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Biomedical subjects

M Namba

Publications and source records attributed to M Namba.

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

Two-dimensional gel electrophoretic analysis of the changes after immortalization of human cells: decrease of intracellular alpha-2-macroglobulin fragment.

To study the mechanisms of immortalization of human cells, an early step in cancer development, we compared the cellular proteins of normal and immortalized human fibroblasts. Two-dimensional gel electrophoresis showed that one spot with a molecular mass 20 kDa and an isoelectric point of 6.0, became significantly smaller after immortalization of human cells. Further, the spot was rarely observed in four human liver cancer cell lines. Investigation of the N-terminal amino acids revealed that the spot was a fragment of alpha-2-macroglobulin. Although the 20 kDa fragment contains methionine, the spot was not labeled with [35S]methionine. Thus we concluded that the spot might be derived from the culture medium. These results indicated that intracellular metabolism of a-2-macroglobulin, which is a multifunctional protease inhibitor, changed after the cells were transformed.

Cell Line↗
PubMed1998

Characteristics of cardiac hypertrophy in the juvenile visceral steatosis mouse with systemic carnitine deficiency.

The juvenile visceral steatosis (JVS) mouse exhibits hereditary systemic carnitine deficiency and develops cardiac hypertrophy. The aim of this study was to clarify the characteristics of cardiac hypertrophy in the JVS mouse. Total carnitine content in IVS mouse heart was about 10% of that of control mouse heart at 4 and 8 weeks of age. The heart weight/body weight ratio was bigger in JVS mice than that in control mice at 2 weeks of age, and this difference in ratio increased with age. The wall areas of both ventricles and septum in JVS mice were larger than those of the control mice at 2 and 8 weeks. The myocyte diameter in both ventricular walls and septum in JVS mice was longer than that of the control mice. On electron microscopy, the percent of mitochondria in the myocyte was 66% in JVS mice, and 37% in control mice. The percent of lipid fraction in JVS mice was six-fold higher than that in control mice. Total content of adenine nucleotides in JVS mouse heart was about 60% of that in control mouse heart. Adenylate energy charge in JVS mouse heart was 63 and 45% of that in the control mouse heart at 4 and 8 weeks, respectively. Overall, the cardiac enlargement observed in this animal model could be accounted for by a proportional increase in the myocyte diameter in the ventricles and septum, accompanied by an increase in mitochondria. Furthermore, this cellular growth is associated with decreases in the levels of ATP and ADP, and adenylate energy charge.

Adenosine Diphosphate↗
PubMed1998

Inflammatory pseudotumor of the abdomen: report of a case.

We report herein the case of a 38-year-old woman in whom a large tumor, 15 cm in diameter, was detected in the right lower quadrant of the abdomen. The tumor was removed and histological examination indicated that it was an inflammatory pseudotumor (IPT). IPT is not completely understood, but according to the few documented cases of IPT of the abdomen, its clinical behavior is benign and no recurrence has ever been reported. Thus, local resection is the most effective treatment.

Abdominal Neoplasms↗
PubMed1998

Establishment of a human fibroblast cell line producing tumor necrosis factor alpha (KMST-6/TNF) and growth inhibitory effects of its conditioned medium on malignant cells in culture.

To develop a new gene therapy model for cancer, a clonal cell line (KMST-6/TNF) which produces human tumor necrosis factor alpha (hTNF-alpha) has been developed by introducing hTNF-alpha cDNA into a human immortal fibroblast cell line (KMST-6). The conditioned medium (CM) of KMST-6/TNF cells inhibited the growth of various malignant human cell lines, but not that of normal human fibroblasts. Although the growth inhibitory effects of KMST-6/TNF CM were neutralized to a considerable degree by anti-TNF-alpha antibody, its inhibitory effects were more marked than the purified human natural TNF-alpha itself in the same units, suggesting that KMST-6/TNF CM contains some growth inhibitory substances other than TNF-alpha. However, interferons alpha, beta, and gamma were undetectable in the KMST-6/TNF CM.

Blotting, Northern↗
PubMed1998

Mutation/polymorphism scanning of glucose-6-phosphatase gene promoter in noninsulin-dependent diabetes mellitus patients.

Glucose-6-phosphatase (G6Pase) catalyzes the rate-limiting step of gluconeogenesis, and hepatic G6Pase activity is increased in diabetes. We have cloned and analyzed the human G6Pase gene promoter region and identified putative regulatory sequences for insulin, cAMP, glucocorticoid, and hepatocyte nuclear factors. The promoter region of the G6Pase gene was analyzed in 154 noninsulin-dependent diabetes mellitus patients and 90 control subjects by PCR-single strand conformation polymorphism and direct sequencing methods. Polymorphisms were not found in any subjects. The results suggested that in noninsulin-dependent diabetic patients, the major cause of the hepatic glucose overproduction was not attributed to dysregulation of the G6Pase gene due to mutation/polymorphism of its promoter region.

Adolescent↗
PubMed1998

Differential localization of two types of transferrin: produced by human fibroblasts or incorporated from culture medium.

In a previous paper, we demonstrated that cultured human fibroblasts synthesize transferrin (Tf). Two types of Tf are present; one is produced by the cells and the other is internalized from the culture medium. To study the metabolism of intracellular Tf, we investigated the subcellular localization of the two types of Tf in human fibroblasts by immunocytochemical and fluorescence-labeling techniques. The internalized Tf was found to be localized in the perinuclear area, and the synthesized Tf was associated with microtubules, forming a fibrous structure in the cytoplasm. When the cells were treated with colchicine which depolymerizes microtubules irreversibly, the synthesized Tf lost its fibrous structure and spread out in cytoplasm, but the internalized Tf remained around the nucleus. These results suggest that the two types of Tf are regulated differently in the cells.

Cell Nucleus↗
PubMed1998

Mutation P291fsinsC in the transcription factor hepatocyte nuclear factor-1alpha is dominant negative.

The type 3 form of maturity-onset diabetes of the young (MODY3) results from mutations in the gene encoding the transcription factor, hepatocyte nuclear factor-1alpha (HNF-1alpha). The mechanism by which mutations in only one allele of the HNF-1alpha gene impair pancreatic beta-cell function is unclear. The functional form of HNF-1alpha is a dimer--either a homodimer or a heterodimer with the structurally related protein HNF-1beta--that binds to and activates transcription of the genes whose expression it regulates. HNF-1alpha is composed of three functional domains: an amino-terminal dimerization domain (amino acids 1-32), a DNA-binding domain with POU-like and homeodomain-like motifs (amino acids 150-280), and a COOH-terminal transactivation domain (amino acids 281-631). Because the dimerization domain is intact in many of the mutant forms of HNF-1alpha found in MODY subjects, these mutant proteins may impair pancreatic beta-cell function by forming nonproductive dimers with wild-type protein, thereby inhibiting its activity; that is, they are dominant-negative mutations. This hypothesis was tested by comparing the functional properties of the frameshift mutation P291fsinsC, the most common mutation identified to date in MODY3 patients, and wild-type HNF-1alpha. P291fsinsC-HNF-1alpha showed no transcriptional transactivation activity in HeLa cells, which lack endogenous HNF-1alpha. Overexpression of P291fsinsC-HNF-1alpha in MIN6 cells, a mouse beta-cell line, resulted in an approximately 40% inhibition of the endogenous HNF-1alpha activity in a dosage-dependent manner. Furthermore, heterodimer formation between wild-type and P291fsinsC mutant proteins were observed by electrophoretic mobility shift assay. These data suggest that the P291fsinsC mutation in HNF-1alpha functions as a dominant-negative mutation. However, other mutations, such as those in the promoter region and dimerization domain, may represent loss of function mutations. Thus mutations in the HNF-1alpha gene may lead to beta-cell dysfunction by two different mechanisms.

Animals↗
PubMed1998

Reduced metastatic potential and c-myc overexpression of colon adenocarcinoma cells (Colon 26 line) transfected with nm23-R2/rat nucleoside diphosphate kinase alpha isoform.

Increased expression of nm23/nucleoside diphosphate kinase (NDP kinase) has been reported to be associated with both reduced metastatic potential in breast carcinoma and tumor progression in colon adenocarcinoma and lung adenocarcinoma. We examined effects of expression of nm23-R2 rat NDP kinase alpha isoform on mouse adenocarcinoma cells (Colon 26 line) and found a significant reduction of metastatic potential along with overexpression of c-myc. We also found that the proliferation rate of the transformed cells was the same as that of the control cells in culture. These results indicate that the cell growth potential in vitro is irrelevant to metastatic potential of the cells in vivo.

Adenocarcinoma↗
PubMed1998

ATP inhibition of proliferation of immortalized human fibroblasts is greater than that of normal human diploid fibroblasts.

It is known that cancers develop by a multi-step process. Normal cells are first immortalized, and then transformed into tumorigenic cells. Normal human cells are very rarely immortalized, but once they are, they are relatively easily transformed into tumorigenic cells. This indicates that the immortalization step plays a critical part in the development of human cancers. Thus, elucidation of the mechanisms of this step would shed light on the process of carcinogenesis in human cells. To understand the causes of immortalization, it is important to determine the differences in cellular phenotype between immortalized and normal human cells. In this study, we found that immortalized human fibroblasts were more sensitive to the growth inhibitory effects of ATP than normal human fibroblasts. ADP was as effective as ATP, but AMP, adenosine, and phosphoric acid were not. These results indicate that a high-energy bound of ATP and ADP may contribute to the growth inhibition of the cells. When the immortalized cells were pulse-labeled with [32P]-ATP, 30-, 31-, 33- and 40-kDa membrane fraction proteins were more prominently labeled in the immortalized cells than in the normal cells. At present, the characteristics of these proteins are being investigated.

Adenosine↗
PubMed1998

Molecular mechanisms of pancreatic beta-cell destruction in autoimmune diabetes: potential targets for preventive therapy.

Mononuclear cell infiltration into the islets of the pancreas (insulitis) is characteristic of autoimmune diabetes. T lymphocytes are the predominant subpopulation seen in insulitis, and are involved in the autoimmune process. Insulin-producing beta cells are thought to be destroyed by cytotoxic T cells, cytokines or nitric oxide, and beta-cell death occurs, at least partly, via apoptosis. Beta-cell death induced by cytokines is inhibited by Bcl-2, suggesting its potential as a tool for gene therapy. The Fas/Fas-ligand system plays a critical role in inducing insulitis and overt diabetes in nonobese diabetic (NOD) mice, a model of autoimmune diabetes. T-cell receptor gene usage in infiltrating T cells is not restricted in NOD mice, but there are some observations indicating relative restriction in human IDDM patients. Preventive strategies might be developed by focusing on these molecules involved in beta-cell destruction. The establishment of screening techniques for detecting prediabetic patients is also necessary to allow successful intervention.

Animals↗
PubMed1998

[Recurrent nerve palsy after endotracheal intubation].

We encountered left recurrent nerve palsy in four patients who had undergone surgery unrelated to the course of the vagus nerve or recurrent nerve, during which they had received endotracheal anesthesia. They were intubated without difficulty and underwent surgery without trouble, but postoperatively they all complained of hoarseness. We used a disposable ENTRASOFT endotracheal tube with high volume, low-pressure cuff in three patients and a disposable PORTEX endotracheal tube with low volume, standard cuff in one patient. In three patients recurrent nerve palsy healed completely within two months after the operation, and one patient was recovering from the palsy on the twentieth day after the operation. We believe that the most plausible explanation of recurrent nerve palsy is the excessive pressure from the inflated endotracheal tube cuff on the intralaryngeal course of the anterior branch of the recurrent nerve. Monitoring cuff pressure is most important to prevent recurrent nerve palsy after endotracheal intubation.

Adult↗
PubMed1998

[Simultaneous MIDCAB and subtotal gastrectomy in an elderly patient with severe ischemic heart disease].

An 81 year old man with severe ischemic heart disease and left ventricular dysfunction was scheduled for a subtotal gastrectomy for his advanced gastric cancer. His cardiac function was so poor that we performed minimally invasive coronary artery bypass grafting (MIDCAB; coronary artery bypass grafting without cardiopulmonary bypass for LAD through a small left thoracotomy), just before the abdominal operation. Anesthesia was induced and maintained with fentanyl, vecuronium and sevoflurane. To control heart rate below 60 bpm during local coronary occlusion for bypass grafting, edrophonium 5 mg was administered just before the occlusion. During the bypass grafting procedure, the patient's heart rate was maintained at 50-60 bpm and his hemodynamic profile slightly declined but was permissible. After bypass grafting, his cardiac performance was improved with low dose dobutamine. Subsequently subtotal gastrectomy was carried out. His postoperative course was uneventful. Combined MIDCAB and abdominal operation may be beneficial for selected patients with severe ischemic heart disease.

Adenocarcinoma↗
PubMed1998

Dose-dependent biphasic effects of phenobarbital on growth and differentiation of primary culture rat hepatocytes.

The actions of phenobarbital, a liver tumour promoter, on growth and differentiation of primary culture normal rat hepatocytes change biphasically as a function of its concentration. At low concentrations of 0.5-2 mmol/L, phenobarbital enhances DNA synthesis of normal adult rat hepatocytes in the presence of epidermal growth factor (EGF) and/or dexamethasone. This is also true for normal suckling (1-2-week-old) rat hepatocytes, without added growth factor(s), in serum-free primary culture. Contrarily, phenobarbital at high concentrations (3-4 mmol/L) suppresses DNA synthesis of suckling rat hepatocytes. Furthermore, phenobarbital inhibits DNA synthesis of transforming growth factor-alpha-stimulated primary hepatocytes from normal adult rats in a dose-dependent manner within a concentration range of 3-6 mmol/L. When normal adult rat hepatocytes are led to undergo multiple proliferative cycles upon stimulation with hepatocyte growth factor (HGF) and EGF in the chemically defined hepatocyte growth medium (HGM), 3 mmol/L phenobarbital also remarkably suppresses DNA synthesis. Phenobarbital at 3 mmol/L effectively keeps these hepatocytes morphologically differentiated and accelerates restoration of the expression of markers characteristic of differentiated cells after the initial cellular growth phase. In addition, phenobarbital efficiently supports prolonged survival of the hepatocytes.

Animals↗
PubMed1997

Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells.

Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence was generated. However, natural hIL-18 has not yet been isolated, and its cellular processing is therefore still unclear. To clarify this, we purified natural hIL-18 from the cytosolic extract of monocytic THP.1 cells. Natural hIL-18 exhibited a molecular mass of 18.2 kDa, and the NH2-terminal amino acid was Tyr37. Biological activities of the purified protein were identical to those of recombinant hIL-18 with respect to the enhancement of natural killer cell cytotoxicity and interferon-gamma production by human peripheral blood mononuclear cells. We also found two precursor hIL-18 (prohIL-18)-processing activities in the cytosol of THP.1 cells. These activities were blocked separately by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. Further analyses of the partially purified enzymes revealed that one is caspase-1, which cleaves prohIL-18 at the Asp36-Tyr37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp71-Ser72 and Asp76-Asn77 to generate biologically inactive products. These results suggest that the production and processing of natural hIL-18 are regulated by two processing enzymes, caspase-1 and caspase-3, in THP.1 cells.

Amino Acid Sequence↗
PubMed1997

Requirement of Fas for the development of autoimmune diabetes in nonobese diabetic mice.

Insulin-dependent diabetes mellitus (IDDM) is assumed to be a T cell-mediated autoimmune disease. To investigate the role of Fas-mediated cytotoxicity in pancreatic beta cell destruction, we established nonobese diabetic (NOD)-lymphoproliferation (lpr)/lpr mice lacking Fas. Out of three genotypes, female NOD-+/+ and NOD-+/lpr developed spontaneous diabetes by the age of 10 mo with the incidence of 68 and 62%, respectively. In contrast, NOD-lpr/lpr did not develop diabetes or insulitis. To further explore the role of Fas, adoptive transfer experiments were performed. When splenocytes were transferred from diabetic NOD, male NOD-+/+ and NOD-+/lpr developed diabetes with the incidence of 89 and 83%, respectively, whereas NOD-lpr/lpr did not show glycosuria by 12 wk after transfer. Severe mononuclear cell infiltration was revealed in islets of NOD-+/+ and NOD-+/lpr, whereas islet morphology remained intact in NOD-lpr/lpr. These results suggest that Fas-mediated cytotoxicity is required to initiate beta cell autoimmunity in NOD mice. Fas-Fas ligand system might be critical for autoimmune beta cell destruction leading to IDDM.

Adoptive Transfer↗
PubMed1997

Transformation of normal human fibroblasts into immortalized cells with the mutant p53 gene and X-rays.

In vitro cell transformation is a valuable approach for studying the mechanisms of multistep carcinogenesis of human cells. Since immortalization is an essential step for in vitro neoplastic transformation of human cells, this study addresses the question of whether mutant p53 contributes to the immortalization process of human cells. The mutant p53 gene (mp53: codon273Arg-His) was introduced into normal human fibroblasts (OUMS-24 line) and a G418-resistant clone, OUMS-24/P6 line, was obtained. This clone showed an extended life span and chromosome abnormalities, but senesced at the 79th population doubling level (PDL). When these cells were subjected to intermittent X-ray treatment, they became an immortalized cell line (OUMS-24/P6X). Although these immortalized cells showed chromosome abnormalities, they were not tumorigenic. On the other hand, normal OUMS-24 cells into which mp53 had not been introduced were not immortalized by the same X-ray treatment. These results indicate that introduction and expression of mp53 alone were not sufficient for immortalization of human cells, and that mutations of the remaining wild-type p53 or other genes may have been necessary for immortalization. In fact, no expression of the 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 an increase in cdk2 and cdc2 kinase activity. These findings indicate that the p53-p21 cascade may play some role in the immortalization of human cells. On the other hand, there was no significant difference in expression of proteins such as Rb, p16, cdk4, cdk6, cyclin A and cyclin D1 between the normal and immortalized human fibroblasts.

Cell Transformation, Neoplastic↗
PubMed1997

Screening the p53 status of human cell lines using a yeast functional assay.

We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promotor in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth. p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30 degrees C but not at 37 degrees C. Four temperature-sensitive p53 mutations were isolated: CAT-->CGT at codon 214 (H214R), TAC-->TGC at codon 234 (Y234C), GTG-->ATG at codon 272 (V272M), and GAG-->AAG (E285K). Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization.

Alleles↗