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A Hatamochi

Publications and source records attributed to A Hatamochi.

At least 55 records · Page 3Linked to original sources
PubMed1991

Analysis of methylation in the c-MYC gene in five human myeloma cell lines.

Genomic alterations of the human c-MYC gene were analysed in five human myeloma cell lines established in Kawasaki Medical School and compared with those of normal lymphocytes, Raji cells from Burkitt's lymphoma, and an Epstein-Barr virus positive lymphoblastoid cell line (LCL). Although no structural chromosome aberrations at 8q24, the c-MYC locus, were distinct, the mRNA level of c-MYC in these myeloma cell lines was 30-50-fold that in normal peripheral blood lymphocytes. Regarding the methylation of c-MYC, DNAs of the myeloma cell lines were digested with MspI plus EcoRI or HpaII plus EcoRI, and hybridized with three genomic 32P-labelled probes; the first, second and third exons of the human c-MYC gene, respectively. The extent of methylation in cytosine at a single CCGG site in the third exon substantially decreased in these myeloma cell lines as compared with that in normal tonsillar B, LCL and Raji cells. No significant differences in hypomethylation between these myeloma, normal B, LCL and Raji cells was detected in the first and second exon of c-MYC. These results suggest that the hypomethylation in the third exon of c-MYC might be related to the enhanced expression of c-MYC in these human myeloma cell lines.

Aged↗
PubMed1990

Increased collagen synthesis accompanying elevated m-RNA levels in cultured Werner's syndrome fibroblasts.

Although Werner's syndrome (WS) is a premature aging disease and its fibroblasts typically grow poorly in culture, WS may cause abnormalities in connective tissue metabolism that are seldom seen in normal aging, such as scleroderma-like skin. In a preliminary report, we described increased collagen synthesis in fibroblasts derived from two WS patients. The present study was undertaken to determine the degree of the regulation of collagen gene expression in dermal fibroblasts from two other patients. Overproduction of collagenase sensitive protein was observed in WS fibroblasts. Collagen m-RNA levels, that were determined by hybridization of RNA blots with specific cDNA were about 2 times greater than those in the control cells. These results suggest that control of collagen synthesis in WS fibroblasts is altered at the transcriptional level.

Cells, Cultured↗
PubMed1989

Collagenase gene expression in fibroblasts is regulated by a three-dimensional contact with collagen.

Collagenase activity in fibroblasts is regulated by cytokines and the interaction with the extracellular matrix. In this study we demonstrate that fibroblasts cultured within a three-dimensional collagen gel show a strong induction of collagenase gene expression. In addition to increased de novo synthesis most of the secreted enzyme was found to be activated leading to a high collagenolytic activity and complete degradation of collagen matrices after removal of fetal calf serum. Collagen I gene expression was found to be reduced under these conditions. These data suggest a specific modulation of cellular metabolism in response to contact with a three-dimensional collagenous matrix resulting in the divergent regulation of collagen and collagenase.

Adult↗
PubMed1989

Down-regulation of alpha 3(VI) chain expression by gamma-interferon decreases synthesis and deposition of collagen type VI.

Treatment of cultured human skin fibroblasts with increasing doses of gamma-interferon produces a distinct reduction of steady-state levels of the alpha 3 chain of collagen VI mRNA by about 60% but not of the alpha 1 and alpha 2 chain mRNAs. A similar decrease was also observed for collagen I and III mRNA while fibronectin mRNA remained at the same level. The decrease in alpha 3(VI) mRNA is accompanied by a reduced synthesis of collagen VI and by a reduced deposition of both collagen VI and fibronectin in urea-insoluble form in the cell matrix. No other gamma-interferon effects were observed for fibronectin biosynthesis. Immunoprecipitation of metabolically labeled collagen VI demonstrated a strongly reduced synthesis (by 65-80%) of intracellular alpha 3(VI) chains with no decrease found for alpha 1(VI) and alpha 2(VI) chains. All three chains were, however, found to be reduced in the culture medium. Pepsin treatment of immunoprecipitated collagen VI showed similar chain ratios for material in the culture medium obtained in the absence or presence of gamma-interferon. It indicates that correctly assembled heterotrimers of the composition [alpha 1(VI) alpha 2(VI) alpha 3(VI)] are formed and secreted also in the absence of an equivalent alpha 3(VI) chain synthesis but at a reduced rate. The data support previous predictions from sequence analyses [Chu et al. (1988) J. Biol. Chem. 263, 18,601-18,606] that collagen VI molecules composed of all three constituent chains are more stable than other assembly alternatives.

Actins↗
PubMed1989

Regulation of collagen VI expression in fibroblasts. Effects of cell density, cell-matrix interactions, and chemical transformation.

Collagen VI expression was studied in cultured human skin fibroblasts and mouse 3T3 cells using cDNA probes specific for alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. A 2-3-fold increase of these mRNAs was observed when fibroblasts were grown at increasing densities while only minimal changes occurred for the mRNA levels of collagens I and III, fibronectin, and beta-actin. Changes in mRNA correlated well with an increased production of corresponding proteins as determined by immunological assays. A comparable increase of alpha 1(VI) and alpha 2(VI) but not of alpha 3(VI) chain mRNAs was found for fibroblasts grown in a three-dimensional collagen gel after gel contraction. These conditions resulted, however, in a decrease of steady-state levels of collagens I and III and actin mRNAs. Transformation of 3T3 cells by phorbol ester did not change collagen VI mRNAs but caused a 3-5-fold reduction in mRNA levels for the other extracellular matrix proteins. These data strongly imply different regulatory mechanisms for the expression of collagen VI compared with collagens I and III and fibronectin. The differences may be correlated to changes in cell shape and reflect the requirement for collagen VI as a cell-binding protein.

Actins↗
PubMed1989

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on collagen synthesis of human skin fibroblasts in vitro.

The effect of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on collagen metabolism by human diploid fibroblasts in confluent monolayer culture was examined. Recombinant TNF alpha reduced collagen mRNA levels 2-fold and stimulated collagenase mRNA levels 5-fold, while recombinant IFN gamma affected only collagen mRNA levels. The combination of TNF alpha (10 ng/ml) and IFN gamma (100 ng/ml) resulted in a much stronger (about 30-fold) reduction of collagen mRNA levels indicating that the two cytokines act synergistically. In contrast no such synergism was observed with respect to collagenase mRNA levels. The effect of TNF alpha and IFN gamma on collagen metabolism reported here indicates a complex interaction of different cytokines in the control of tissue remodeling that occurs during inflammation, repair, or atrophy.

Adult↗
PubMed1989

Localization of collagen alpha 1(I) gene expression during wound healing by in situ hybridization.

The cellular localization of collagen alpha 1(I) chain gene expression during wound healing in rats was investigated using in situ hybridization. Activation of collagen gene expression was first found within fibroblastic cells around the deep layers of the granulation tissue as early as 16 h post wounding. Heavily labeled cells were also detected near the intact wound edge and around hair follicles. At day 6 intense alpha 1(I) collagen gene expression was found within most cells of the granulation tissue and after day 8 most of the activity was localized to cells directly underneath the epidermis. 26 d after the induction of the wounds hardly any alpha 1(I) collagen gene expression could be demonstrated, which indicates a close, time-dependent control of collagen synthesis during repair processes.

Animals↗
PubMed1988

Influence of corticosteroids on chemotactic response and collagen metabolism of human skin fibroblasts.

Following chronic administration of corticosteroids in vivo, a number of complications occur, which mainly involve the metabolism of connective tissue cells. Therefore, several attempts have been made to develop corticosteroids, which show less pronounced side effects. Fibroblasts were kept in monolayer cultures and were exposed to corticosteroids demonstrating similar anti-inflammatory activity (prednicarbate, desoximetasone). Chemotaxis of fibroblasts was studied over 4 hr, protein and collagen synthesis were estimated using proteinchemical methods and also by dot blot hybridization. Corticosteroids used in a high dosage (10 microM) affected all biosynthetic capacities of the investigated fibroblasts. Protein synthesis and production of collagen types I and III were reduced and a similar decrease of mRNA levels for collagen type I could be found indicating an influence on the pretranslational control. In the same concentrations desoximetasone was much more active than prednicarbate. Fibroblast migration was dosage dependently inhibited from 10(-9) M to 10(-5) M for desoximetasone, while incubation with prednicarbate did not cause a reduction of the chemotactic response at concentrations lower than 10(-7) M. These data suggest that modifications of corticosteroids might result in a dissociation of some of their biological activities and can specifically influence their effects on biosynthetic capacities of fibroblasts.

Adrenal Cortex Hormones↗
PubMed1988

A CCAAT DNA binding factor consisting of two different components that are both required for DNA binding.

A CCAAT-binding activity present in nuclear extracts of rat liver and NIH 3T3 fibroblasts was purified using, as assay, DNA binding to a segment of the mouse alpha 2(I) collagen promoter. The activity consists of two components, designated factors A and B, which are separated by ion exchange chromatography on either Mono Q or Mono S columns. Factor A is heat-sensitive, whereas factor B is heat-resistant. Both factors are required for DNA binding and both are present in the DNA protein complex. The A + B complex was extensively purified by heparin-agarose and sequence-specific affinity chromatography. The Mr of factor A is 39,000, whereas the Mr of factor B is 41,000 as determined by renaturation of a highly purified preparation after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Competition experiments indicate that this CCAAT-binding complex has a DNA sequence specificity that is different from those of other CCAAT-binding proteins.

Animals↗
PubMed1988

Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel.

Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.

Blotting, Northern↗
PubMed1987

Separate binding sites for nuclear factor 1 and a CCAAT DNA binding factor in the mouse alpha 2(I) collagen promoter.

A factor present in nuclear extracts of NIH-3T3 fibroblasts was identified which binds to a region between -315 and -295 with respect to the start of transcription in the mouse alpha 2(I) collagen promoter. This factor was assayed using three different DNA binding assays. Evidence that this factor is nuclear factor 1 or a related protein is based on competition experiments using fragments of another promoter that contains a nuclear factor 1 binding site as well as a fragment containing a consensus sequence for nuclear factor 1 binding sites. The nucleotide sequence of the binding site in the alpha 2(I) promoter. TCGN5GCCAA, presents similarities to the sequence published previously as a consensus recognition sequence for nuclear factor 1. Methylation interference experiments indicate that the factor interacts at least with two successive G residues in the major groove complementary to the two successive C residues in the GCCAA motif. This factor is different from a factor that binds to a CCAAT element between -84 and -80 in the mouse alpha 2(I) collagen promoter. This was shown by competition experiments using both crude NIH-3T3 fibroblast nuclear extracts and a partially purified CCAAT binding factor. These results suggest that in the alpha 2(I) collagen promoter nuclear factor 1 and a CCAAT binding factor bind to separate sites, despite analogies in their recognition sequences.

Animals↗
PubMed1986

Differential binding of a CCAAT DNA binding factor to the promoters of the mouse alpha 2(I) and alpha 1(III) collagen genes.

An exonuclease III assay (Wu, C. (1985) Nature 317, 84-87) was used to identify in nuclear extracts of NIH 3T3 cells a factor which binds to the CCAAT segment of the alpha 2(I) collagen promoter between -80 and -84. This sequence is located on the coding strand in the alpha 2(I) collagen promoter. Binding is specific since only promoter fragments which contain the CCAAT box sequences on one or the other DNA strand inhibit binding to the alpha 2(I) collagen CCAAT box. The CCAAT binding factor protects approximately 26 base pairs of the alpha 2(I) collagen promoter from exonuclease III digestion. Binding to the alpha 2(I) collagen promoter CCAAT box is not inhibited by a fragment of the alpha 1(III) collagen promoter (from -396 to +16), which does not contain a CCAAT sequence on either one or the other strand. Our data suggest that two genes such as the alpha 2(I) and alpha 1(III) collagen genes, which are coordinately expressed in many tissues, are not necessarily regulated by the same trans-acting DNA binding proteins.

Animals↗
PubMed1985

Effects of histamine on collagen synthesis by cultured fibroblasts derived from guinea pig skin.

Fibroblast-like cells derived from guinea-pig skin were cultured for 3 h in the presence of various concentrations of histamine. The total protein synthesized was determined by the incorporation of radioactive proline, and the collagenous protein synthesized was measured by the incorporation of labeled hydroxyproline in the cell layer and medium. Synthesis of both total and collagenous protein increased in the presence of histamine in the concentration range of 10(1)-10(3) micrograms/ml. The ratio of collagen to total protein synthesized also increased at these concentrations. However, in no case was an increase found when H1 antagonist (chlorpheniramine) and H2 antagonist (cimetidine) were added with the histamine. DNA synthesis was not affected by histamine at the concentrations used. These results suggest that histamine increases the synthesis of collagen by fibroblast-like cells through H1 and H2-receptors.

Animals↗
PubMed1985

Increased number of OKT6-positive dendritic cells in the hair follicles of patients with alopecia areata.

In 6 patients with untreated alopecia areata in the progressive stage, 6 in the stationary stage, and 6 normal individuals as controls, an in situ analysis of OKT6-positive dendritic cells in hair follicles, and peribulbar and intrabulbar infiltrates was performed using the avidin-biotin-peroxidase method with monoclonal antibodies. In controls, OKT6-positive dendritic cells were distributed only in the upper portions of hair follicles and were not observed in the bulbar area, and the percentage of these cells among all epithelial cells of the hair follicles was 1.0 +/- 0.1% (mean +/- SE). In stationary-stage patients, the distribution and the percentage of positive cells were the same as those for the controls (1.1 +/- 0.1%). In the progressive stage, however, positive cells were distributed in both the upper portions of the hair follicles and the bulbar area, and the percentage of positive cells (4.9 +/- 0.3%) was significantly higher than that of controls. Staining for T, B lymphocytes and T cell subsets in the peribulbar infiltrates revealed a predominance of OKT4-positive cells (the OKT4/OKT8 ratio was from 3:1 to 4:1). This indicates that the number of OKT6-positive dendritic cells increases in the hair follicles of progressive alopecia areata and that these cells may play an important role in cooperation with T cells in the pathogenesis of alopecia areata.

Alopecia Areata↗