism that is present in 30% of normal individuals. We found that two ALT cell lines have no detectable expression of the hTR gene. This shows that the ALT mechanism in these cell lines is not dependent on hTR. Expression of exogenous hTR via infection of these cells with a recombinant hTR-adenovirus vector did not result in telomerase activity, indicating that their lack of telomerase activity is not due to absence of hTR expression. We conclude that the ALT mechanism is not dependent on the expression of hTR, and does not involve mutations in the hTR sequence.

Base Sequence↗

PubMed1997

Comparison of cellular characteristics between human hepatoma cell lines with wild-type p53 and those with mutant-type p53 gene.

Characteristics of human hepatoma cell lines with the wild-type p53 were compared with those of human hepatoma cell lines with the mutant-type p53. The p21 protein located downstream of p53 was expressed in cell lines with the wild-type p53 but was not expressed in cell lines with the mutant-type p53. As to other tumor suppressor genes such as p16 and p27, there was no difference in their expression between both types of cell lines. In addition, no marked difference was observed in the activities of CDK2 and CDK4 between cell lines with the wild-type and the mutant-type p53. Phosphorylated Rb protein was detected in all cell lines except the HLE line, indicating that this cell line may have a deletion of and/or a mutation of the Rb gene. These results indicate that abnormalities of tumor suppressor genes other than p53, p16, p27, and Rb may be involved in hepatocarcinogenesis. The population doubling time of the wild-type p53 cells was significantly longer than that of the mutant p53 cells. Neither type of cell line showed a specific chromosome distribution which would indicate karyotype instability. The cell lines expressing the wild-type p53 produced tumors at lower frequency than those with the mutant p53 gene. Although there was no significant difference in effects of TGF-beta 1, EGF, cholera toxin, and db-cAMP on cell growth between the two types of cells, all three cell lines with the wild-type p53 were resistant to cytotoxicity of TNF-alpha, while two of the three with the mutant p53 were very sensitive to its cytotoxic effects.

Animals↗

PubMed1997

Yeast functional assay of the p53 gene status in human cell lines maintained in our laboratory.

We used a yeast functional assay (functional analysis of separated alleles in yeast: FASAY) to determine the p53 gene status of human cell lines maintained in our laboratory. This assay enables the researcher to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS 3 via the p53-responsive GAL 1 promoter in Saccharomyces cerevisiae. The cell lines examined were ten hepatoma, two hepatoblastoma, three in vitro immortalized fibroblast, two osteosarcoma, a chondrosarcoma, an ovarian teratocarcinoma and a colon cancer cell line. Out of 20 cell lines, 11 cell lines had mutations in both alleles of the p53 gene, and another 8 cell lines had no mutation in the p53 gene. Thus, 55% of the cell lines examined had mutations in the p53. Interestingly, PA-1 cells had both the normal and the mutant p53 alleles, showing that FASAY is a useful method for detecting the wild-type and mutated p53 genes simultaneously. As for the three liver cell lines harboring HBsAg, there was no relationship between their p53 gene status and the presence of HBsAg. Two cell lines were normal for p53 status, while the other had a mutation of the p53 gene.

Alleles↗

PubMed1997

Effects of simulated microgravity on human osteoblast-like cells in culture.

Physiological strain plays an important role in maintaining the normal function and metabolism of bone cells. It is well know that the mineral content of astronauts' bones decreases during spaceflight. Thus, gravity is one of the important factors in the musculoskeletal system. The vector-free horizontal clinostat has been used to simulate conditions of microgravity for examining such effects on cells in culture. We analyzed the effects of simulated microgravity using a horizontal clinostat on cultured osteoblast-like cells (HuO9 cell line). Total cellular protein, which was measured as an indication of cell proliferation, was not significantly inhibited under simulated microgravity conditions. No morphological changes were detected under microgravity conditions by phase-contrast microscopy. However, the alkaline phosphatase (ALP) activity and osteocalcin production of the HuO9 cells decreased significantly under microgravity conditions. Our data indicate that simulated microgravity directly inhibits some differentiation phenotypes and some functions of osteoblasts. On the other hand, the addition of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) increased ALP activity under simulated microgravity conditions, although the total activity of ALP in the cells treated with 1,25-(OH)2-D3 was still lower under simulated microgravity conditions than that in the control cells. However, the cells under simulated microgravity conditions showed a greater enhancement of ALP activity by treatment with 1,25-(OH)2-D3.

Alkaline Phosphatase↗

PubMed1997

Enhancement of DNA-transfection frequency by X-rays.

This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy.

Cell Line↗

PubMed1997

Demonstration of two different processes of beta-cell regeneration in a new diabetic mouse model induced by selective perfusion of alloxan.

To clarify the regeneration process of pancreatic beta-cells, we established a new mouse model of diabetes induced by selective perfusion of alloxan after clamping the superior mesenteric artery. In this model, diabetes could be induced by the destruction of beta-cells in alloxan-perfused segments, while beta-cells in nonperfused segments were spared. Intraperitoneal glucose tolerance tests showed glucose intolerance, which gradually ameliorated and was completely normalized in 1 year with a concomitant increase of insulin content in the pancreas. Histological examination showed neo-islet formation in the alloxan-perfused segment and the proliferation of spared beta-cells in the nonperfused segment. In the alloxan-perfused segment, despite a marked reduction of islets in size and number at an early stage, both the number of islets, including islet-like cell clusters (ICCs), and the relative islet area significantly increased at a later stage. Increased single beta-cells and ICCs were located in close contact with duct cell lining, suggesting that they differentiated from duct cells and that such extra-islet precursor cells may be important for beta-cell regeneration in beta-cell-depleted segment. In addition to beta-cells, some nonhormone cells in ICCs were positive for nuclear insulin promoter factor 1, which indicated that most, if not all, nonhormone cells positive for this factor were beta-cell precursors. In the nonperfused segment, the islet area increased significantly, and the highest 5-bromo-2-deoxyuridine-labeling index in beta-cells was observed at day 5, while the number of islets did not increase significantly. This indicated that the regeneration of islet endocrine cells occurs mostly through the proliferation of preexisting intra-islet beta-cells in the nonperfused segment. In conclusion, the regeneration process of beta-cells varied by circumstance. Our mouse model is useful for studying the mechanism of regeneration, since differentiation and proliferation could be analyzed separately in one pancreas.

Alloxan↗

PubMed1997

Gene regulation by decoy approach (I): synthesis and properties of photo-crosslinked oligonucleotides.

To explore gene regulation by double stranded oligonucleotides as decoy molecules for transcriptional factors, oligodeoxyribonucleotides (ODN) and the complementary oligo(deoxyribonucleoside phosphorothioate)s (OPT) were photo-crosslinked by use of psoralen. UV melting curves showed that the thermal stability of crosslinked duplexes increased compared with that of non-crosslinked ones. CD spectra showed that the photo-crosslinked duplexes formed B-DNA structure. In addition, cross-linked duplexes were resistant against digestion by snake venom phosphodiesterase. These results suggest that the photo-crosslinked duplexes have desirable characteristics as decoy molecules.

Base Sequence↗

PubMed1996

Down regulation of protein kinase C during growth enhancement induced by interleukin-6 on a human myeloma cell line, KMS-11.

Interleukin-6 (IL-6) is one of the most important growth factors for myeloma cells. We examined the effect of recombinant IL-6 on the proliferation of five human myeloma cell lines, which were independently established AT Kawasaki Medical School. Only the KMS-11 cell line among these five lines showed growth enhancement induced by IL-6. Based on the results, a possible contribution of Ca(2+)-phospholipid-dependent protein kinase C (PKC) to the signal transduction in KMS-11 cells during growth enhancement was studied, since PKC may play an important role in malignant transformation or cell proliferation induced by some growth factors, such as IL-6. When exogenous IL-6 was added to KMS-11 culture, we observed (1) reduction of total PKC activity, and (2) translocation of PKC activity from its cytosol fraction to the membrane fraction. These findings may indicate that down regulation of PKC occurred during the myeloma cell proliferation induced by IL-6. However, IL-6 does not appear to be involved in cell proliferation and differentiation in the other cell lines studied.

Cell Division↗

PubMed1996

Carnitine transport defect in fibroblasts of juvenile visceral steatosis (JVS) mouse.

Juvenile visceral steatosis (JVS) mice are associated with systemic carnitine deficiency (Kuwajima, et al., 1991). In order to investigate the cause of this deficiency, we compared fibroblast carnitine transport activities in normal mice and JVS mice. The kinetic analysis showed that in formal fibroblasts, the Km and Vmax values for saturable uptake was 15.6 microM and 2.56 pmol/min/mg protein, respectively. In JVS fibroblasts, however, saturable uptake was not observed. There was no great difference in the linear component of uptake between normal and JVS fibroblasts. At the physiological concentration (50 microM) of carnitine, the fibroblast carnitine transport activity in JVS mice was decreased to 18% of that in normal mice. Thus there is hardly any carnitine transport activity in the fibroblasts of JVS mice, indicating that the JVS mouse can be regarded as an animal model of primary carnitine deficiency.

Animals↗

PubMed1996

Glucagon-like peptide-1(7-36)amide enhances insulin-stimulated glucose uptake and decreases intracellular cAMP content in isolated rat adipocytes.

We investigated the effect of GLPs on glucose uptake in isolated rat adipocytes. GLP-1(7-36)amide significantly enhanced glucose uptake in the presence of 1 nM insulin. GLP-1(7-36)amide at 15 nM increased glucose uptake maximally by 56.4% as compared with 1 nM insulin alone (P < 0.01). In contrast, with less than 1 nM insulin or without insulin GLP-1(7-36)amide showed no effect on glucose uptake. Full-sequence GLP-1(1-37) at 15 nM in the presence of 1 nM insulin increased glucose uptake by 24.6% as compared with 1 nM insulin alone (P < 0.05). GLP-2 showed no effect on glucose uptake. Further, we examined the effect of GLP-1(7-36)amide on cAMP content in isolated rat adipocytes. Insulin at 1 nM caused a significant decrease of cAMP content. The combination of 15 nM GLP-1(7-36)amide and 1 nM insulin caused a further reduction of cAMP content. These data indicate that GLP-1(7-36)amide possesses augmentative effects on insulin action in isolated rat adipocytes. Furthermore, it is suggested that the stimulatory effect of GLP-1(7-36)amide occurs through the reduction of intracellular cAMP content.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.

We have recently reported that a novel molecule, murine IFN-gamma-inducing factor (IGIF) produced by mouse liver cells, possesses potent biologic activities, including the induction of IFN-gamma production by spleen cells and the enhancement of NK cell cytotoxicity. In this paper, we report on the isolation of human IGIF cDNA clones from normal human liver cDNA libraries using murine IGIF cDNA as a probe. The amino acid sequence deduced from the human cDNA clones indicated a 193-amino acid precursor peptide and revealed 65% homology with that of murine IGIF. The amino acid sequence of IGIF also included an IL-1 signature-like sequence. Subsequently, the cloned cDNA was expressed in Escherichia coli, and preliminary studies on the biologic activities of the recombinant protein were performed. The recombinant human IGIF induced IFN-gamma production by mitogen-stimulated PBMC and enhanced NK cell cytotoxicity, in a manner similar to murine IGIF. In addition, recombinant human IGIF also augmented granulocyte-macrophage-CSF production and decreased IL-10 production, but had no effect on IL-4 production by Con A-stimulated PBMC. Based on these pleiotropic effects of IGIF, we propose that this novel cytokine be designated as IL-18.

Adult↗

PubMed1996

Mutation in p53 and de-regulation of p53-related gene expression in three human cell lines immortalized with 4-nitroquinoline 1-oxide or 60Co gamma rays.

In vitro models of malignant transformation of human cells may provide considerable insight into the mechanisms of multi-step carcinogenesis. It is well established that normal human cells must be immortalized before they can be malignantly transformed; however, they are stringently destined for aging and are rarely immortalized. The mechanism of cellular aging and immortalization is still unknown. We detected expression of only mutated p53 mRNA by direct sequencing of the reverse-transcribed mRNA in 3 human cell lines immortalized either with 4-nitroquinoline 1-oxide or with 60Co gamma rays. Consequently, only the mutated pS3 protein was expressed in each immortalized cell line. The expression of sdiI/p21 and mdm2, both of which are positively regulated by wild-type p53, was significantly down-regulated in the immortalized cell lines, resulting in over-expression of cdk2 and cdk4. Introduction of the sdiI/p21 gene into these cells was followed by a remarkable decrease in their ability to synthesize DNA. These results indicate that the p53 cascade may play an important role in the immortalization of human cells.

4-Nitroquinoline-1-oxide↗

PubMed1996

Altered expression of carnitine palmitoyltransferase II in liver, muscle, and heart of mouse strain with juvenile visceral steatosis.

We conducted a quantitative study of the effect of carnitine deficiency on the mRNA level of carnitine palmitoyltransferase II in the liver, muscle and heart of mice with juvenile visceral steatosis, a strain that is systematically deficient in carnitine. The amount of carnitine palmitoyltransferase II mRNA was increased in liver and muscle of homozygotes, as compared with heterozygotes and normal controls, at 2, 4, and 8 wk of age. The mRNA levels of this enzyme were normalized after carnitine administration. The mRNA level of carnitine palmitoyltransferase II in the heart was increased only at 8 wk, and was not affected by carnitine administration. These results suggest that carnitine displays some effect on the mRNA level of the carnitine palmitoyltransferase II gene in liver and muscle, probably through fatty acid metabolic change.

Ammonia↗

PubMed1996

Disordered expression of hepatic glycolytic and gluconeogenic enzymes in Otsuka Long-Evans Tokushima fatty rats with spontanteous long-term hyperglycemia.

Expression of key regulatory enzymes involved in glucose metabolism was studied in the livers of Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of non-insulin dependent diabetes mellitus. The activity and mRNA levels of glucokinase and L-type pyruvate kinase was increased in the liver of OLETF rats compared with control rats. There was no such remarkable change in liver-type phosphofructokinase. The activities of glucose-6-phosphatase and fructose-1,6-biphosphatase also increase despite high plasma levels of glucose and insulin. The activity of phosphoenolpyruvate carboxykinase did not show any significant change. The mRNA levels for fructose-1,6-biphosphatase, and phosphoenolpyruvate carboxykinase exhibited no marked changes. These results suggest that the expression of glucose-6-phosphatase and fructose-1,6-biphosphatase is disordered in OLETF rats.

Animals↗

PubMed1996

Persistence of hepatitis C virus RNA in established human hepatocellular carcinoma cell lines.

The persistence of the viral RNA of hepatitis C virus (HCV) was examined in 13 hepatocellular carcinoma (HCC) and two hepatoblastoma cell lines by reverse transcription followed by the polymerase chain reaction (RT-PCR). HCV RNA was detected in three HCC lines (JHH-1, JHH-4, and JHH-6) and negative-strand viral RNA was found in JHH-4, indicating that there is a putative replicative intermediate of HCV in JHH-4 cells. To rule out the possibility of contamination, the partial nucleotide sequences of HCV-specific PCR products of these three cell lines were determined. The clone from JHH-1 belonged to genotype 1 (1a or 1b), and the clones from JHH-4 and JHH-6 belonged to genotype 2b, but their sequences differed from each other. These cell lines may be useful for studies related to HCV.

Adolescent↗

PubMed1996

Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-gamma production.

The novel cytokine interferon-gamma-inducing factor (IGIF) augments natural killer (NK) cell activity in cultures of human peripheral blood mononuclear cells (PBMC), similarly to the structurally unrelated cytokine interleukin (IL)-12. IGIF has been found to enhance the production of interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) while inhibiting the production of IL-10 in concanavalin A (Con A)-stimulated PBMC. In this study, when anti-CD3 monoclonal antibody (mAb)-stimulated human enriched T cells were exposed to IGIF, the cytokine dose-dependently enhanced the proliferation of the cells and this could be completely inhibited by a neutralizing antibody against IL-2 at lower concentrations of IGIF. Neutralizing antibody against IFN-gamma had only insignificant inhibitory effects on T cell proliferation at higher concentrations of IGIF. Enzyme-linked immunosorbent assays (ELISA) revealed that, like PBMC, T cells exposed to IGIF produced large amounts of IFN-gamma; however, changes in the production of IL-4 and IL-10 were minimal. IGIF, but not IL-12, significantly enhanced IL-2 and GM-CSF production in T cell cultures, as determined by CTLL-2 bioassay and ELISA, respectively; however, both IGIF and IL-12 enhanced IFN-gamma production by the T cells. When T cells were exposed to a combination of IGIF and IL-12, a synergistic effect was observed on the production of IFN-gamma, but not on production of IL-2 and GM-CSF. In conclusion, IGIF enhances T cell proliferation apparently through an IL-2-dependent pathway and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL-12 in terms of enhanced IFN-gamma production but not IL-2 and GM-CSF production. Based on structural and functional differences from any known cytokines, it was recently proposed that this cytokine be designated interleukin-18.

Adult↗