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M Ueda

Publications and source records attributed to M Ueda.

At least 415 records · Page 23Linked to original sources
PubMed1997

Gene analysis of an NADP-linked isocitrate dehydrogenase localized in peroxisomes of the n-alkane-assimilating yeast Candida tropicalis.

In n-alkane-utilizing yeast, Candida tropicalis, two NADP-linked isocitrate dehydrogenase (NADP-IDH) isozymes are present, one in mitochondria (Mt-NADP-IDH) and the other in peroxisomes (Ps-NADP-IDH). Here we report the isolation, sequencing, and expression of the gene encoding Ps-NADP-IDH (CtIDP2), distinct from the Mt-NADP-IDH gene (CtIDP1). Based on the N-terminal amino acid sequence of purified Ps-NADP-IDH, a cDNA fragment specific for Ps-NADP-IDH was obtained by the 5'-RACE method. Using this fragment as a probe, the genomic CtIDP2 gene was isolated. Nucleotide sequence analysis of CtIDP2 disclosed that the region encoding CtIdp2p had a length of 1233 bp, corresponding to 411 amino acid residues. The deduced N-terminal amino acid sequence matched the results obtained from the purified protein. When this CtIDP2 was expressed in Saccharomyces cerevisiae using the C. tropicalis isocitrate lyase gene promoter (UPR-ICL), high intracellular NADP-IDH activity was observed. Comparison of amino acid sequences and phylogenetic tree analysis with NADP-IDH enzymes from all reported eukaryotic sources revealed that mammalian mitochondrial NADP-IDHs formed a cluster, as did plant NADP-IDHs. CtIdp2p and other yeast NADP-IDHs were not included in these clusters and seemed to diverge at an early stage from all other enzymes of higher eukaryotes. Ps-NADP-IDH had no typical C-terminal peroxisomal targeting signal and no processing was demonstrated at the N-terminus. However, we could find a region near the N-terminus of the protein with high similarity to both the putative N-terminal peroxisomal targeting signal sequence of Fox3p of S. cerevisiae and an internal region of Pox4p of C. tropicalis. The results of northern blot analysis indicated that the biosynthesis of CtIdp2p was induced in a medium containing alkanes as a carbon source, where profuse proliferation of peroxisomes is observed.

Alkanes↗
PubMed1997

Patched (ptch)-associated preferential expression of smoothened (smoh) in human basal cell carcinoma of the skin.

The discovery of specific overexpression of a gatekeeper gene, ptch, in basal cell carcinoma (BCC) led to a hypothesis that the human homologue of patched (PTCH) normally functions as a negative regulator of the signaling pathway that is initiated by hedgehogs (HHs) and activated by the human homologue of smoothened (SMOH); however, no evidence for the involvement of smoh and hhs has been provided. Here, we show novel evidence that smoh is also preferentially overexpressed in BCC, together with ptch (P < 0.002), and that Sonic hh was expressed in only some BCCs. Our data, therefore, indicate that such overexpression of smoh may be associated with overexpression or mutation of PTCH and that this overexpression subsequently stimulates the PTCH/SMOH signaling pathway. In an investigation of a possible regulation of ptch and smoh, we demonstrated that expression of exogenous p21WAF1 in immortalized keratinocytes down-regulates both ptch and smoh and that the down-regulation is accompanied by growth arrest, which suggests the involvement of p21WAF1 in regulation of the PTCH/SMOH signaling pathway.

Basal Cell Carcinoma↗
PubMed1997

Benzoquinones inhibit the expression of inducible nitric oxide synthase gene.

Benzoquinones inhibited the production of NO induced by LPS and gamma-IFN in C6 glia cells. The mechanistic studies showed that benzoquinones inhibited the expression of iNOS mRNA through inactivation of NF kappa B. Benzoquinones may be a useful candidate for the development of a drug to treat disease due to iNOS gene over-expression.

Animals↗
PubMed1997

L-DOPA neurotoxicity is mediated by glutamate release in cultured rat striatal neurons.

The exposure of cultured rat striatal neurons to L-DOPA caused marked cell death. The L-DOPA cytotoxicity was inhibited by the addition of Mg2+ to and by the removal of Ca2+ from the culture medium, and also by the application of tetrodotoxin. Moreover, prolonged application of L-DOPA increased the glutamate content in the culture medium. These results indicate that L-DOPA produces neurotoxicity by facilitating glutamate release.

Animals↗
PubMed1997

Bactericidal activity of human lysozymes carrying various lengths of polyproline chain at the C-terminus.

The amphiphilic polypeptide polyproline having different chain lengths was connected to the C-terminus of human lysozyme by the recombinant DNA technique. The hydrophobicity of human lysozyme increased with increasing length of the polyproline chain. Although the bactericidal activity of wild-type lysozyme is limited to gram-positive bacteria and the hydrolytic activity of the mutant lysozyme decreased with increasing chain length of polyproline, the mutant lysozymes showed bactericidal activity to gram-negative bacteria and the activity increased with increasing hydrophobicity of the mutant enzyme. Experiments with Escherichia coli phospholipid liposomes revealed that the mutant human lysozymes dissipated the valinomycin-induced transmembrane electrochemical potential, and the dissipation increased with increasing hydrophobicity. The increased hydrophobicity of the mutant enzyme may induce interaction of lysozyme with the outer membrane and subsequent penetration into the inner membrane of E. coli, resulting in an increase of bactericidal activity.

Anti-Bacterial Agents↗
PubMed1997

Stimulatory effects of EGF and TGF-alpha on invasive activity and 5'-deoxy-5-fluorouridine sensitivity in uterine cervical-carcinoma SKG-IIIb cells.

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha on migration, invasion and matrix metalloproteinase (MMP) expression of uterine cervical-carcinoma SKG-IIIb cells, and whether these growth factors affect pyrimidine-nucleoside-phosphorylase (PyNPase) activity and 5'-deoxy-5-fluorouridine (5'-dFUrd) sensitivity of tumor cells. Tumor-cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were stimulated by 0.1 to 100 ng/ml of EGF and TGF-alpha in a concentration-dependent manner. The zymography of tumor-conditioned medium showed that the treatment of tumor cells with EGF and TGF-alpha resulted in an increase of the 92-kDa type-IV collagenase (MMP-9), which was confirmed by immunoblot analysis. These growth factors also up-regulated the expression of PyNPase activity of tumor cells and consequently enhanced the anti-proliferative action of 5'-dFUrd, a cytostatic that is biotransformed to 5-fluorouracil (5-FUra) by PyNPase. However, EGF and TGF-alpha did not have significant effects on the 5-FUra sensitivity of tumor cells. These results suggest that EGF and TGF-alpha, tumor environmental factors, simultaneously up-regulate the potential of uterine cervical-carcinoma cells to invade extracellular matrices and their PyNPase activity, which are subsequently associated with the specific action of 5'-dFUrd selectively killing tumor cells of gynecological origin with high invasive and metastatic potential.

Basement Membrane↗
PubMed1997

Centrosome positioning and directionality of cell movements.

In several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell movement: the centrosome is located behind the leading edge, suggesting that it serves as a steering device for directional movement. A logical extension of this suggestion is that a change in the direction of cell movement is preceded by a reorientation, or shift, of the centrosome in the intended direction of movement. We have used a fusion protein of green fluorescent protein (GFP) and gamma-tubulin to label the centrosome in migrating amoebae of Dictyostelium discoideum, allowing us to determine the relationship of centrosome positioning and the direction of cell movement with high spatial and temporal resolution in living cells. We find that the extension of a new pseudopod in a migrating cell precedes centrosome repositioning. An average of 12 sec elapses between the initiation of pseudopod extension and reorientation of the centrosome. If no reorientation occurs within approximately 30 sec, the pseudopod is retracted. Thus the centrosome does not direct a cell's migration. However, its repositioning stabilizes a chosen direction of movement, most probably by means of the microtubule system.

Animals↗
PubMed1997

Three-dimensional cultures of keratinocytes and an application to in vitro-amyloid production of cutaneous amyloidosis.

Some three-dimensional culture models of the skin were reviewed including our systems using a collagen dermal substitute and a matrix dermal substitute. No obvious junctional structures, such as hemidesmosomes and the lamina densa, were formed between the basal keratinocytes and the dermal substitutes, when the cytoplasmic membrane of the basal keratinocytes directly faced the collagenous materials. On the other hand, when the cytoplasmic membrane of the basal keratinocytes faced the preformed basement membrane, the type IV collagen film, or the extracellular matrix gel, an organized interaction occurred between the basal keratinocytes and the dermal substitute through hemidesmosomes and a rudimentary lamina densa. Keratinocyte differentiation in the suprabasal layers seemed to be closely related to such a basal cell differentiation. Our preliminary examination of the experimental amyloid production by the epidermal sheet from the lesional skin of patients with primary localized cutaneous amyloidosis suggested that the basal cells in the transplanted lesional epidermis produced amyloid fibrils in our in vitro culture model. This is another use of the three-dimensional culture models of the skin in addition to the application of the systems to wound treatment.

Amyloid↗
PubMed1997

Redifferentiation of smooth muscle cells after coronary angioplasty determined via myosin heavy chain expression.

BACKGROUND: The pathophysiology of phenotypic modulation of smooth muscle cells (SMCs) involved in restenosis after angioplasty is not well understood. Smooth muscle myosin heavy chain (SM MHC) isoforms (SM1 and SM2) are specific markers for SMC differentiation. In particular, SM2 is useful as a marker of mature SMCs. SMemb is a nonmuscle myosin heavy chain (NM MHC) whose expression is upregulated in immature or activated SMC. METHODS AND RESULTS: To determine SMC phenotypes in neointimal tissues after percutaneous transluminal coronary angioplasty (PTCA), we performed immunohistochemistry on human coronary arteries with antibodies against alpha-SM actin, SM1, SM2, and SMemb. Tissues were obtained from six autopsied patients and from atherectomy specimens from 16 patients who had undergone PTCA. Medial SMCs were positive for alpha-actin, SM1, and SM2. Expression of SM1 and SM2 in the neointima varied with the time after intervention, whereas alpha-actin was constitutively expressed in all cases studied. Neointimal cells at 16 and 20 days after PTCA contained alpha-actin but little or no SM1 or SM2, indicating that these cells modulated their phenotype to the immature state. Neointimal SMCs recovered SM MHC expression, first SM1 and then SM2, by 6 months after PTCA. Increased expression of SMemb was found in the neointima but without apparent relationship to the time after PTCA. CONCLUSIONS: Neointimal SMCs show features of an undifferentiated state, indicated by altered expression of SM MHC, and undergo redifferentiation in a time-dependent manner. The expression of SM MHC isoforms provides insight into the biology of healing after angioplasty and furnishes useful tools for the understanding of the roles of differentiation and phenotypic modulation of SMCs in human vascular lesions.

Aged↗
PubMed1997

Integrin alpha6 is involved in follicular growth in mice.

We previously reported that integrin alpha6 is expressed on granulosa cells in the inner layers of the human and porcine ovarian follicles, where granulosa cells have no direct contact with basal lamina. In this study, we examined the physiological role of integrin alpha6 on follicular growth with an immature superovulated mice model using the anti-integrin alpha6 monoclonal antibody, GoH3. In the ovaries of 9- to 20-day-old mice, integrin alpha6 was detected on all the layers of granulosa cells in the primordial, primary, and secondary follicles by immunohistochemistry. The 13-day-old female mice were superovulated by pregnant mare serum gonadotropin and human chorionic gonadotropin with the treatment of intraperitoneal administration of GoH3, or a control antibody, or PBS. In the group of GoH3 treatment, successful ovulation was observed in 57+/-25.7% of the animals, whereas no ovulation was observed in the control groups (p<0.01). These findings indicate that integrin alpha6 is involved in gonadotropin-induced follicular growth.

Animals↗
PubMed1997

Induction of cancer, actinic keratosis, and specific p53 mutations by UVB light in human skin maintained in severe combined immunodeficient mice.

To study the mechanism and risk of human skin cancer from solar light, we exposed human skin transplanted to severe combined immunodeficient mice to daily doses of UVB for periods of approximately 2 years. We have succeeded for the first time in inducing cancer and solar (actinic) keratosis in human skin by UVB. Of 18 normal skins exposed to doses of 7.3 x 10(5) to 1.8 x 10(6) J/m2, 14 actinic keratoses (77.8%) and 3 squamous cell carcinomas (16.7%) developed, whereas neither actinic keratosis nor cancer was observed in 15 human skins not exposed to UVB. Each human skin showed a different susceptibility, and skins sensitive for actinic keratosis were also sensitive for cancer induction. Among p53 mutations at various sites, mutation at codon 242 (C TGC --> C CGC; Cys --> Arg) was specifically observed in both skin cancers and actinic keratoses. Furthermore, double or triple mutations were induced in all UVB-induced skin cancers and in three of eight actinic keratoses. Most of the mutations (17 of 20) occurred at dipyrimidine sites.

Animals↗
PubMed1997

Growth inhibition and apoptotic cell death in uterine cervical carcinoma cells induced by 5-fluorouracil.

We have investigated the effects of 5-fluorouracil (5-FU) on cell growth, DNA synthesis, morphological changes, DNA fragmentation and Fas antigen expression of cultured human uterine cervical carcinoma cells (OMC-1 squamous-cell carcinoma and OMC-4 adenocarcinoma). Apoptotic cell death in cervical carcinoma tissues from the patients after an intravenous administration of 5-FU was also examined. Treatment of OMC-1 and OMC-4 cells with 0.1-10 microg/ml of 5-FU (1 microg/ml = 7.7 microM) resulted in concentration-dependent inhibition of the number of cumulative viable cells and 6-3H-deoxyuridine uptake into the DNA. These effects of 5-FU were well correlated with apoptotic indices of the cells determined in situ by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end-labeling (TUNEL). A TUNEL signal was detectable in nuclei of normal-looking cells and in a lobulated nucleus with dense chromatin. DNA fragmentation was observed in the cells exposed to 10 microg/ml of 5-FU according to the nucleosomal ladder detected by electrophoresis. Flow cytometric analysis showed that Fas antigen expression of the cells increased upon incubation with 10 microg/ml of 5-FU. Moreover, TUNEL of tissue sections of resected uterus revealed that apoptosis occurred more frequently in patients treated pre-operatively with 500 mg/m2/day of 5-FU for 8 days than in those who did not receive 5-FU. These results suggest that achievable therapeutic levels of 5-FU inhibit cell growth and DNA synthesis and induce apoptotic cell death in uterine cervical carcinoma cells. However, the relationship between 5-FU-mediated apoptosis and the Fas ligand/Fas system remains to be explored.

Adenocarcinoma↗
PubMed1997

Isolation of a T-cell clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia.

The existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vbeta21+ T-cell clone that was most dominant among Vbeta21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti-HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

Aged↗
PubMed1997

Transgenic rescue from embryonic lethality and renal carcinogenesis in the Eker rat model by introduction of a wild-type Tsc2 gene.

We recently reported that a germ-line insertion in the rat homologue of the human tuberous sclerosis gene (TSC2) gives rise to dominantly inherited cancer in the Eker rat model. In this study, we constructed transgenic Eker rats with introduction of a wild-type Tsc2 gene to ascertain whether suppression of the Eker phenotype is possible. Rescue from embryonic lethality of mutant homozygotes (Eker/Eker) and suppression of N-ethyl-N-nitrosourea-induced renal carcinogenesis in heterozygotes (Eker/+) were both observed, defining the germ-line Tsc2 mutation in the Eker rat as embryonal lethal and tumor predisposing mutation. To the best of our knowledge, this is the first report of rescue from a naturally occurring dominantly inherited cancer. This transgenic rescue system will be useful to analyze Tsc2 gene function, its relation to tumorigenesis in vivo, and genetic-environmental interactions in carcinogenesis.

Animals↗
PubMed1997

Derepression of gene expression mediated by the 5' upstream region of the isocitrate lyase gene of Candida tropicalis is controlled by two distinct regulatory pathways in Saccharomyces cerevisiae.

The 5' upstream region of the gene encoding isocitrate lyase of Candida tropicalis (UPR-ICL) is functional as a promoter in Saccharomyces cerevisiae, and it is regulated by carbon source; the expression of the gene is repressed when cells are grown on glucose, while it increases to a higher level in acetate-grown cells. Therefore, we have investigated regions in UPR-ICL responsible for gene expression in glucose-grown and acetate-grown cells. In glucose-grown cells, a deletion of the region between -801 and -569 (region G1) significantly decreased gene expression compared with that observed with the complete UPR-ICL. The region from -421 to -379 (region G2) also repressed gene expression in glucose-grown cells. In acetate-grown cells, two regions were found to strongly enhance gene expression, one between -728 and -569 (region A1) and the other between -370 and -356 (region A2). Whereas region A2 contained a sequence motif similar to the carbon-source-responsive element (CSRE), which mediates regulation by carbon source of S. cerevisiae ICL1, region A1 did not show similarity to any reported cis-acting elements. Deletion mutants of UPR-ICL containing only one of these regions showed that each region could independently activate gene expression to a similar level when the cells were grown on acetate. The influences of null mutations in the MIG1, SNF1 and CAT8 genes on regulation of UPR-ICL-mediated gene expression were examined. Expression of the ICL gene with full-length UPR-ICL increased about tenfold in mig1 cells grown on glucose, while little difference was observed in acetate-grown cells. The effects of snf1 and cat8 mutations were different between region-A1-mediated and region-A2-mediated gene expression in acetate-grown cells. Region-A2-mediated expression decreased 95% and 86% in snf1 and cat8 cells, respectively, while region-A1-mediated expression decreased 72% in snf1 cells and was not affected by the cat8 mutation. This finding indicates that region-A1-mediated gene expression is regulated by a pathway independent of CAT8, which is necessary for derepression of CSRE-mediated gene expression in S. cerevisiae.

Base Sequence↗
PubMed1997

Evidence for UV-associated activation of telomerase in human skin.

Telomerase activation plays a crucial role in the immortalization of human cells and carcinogenesis; however, the temporal and pathophysiological aspects of the activation in vivo are poorly understood. We found telomerase activity not only in malignant tumors (91%) but also in most benign (60%) and premalignant (89%) skin tumors. This suggests the involvement of telomerase activation in a crucial biological step of human skin carcinogenesis. Because UV light is a major factor in skin carcinogenesis, we further examined telomerase activity in normal skin samples and in normal skin samples adjacent to benign, premalignant, and malignant skin lesions. Data for chronically sun-exposed body sites were compared with those for covered sites. Among normal skin samples, 39% (26 of 67) had telomerase activity, and this activity was unrelated to neighboring lesions but strongly associated with the level of sun exposure. Fifty-four % (21 of 39) of normal skin samples from chronically sun-exposed sites were telomerase-positive, compared with only 12% (3 of 26) of samples from covered sites. When we examined telomerase activity and CC to TT mutations at codons 247/8 of the p53 gene (which are considered to be UV specific) in the same normal skin samples, only 43% (7 of 16) of telomerase-positive normal skin samples at sun-exposed sites contained the p53 mutations, whereas all (7 of 7) of the samples with UV-specific p53 mutations showed telomerase activity (P = 0.019). These data suggest that telomerase activation is involved at an early stage of human skin carcinogenesis and that activation may precede the acquisition of UV-associated p53 mutations in the skin. Telomerase activity was also found in plucked hair follicles and enzymatically separated epidermis, which may be associated with the presence of stem cells in the skin.

Enzyme Activation↗