Search PubMed⌕ Search

Biomedical subjects

K Sogawa

Publications and source records attributed to K Sogawa.

At least 37 records · Page 2Linked to original sources
PubMed1998

The gut-enriched Krüppel-like factor suppresses the activity of the CYP1A1 promoter in an Sp1-dependent fashion.

The gut-enriched Krüppel-like factor (GKLF) is a newly identified zinc finger-containing transcription factor. Recent studies indicate that GKLF binds to a core DNA sequence of 5'-(G/A)(G/A)GG(C/T)G(C/T)-3', which is found in an endogenous cis element, the basic transcription element (BTE) of the cytochrome P-450IA1 (CYP1A1) promoter. The present study characterizes the ability of GKLF to regulate CYP1A1 expression. By electrophoretic mobility gel shift assay (EMSA) and methylation interference assay, GKLF was found to bind BTE in a manner similar to several other transcription factors known to interact with BTE including Sp1 and BTEB. Cotransfection studies in Chinese hamster ovary cells showed that GKLF inhibited the CYP1A1 promoter in a dose- and BTE-dependent manner. The same experiments also revealed that BTE was responsible for a significant portion of the CYP1A1 promoter activity. EMSA of nuclear extracts from Chinese hamster ovary cells showed that Sp1 and Sp3 were two major proteins that interacted with BTE. Additional cotransfection studies showed that GKLF inhibited Sp1-mediated activation of the CYP1A1 promoter. In contrast, GKLF enhanced Sp3-dependent suppression of the same promoter. Moreover, the ability of GKLF to inhibit Sp1-dependent transactivation was in part due to physical interaction of the two proteins. These findings indicate that GKLF is a negative regulator of the CYP1A1 promoter in a BTE-dependent fashion and that this inhibitory effect is in part mediated by physical interaction with Sp1.

Animals↗
PubMed1998

Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia.

Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O2 radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-L-glutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 microM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 = 6.6 microM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 = 2.5 microM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1alpha or HIF-1alpha-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1alpha to a DNA-binding form.

Cell Hypoxia↗
PubMed1998

Direct measurement of DNA molecular length in solution using optical tweezers: detection of looping due to binding protein interactions.

DNA looping is caused by the interaction between DNA binding proteins located at separate positions on a DNA molecule and may play an important role in transcription regulation. We have developed a system to stretch single DNA molecules and to measure changes in molecular length. DNA molecules were prepared and 5' end-labeled by PCR amplification. Two beads and the intervening DNA molecule were trapped and manipulated independently with dual trap optical tweezers. The trapped DNA molecule was then stretched and the extension (the distance between the two beads) was measured. The extension at the specific tension force of 30 pN was calculated and used as a molecular length. The molecular length was found to be proportional to the base pair number. The rise per residue was calculated to be 3.31 +/- 0.05 A. The length measurement was applied to DNA fragments containing GC box sequences at two different locations separated by a distance of 2.428 kbp. The addition of GC box binding transcription factor Sp1 shortened the molecular length, suggesting DNA looping forms as a result of interaction between transcription factors.

Base Sequence↗
PubMed1998

Detection of telomerase activity in oral lesions.

Telomerase is a ribonucleoprotein complex intimately associated with cell immortalization and neoplastic transformation. In almost all types of cancer this enzyme is reactivated and stabilizes telomere length. It may be necessary for continuous cell proliferation. In this study we used a non-radioactive polymerase chain reaction assay to analyze telomerase activity in various tissue specimens taken from the oral cavity. Four of 4 (100%) squamous cell carcinoma cell lines, 28 of 29 (96%) malignant tumors, 10 of 28 (36%) benign lesions, and none of the 14 (0%) oral control tissues possessed telomerase activity. Moreover, 4 of 15 (27%) oral rinses and 3 of 3 (100%) samples of ascites and pleural effusion taken from patients with oral malignancy were telomerase positive. These findings indicate that the evaluation of telomerase activity in tissue and body fluid specimens may provide information useful in the diagnosis of oral malignancy.

Ameloblastoma↗
PubMed1998

Inhibitory effect of a marine microalgal polysaccharide on the telomerase activity in K562 cells.

An extracellular polysaccharide from marine microalga, dinoflagellate Gymnodinium sp. A3 (GA3), showed cytotoxicity to human myeloid leukemia K562 cells. We measured telomerase activity in K562 cells cultured with GA3 polysaccharide. 10.0 micrograms/ml of GA3 polysaccharide inhibited the telomerase activity in the cells completely. Also, we found a decrease in expression of the catalytic subunit of protein phosphatase (PP) type 1, PP1 gamma 1, in K562 cells cultured with GA3 polysaccharide.

Animals↗
PubMed1998

Decrease of nuclear protein phosphatase 1 activity and induction of mitotic arrest and apoptosis by a marine microalgal polysaccharide in human myeloid leukemia U937 cells.

An extracellular polysaccharide, which we designate GA3P, produced from a marine microalga dinoflagellate Gymnodinium sp. A3, has been previously reported to induce apoptosis in lymphoid and myeloid cell lines. We found that the GA3P accumulates cells into the mitotic phase of the cell cycle and decreases nuclear protein phosphatase 1 (PP1) activity in a dose-dependent manner in myeloid leukemia U937 cells. Dose-dependent patterns in the decrease of nuclear PP1 activity and in the accumulation of cells into mitotic phase or apoptotic status by the GA3P were concordant with each other, indicating that the decrease of nuclear PP1 activity at least mediates some of the etiological steps in development of mitotic arrest and apoptosis induced by the GA3P. In addition, the GA3P repressed the expression of protein levels of the PP1 catalytic subunit isoform PP1 gamma 1 gamma 1. We thus suggest that the decrease of nuclear PP1 activity is due to down-regulation of the protein levels of the PP1 gamma 1.

Animals↗
PubMed1998

Increased polyploidy, delayed mitosis and reduced protein phosphatase-1 activity associated with excess copper in the Long Evans Cinnamon rat.

Until now, it is not known whether copper hepatotoxicity impairs mitosis. Enlarged hepatocytes with huge nuclei considered as polyploids are frequently observed in the Long Evans Cinnamon (LEC) rat which exhibits an abnormal accumulation of hepatic copper due to a defect in the gene homologous to human Wilson's disease gene responsible for intracellular copper delivery. This defect may lead to a abnormal mitotic progression in increased polyploidization and is associated with excessive hepatic copper. This study was designed to examine whether excess copper impairs mitotic progression and results in increased polyploidization using a model of LEC rat liver. Polyploidy was analyzed by flow cytometry. The rate of mitotic progression was investigated using the fraction of mitotic hepatocytes or a mitosis-specific phosphoprotein retained in regeneration. Nuclear protein phosphatase-1 (PP-1) activity essential to mitotic progression was measured. The effect of excess copper on incidence of polyploidy, the rate of mitotic progression and nuclear PP-1 activity was investigated using age- or copper overload-dependent changes in them in LEC rat, or genetic profile-dependent changes of them in backcrosses. LEC rat liver showed an increase of polyploidy, a delay of mitotic progression, and a reduction of nuclear PP-1 activity. These abnormal features concurred with increase of copper concentration accompanied by changes of age in LEC rats from 2 to 4 months of age, induced by dietary copper overload in LEC rat, or caused by single genetic defect in backcrosses. Excess copper impairs mitotic progression, resulting in increased polyploidization. Nuclear PP-1 activity is likely to be at least one of targets of copper hepatotoxicity leading to impairment of mitotic progression.

Age Factors↗
PubMed1998

Extraction of cytokeratin from the human submandibular gland and its electrophoretic analysis.

We evaluated a method for extracting cytokeratin (CK) from the normal human submandibular gland and analyzed CK distribution by one- and two-dimensional electrophoresis. Four submandibular glands without histological changes under a light microscope were used as specimens. Intermediate filamentous protein was extracted by a prior modified method CK was not adequately extracted in 1-2% sodium dodecyl sulfate -5% 2-mercaptoethanol solution. On the other hand, 11 bands with molecular weights of 40-58 K were obtained after extraction in 9.5 M urea -5% 2-mercaptoethanol solution. Although fragmentation of cytoskeletal protein was observed, there were no differences in bands according to the strength of homogenization. This fragmentation seemed to be due to restriction degradation by protease. Immunoblotting revealed the reaction of this CK extract to 2 pan-CK antibodies, i.e., K8.13, which recognizes type II basic CK, and C-11, which recognizes CK4, CK5, CK6, CK8, CK10, CK13, CK18. Among monospecific antibodies, anti-CK7 (LDS-68), CK8 (M20), CK13 (KS-1A3), CK18 (CY-90), and CK19 (BA17) antibodies showed positive reactions. Glial fibrillary acidic protein, neurofilament (NF) 68 K, NF160 K, or NF200 K showed no reactions. These results of one- and two-dimensional electrophoretic analysis suggest the presence of CK5, CK7, CK8, CK13, CK14, CK15, CK17, CK18, and CK19 in the normal submandibular gland.

Antibodies, Monoclonal↗
PubMed1998

Induction of apoptosis by a marine microalgal polysaccharide in a human leukemic cell line.

An extracellular polysaccharide produced by a marine microalga Gymnodinium A3 OKU-1 strain showed a strong cytotoxicity for the human leukemia K-562 cells (CC50 = 11.6 µg/ml). We revealed that this polysaccharide induced apoptosis of K-562 cells, as shown by cell morphology, nuclear fragmentation, in situ end-labeling of fragmented DNA, and internucleosomal DNA fragmentation, suggesting that the mechanism of cytotoxicity of this polymer was based on the induction of apoptosis.

Journal Article↗
PubMed1997

A point mutation responsible for defective function of the aryl-hydrocarbon-receptor nuclear translocator in mutant Hepa-1c1c7 cells.

A 3,4-benzopyrene-resistant mutant clone (c4) of the mouse hepatoma Hepa-1c1c7 cell line was examined for the mutation that causes the defective function of aryl-hydrocarbon receptor (AHR) nuclear translocator (Arnt). Arnt dimerizes with AHR and mediates the induction signal of aryl-hydrocarbon hydroxylase activity. The Arnt cDNAs of c4 cells were cloned by reverse-transcription/PCR to compare the sequences with that of wild-type Arnt cDNA. The Arnt cDNA of c4 cells was found to have a single point mutation, leading to replacement of Gly326 with Asp between two internal repeats in the highly conserved Per-Arnt-Sim (PAS) domain, PAS A and PAS B. The inability of [Asp326]Arnt/AHR heterodimers to enhance reporter gene transcription under the control of the CYP1A1 gene promoter and enhancer confirmed that the G326-->D substitution was a causative mutation. While fluorescence microscopy and coimmunoprecipitation experiments showed that this mutant form of Arnt was not changed from wild-type Arnt in terms of nuclear localization or heterodimer formation with AHR, the binding activity of the [Asp326]Arnt x AHR heterodimer to the xenobiotic-responsive element was reduced markedly. Determination of the turnover rate in COS-7 cells transfected with expression plasmids for mutant Arnt or normal Arnt showed that the mutant protein turned over with an accelerated rate compared with that of the normal. Moreover, the mutant protein displayed increased proteolytic digestibility in vitro with various proteases.

Amino Acid Sequence↗
PubMed1997

A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development.

We have isolated and characterized a cDNA for a novel Per-Arnt/AhR-Sim basic helix-loop-helix (bHLH-PAS) factor that interacts with the Ah receptor nuclear translocator (Arnt), and its predicted amino acid sequence exhibits significant similarity to the hypoxia-inducible factor 1alpha (HIF1alpha) and Drosophila trachealess (dTrh) gene product. The HIF1alpha-like factor (HLF) encoded by the isolated cDNA bound the hypoxia-response element (HRE) found in enhancers of genes for erythropoietin, vascular endothelial growth factor (VEGF), and various glycolytic enzymes, and activated transcription of a reporter gene harboring the HRE. Although transcription-activating properties of HLF were very similar to those reported for HIF1alpha, their expression patterns were quite different between the two factors; HLF mRNA was most abundantly expressed in lung, followed by heart, liver, and other various organs under normoxic conditions, whereas HIF1alpha mRNA was ubiquitously expressed at much lower levels. In lung development around parturition, HLF mRNA expression was markedly enhanced, whereas that of HIF1alpha mRNA remained apparently unchanged at a much lower level. Moreover, HLF mRNA expression was closely correlated with that of VEGF mRNA. Whole mount in situ hybridization experiments demonstrated that HLF mRNA was expressed in vascular endothelial cells at the middle stages (9.5 and 10.5 days postcoitus) of mouse embryo development, where HIF1alpha mRNA was almost undetectable. The high expression level of HLF mRNA in the O2 delivery system of developing embryos and adult organs suggests that in a normoxic state, HLF regulates gene expression of VEGF, various glycolytic enzymes, and others driven by the HRE sequence, and may be involved in development of blood vessels and the tubular system of lung.

Amino Acid Sequence↗
PubMed1997

Enhanced expression of PP1 gamma 1, a catalytic subunit isoform of protein phosphatase type 1, in invasive ductal carcinoma of the breast.

Breast cancer is one of the most common malignancies of women. Assessing the biological parameters of malignant tumors may facilitate predictions of clinical outcome. The expression of the three catalytic subunits of protein phosphatase (PP) type 1, PP1 alpha, PP1 gamma 1 and PP1 delta, as well as the one catalytic subunit of PP type 2, PP2AC, were examined in ten cases of mammary dysplasia, ten cases of fibroadenoma and 12 cases of invasive ductal carcinoma, using immunohistochemical analysis. Moreover, we measured the S-phase fraction of the cell cycle for use as a marker value of cell growth, using flow cytometric analysis. The percentage of proliferating cells that stained positive with antisera against PP1 gamma 1 was significantly higher in invasive ductal carcinoma than in mammary dysplasia and fibroadenoma. Furthermore, invasive ductal carcinoma showed a markedly high number of tumor cells in the S-phase of the cell cycle, as compared to mammary dysplasia and fibroadenoma. Our results indicate that PP1 gamma 1 may be involved in the accelerated growth of malignant cells in breast tumors.

Adult↗
PubMed1997

Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor.

BACKGROUND: The aryl hydrocarbon receptor (AhR or dioxin receptor) is a ligand-activated transcription factor that is considered to mediate pleiotropic biological responses such as teratogenesis, tumour promotion, epithelial hyperplasia and the induction of drug-metabolizing enzymes to environmental contaminants usually represented by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast to the role of AhR in the regulatory mechanism of xenobiotic-metabolizing enzymes, there is no direct proof that the AhR is involved in the teratogenic effects of TCDD. RESULTS: To gain insight into the physiological and teratogenic role of the AhR, we have used gene targeting in mice to disrupt the murine Ahr gene by homologous recombination. Ahr-null mice were viable and fertile and were apparently normal at birth, but displayed a slightly slower growth rate than wild-type mice for the first few weeks of life. When pregnant dams were administered with TCDD by gavage, at a dose of 40 microg/kg body weight at gestation day 12.5, none of the Ahr-null mutant foetuses were sensitive to the teratogenic effects of TCDD, although almost all wild-type foetuses suffered from cleft palate and hydronephrosis. In heterozygous Ahr+/- genotypes, nearly all foetuses suffered from hydronephrosis in response to TCDD treatment, while haplo-insufficiency was observed in the incidence of cleft palate. CONCLUSION: These results clearly show that the AhR is involved in the malformation of the palate and kidney in mouse embryos caused by TCDD and suggests that the mechanism of its involvement differs between the cleft palate and hydronephrosis.

Animals↗
PubMed1997

Transcriptional activation domain of human BTEB2, a GC box-binding factor.

BTEB2 is a GC box-binding transcription factor containing proline-rich and zinc finger domains as transactivation and DNA binding domains, respectively. We have identified a small region in the proline-rich domain indispensable for its transcription-enhancing activity by transfection experiments using various expression plasmids with point mutations or small deletions in the domain. This region comprising about 10 amino acids was relatively hydrophobic and rich in proline and alanine residues. BTEB2 purified from a baculovirus expression system could enhance transcription, depending on the presence of GC boxes in the promoter region of templates in an in vitro transcription assay. BTEB2 deleted of the hydrophobic region, however, lost the transcription-enhancing activity, in confirmation of the above results. Basic transcription factors which possibly interact with BTEB2 were examined. Initiation factors, TFIIB, TFIIE beta, and TFIIF beta as well as the TATA box-binding protein (TBP) were found to interact with BTEB2 when analyzed by in vitro binding experiments, although these interactions could not be attributed to the proline-rich domain. We discussed factors which interact with and transmit the transcriptional activity of the BTEB2 activation domain to basic transcriptional machinery.

Amino Acid Sequence↗
PubMed1997

CBP/p300 functions as a possible transcriptional coactivator of Ah receptor nuclear translocator (Arnt).

A heterodimer of AhR (aryl hydrocarbon receptor) and Arnt (AhR nuclear translocator) conveys a transactivation signal of aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the genes for a group of drug-metabolizing enzymes. This inducible expression of the genes is inhibited by adenovirus E1A, suggesting that CBP/p300 is somehow involved in the transactivation of the genes by the AhR and Arnt heterodimer. Yeast and mammalian two hybrid systems revealed that CBP/p300 interacted with the transactivation domain of Arnt, but not with that of AhR, via the CREB-binding domain. The pull down assay using GST-Arnt hybrid protein confirmed the interaction between Arnt and CBP/p300. Considering these results and that Arnt or Arnt2 functions as a common partner in the formation of transcriptional regulators with other bHLH/PAS proteins such as AhR, HLF, and HIF-1alpha, the possibility arises that CBP/p300 is extensively involved as a coactivator in the transactivation process by bHLH/PAS (a conserved sequence motif among Per, Arnt, and Sim) heterodimer transcription factors through interaction with Arnt or Arnt2.

Acetyltransferases↗
PubMed1997

Ah receptor, a novel ligand-activated transcription factor.

The aryl hydrocarbon receptor (AhR) is widely distributed in vertebrates and is known to be involved in metabolism of xenobiotics including man-made chemicals, most of which act as a ligand for the receptor, although no endogenous ligand has yet been known. Upon binding a ligand, the receptor is activated to translocate to the nuclei, and during the nuclear translocation process, it is dissociated from the 90 kDa heat shock protein (Hsp90) to form a heterodimer with Arnt (Ah receptor nuclear translocator). The heterodimer complex binds a DNA response element termed xenobiotic responsive element (XRE) localized upstream of the target genes of many drug-metabolizing enzymes including cytochrome P4501A1 and glutathione S-transferase to activate their transcription. Recent cDNA cloning has revealed that the AhR, like Arnt, possesses characteristic structural motifs of basic helix-loop-helix and PAS domains responsible for DNA recognition, heterodimerization, and ligand binding, and functions as a novel receptor-type transcription factor.

Animals↗