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Biomedical subjects

M Obinata

Publications and source records attributed to M Obinata.

At least 91 records · Page 5Linked to original sources
PubMed1995

Differentiation capacity toward mesenchymal cell lineages of bone marrow stromal cells established from temperature-sensitive SV40 T-antigen gene transgenic mouse.

Stromal cell lines were established from bone marrow of temperature-sensitive T-antigen gene transgenic mice. These stromal cell lines consisted of fibroblasts, endothelial cells, and preadipocytes. We found that these stromal cell lines exhibited phenotypic changes depending on the inactivation of T-antigen and growth condition; one preadipocyte line was induced toward adipocytes and osteogenic cells, and several preadipocyte and endothelial cell lines were induced toward muscle cells and adipocytes. Some cell lines showed bipotential characters. These results indicated that stromal cells consisting of bone marrow hematopoietic microenvironment are derived from multipotent mesenchymal stem cells.

Adipocytes↗
PubMed1995

Properties of incompletely immortalized cell lines generated from a line established from temperature-sensitive SV40 T-antigen gene transgenic mice.

Kidney tubule cell line (TKC2) established from temperature-sensitive (ts) SV40 T-antigen gene transgenic mice exhibited cell death by apoptosis at nonpermissive temperature (39 degrees C). To learn how immortalization occurs, we generated T-antigen-independent immortalized cells (resistant clones) by culturing TKC2 cells at 39 degrees C. These independently isolated resistant clones exhibited ts properties in T-antigen and contained only wild-type p53. They grew similarly at 39 degrees and at 33 degrees C, but cell death was induced soon after they reached confluency at 39 degrees C. However, cell death was equally prevented by transfecting the bcl-2 gene. While TGF-beta showed no growth-suppressive effect in the sensitive and resistant clones at 33 degrees C, it induced significantly earlier cell death in the resistant clones at 39 degrees C. Thus, the resistant clones may be incompletely immortalized cell lines which induce cell death in response to the growth-suppressive effects of contact inhibition.

Animals↗
PubMed1995

Inducible expression of erythroid-specific mouse glycophorin gene is regulated by proximal elements and locus control region-like sequence.

Cis-acting elements of the gene for mouse glycophorin, an erythroid-specific membrane glycoprotein, were determined by transient and stable transfection assays using murine erythroleukemia (MEL) cells. Cis-acting elements proximal to the transcription start site of the gene can be separated into the basal promoter (-1 to 191 bp) and the distal element (-133 to -92). The basal promoter contained GGTGG and GATA motifs and the distal element contained GATA-1 and NF-E2 motifs. Deletion analysis of the distal GATA site and its neighboring sequence and DNase-I footprinting/EMSA (electrophoretic mobility shift assay) analysis indicated that induced nuclear factor binding to GATA-1 and its neighboring sequence may be required for expression during MEL cell differentiation induced by dimethyl sulfoxide treatment. The NF-E2 site was also shown to be essential for the promoter activity. An approximately 400 bp far upstream region (-1325 to -948bp) containing the binding motifs for GGGTGG, GATA-1 and NF-E2 showed no enhancing activity when this region was examined by transient transfection assay, but it did show enhancement of the differentiation-specific promoter activity in the stable transfection assay. The far upstream region of mouse glycophorin gene may have a function similar to that of the locus control region (LCR) of human beta-globulin gene cluster.

Animals↗
PubMed1995

Establishment and characterization of bone marrow stromal cell lines that support osteoclastogenesis.

We established bone marrow stromal cell lines that support tartrate-resistant acid phosphatase-positive multinucleated cell [TRAcP(+)MNC] formation by using transgenic mice harboring simian virus 40 large T antigen gene. The morphology of these TM cell lines (large T-immortalized marrow cells) was spindle-like at sparse cell density, whereas it became smaller and cuboidal at confluence. The TM cell lines showed diverse ranges of activity in supporting TRAcP(+)MNC formation when they were examined in the cocultures with spleen cells in the presence of 1 alpha,25-dihydroxyvitamin D3. Among these cell lines, TM8 supported the TRAcP(+)MNC formation most efficiently (from 400-1500 cells/well) when cocultured with spleen cells. Another bone marrow-derived cell line, TM5, supported TRAcP(+)MNC formation in the coculture assay, whereas the efficiency was approximately one fifth that of TM8. Interestingly, TM8 cells also supported TRAcP(+)MNC formation even in the cocultures at low serum concentration (0.5% fetal bovine serum) with an efficiency yielding over 200 TRAcP(+)MNCs/well. TM8 cells expressed certain levels of macrophage colony-stimulating factor and stem cell factor messenger RNAs (mRNAs), but low levels of c-fms mRNA. Expression of c-kit mRNA in TM5 and TM8 cells was undetectable. 1 alpha,25-Dihydroxyvitamin D3 treatment enhanced the expression of osteopontin mRNA more than 10-fold in these cells, indicating the presence of the receptor for this steroid. These TRAcP(+)MNCs, which developed in the cocultures of the TM8 and spleen cells, formed pits when cultured on bone slices, indicating that they were capable of resorbing bone. The various levels of expression of these genes and the difference in the supporting activities for the TRAcP(+)MNC development in the diverse TM cell lines suggest the heterogeneity in the marrow cell populations in vivo regarding their activity in supporting osteoclastogenesis.

Acid Phosphatase↗
PubMed1995

Establishment of vasopressin-responsive early proximal tubular cell lines derived from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

A little is known about vasopressin receptor in early proximal tubule (S1). The purpose of this study is to establish the vasopressin-responsive S1 cell line derived from transgenic mice harboring temperature-sensitive(ts) simian virus (SV) 40 large T-antigen gene. The cells showed a temperature-sensitive cell growth characteristic of encoding tsSV40. The S1 cells retained a unique morphology specific to proximal tubule. The cells showed the vasopressin-induced increase in intracellular calcium concentration ([Ca2+]i) mediated by both V1a and the putative Vp receptor. Of nineteen clonal cell lines established from the parental cells, three expressed only V1a receptor, and five retained both V1a and Vp receptor. In conclusion, these immortalized S1 cell lines may be useful for studying vasopressin receptor subtypes in S1.

Animals↗
PubMed1994

Different cellular mechanisms of vasopressin receptor V1 and V2 subtype in vasopressin-induced adenosine 3', 5'-monophosphate formation in an immortalized renal tubule cell line, TKC2.

Vasopressin (VP) stimulates adenosine 3',5'-monophosphate (cAMP) formation in an immortalized renal tubule cell line, TKC2, which is derived from transgenic mouse harboring temperature-sensitive SV40 T-antigen gene. VP (10(-8) M)-induced cAMP formation was significantly attenuated by either non-peptide vasopressin receptor V1 or V2 subtype antagonist, OPC-21268 (10(-8) and 10(-6) M) or OPC-31260 (10(-8) and 10(-6) M), respectively, and it was completely abolished by combination of both agents (10(-6) M). VP (10(-8) M) also induced an increase in cytosolic free Ca2+ and prostaglandin (PG) E2 synthesis, both of which were significantly inhibited by OPC-21268 (10(-8) M), but not by OPC-31260 (10(-6) M). Either OPC-21268 (10(-8) M), depletion of extracellular Ca2+ or inhibition of cyclooxygenase attenuated both VP-induced PGE2 synthesis and cAMP formation. In conclusion, both V1 and V2 receptors can stimulate cAMP formation. V1 receptor, however, stimulates cAMP formation via Ca(2+)-dependent PGE2 synthesis, whereas V2 receptor may stimulate it directly.

Animals↗
PubMed1994

Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis.

Adhesion molecules are required for development of hematopoietic stem and progenitor cells in the respective hematopoietic microenvironments. We previously showed that development of the erythroid progenitor cells is dependent on their direct adhesion to the stroma cells established from the erythropoietic organs. In this stroma-dependent erythropoiesis, we examined the role of adhesion molecules in erythropoiesis by blocking antibodies. The development of the erythroid cells on stroma cells was inhibited by anti-very late activation antigen-4 (VLA-4 integrin) antibody, but not by anti-VLA-5 antibody, although the erythroid cells express both VLA-4 and VLA-5. Whereas high levels of expression of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, ligands for VLA-4, were detected in the stroma cells, the adhesion and development of the erythroid progenitor cells were partly inhibited by the blocking antibody against VCAM-1. VLA-5 and fibronectin could mediate adhesion of the erythroid progenitor cells to the stromal cells, but the adhesion itself may not be sufficient for the stroma-supported erythropoiesis. The stromal cells may support erythroid development by the adhesion through a new ligand molecule(s) for VLA-4 in addition to VCAM-1, and such collaborative interaction may provide adequate signaling for the erythroid progenitor cells in the erythropoietic microenvironment.

Animals↗
PubMed1994

The helix-loop-helix protein Id inhibits differentiation of murine erythroleukemia cells.

Id is considered to be a negative regulator of basic helix-loop-helix proteins, which play important roles in cell type-specific transcription and cell lineage commitment. The Id gene was first cloned in murine erythroleukemia (MEL) cells, which can be induced to differentiate toward erythrocytes with Me2SO, and its mRNA decreases after differentiation in various types of cells. In this report, we demonstrate that overexpression of Id interferes with MEL cell differentiation and that inhibition of differentiation is accompanied by reduction in expression of three erythroid-specific genes. While down-regulation of Id is an early event in the differentiation process of MEL cells, E-box binding activity of these cells increases only at a later stage of differentiation, and this late increase is reduced by the overexpression of Id in the early stage. Sequential changes in the activity of several basic helix-loop-helix proteins thus appeared to be involved in erythroid differentiation.

Animals↗
PubMed1994

Apoptosis is induced at nonpermissive temperature by a transient increase in p53 in cell lines immortalized with temperature-sensitive SV40 large T-antigen gene.

Hepatocyte (TLR2) and kidney tubule (TKC2) cell lines established from temperature-sensitive (ts) SV40 T-antigen gene transgenic mice not only were arrested in growth, but also exhibited cell death at nonpermissive temperature (39 degrees C). The cell death was determined to be caused by apoptosis from observations of nuclear fragmentation and DNA fragmentation. These cell lines contained relatively high levels of wild-type p53 which formed complexes with T-antigen at permissive temperature (33 degrees C), but after shift to a nonpermissive temperature, the inactivation of T-antigens led to the liberation of an abundance of p53 proteins from the complexes, apparently inducing apoptosis.

Animals↗
PubMed1994

Structural organization of the mouse glycophorin A gene.

The human glycophorin gene has been extensively studied, but information on the homologous gene from other species has been unavailable. Here, we determined the structural organization of mouse glycophorin A gene and compared it with the human gene. The mouse glycophorin gene is a single copy gene while in humans, there are two highly related genes (A and B) that were generated by homologous recombination. Chromosomal mapping indicated that the mouse gene is located in the central region of the mouse chromosome 8, which is syntenic with human chromosomes 4q28-31 where the human glycophorin A gene has been mapped. The mouse gene consists of 8 exons, while the human gene consists of 7 exons and the length of each exon is quite short except for the last exon. The last 4 exons showed extensive homology between the mouse and human genes but divergence in the 5'-exons of the two genes was high. The results suggest that glycophorin genes of mouse and human may have been generated from the same ancestor, but diverged greatly during evolution. The upstream regulatory region of the mouse gene consists of multiple motifs for DNA binding factors that may be required for its erythroid-specific expression.

Animals↗
PubMed1994

The s-Myc protein having the ability to induce apoptosis is selectively expressed in rat embryo chondrocytes.

Gene transfection experiments demonstrated that over-expression of the s-myc gene under the control of a human metallothionein promoter induced apoptosis in cells such as rat and human glioma cells. In contrast to c-Myc-mediated apoptosis requiring withdrawal of serum growth factors, s-myc expression induced apoptosis in glioma cells in the presence of 10% fetal calf serum. Whereas, s-Myc-mediated apoptosis was suppressed in proportion to the increase of bcl-2 expression as seen in c-Myc mediated apoptosis. The s-myc gene was expressed in rat embryo cells being committed to differentiate to hypertrophic chondrocytes which undergo programmed cell death. CAT assay demonstrated that in the NH2-terminal region, the s-Myc protein contains a domain structure required for expression of transactivation activity that is approximately six times higher than that of c-Myc. Therefore, these findings strongly suggest that s-Myc may play an important role in transcription regulation of a set of genes whose expression induces programmed cell death in vitro and in vivo.

3T3 Cells↗
PubMed1993

Erythropoietin as a mitogen for fetal liver stromal cells which support erythropoiesis.

Fetal liver stromal cells established from 13-day-old embryos support erythropoiesis in vitro in combination with erythropoietin (Epo). Epo stimulated growth of these stromal cells in a dose-dependent manner and mRNA for Epo receptor was detected. These results suggest the possible involvement of Epo in the generation of the fetal erythropoietic microenvironment during development.

Animals↗
PubMed1993

Establishment of gastric surface mucous cell lines from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

We succeeded in establishing two gastric surface mucous cell lines (designated as GSM06 and GSM10), which produce periodic acid-Schiff (PAS)- and concanavalin A (Con A)-positive glycoproteins, from a primary culture of gastric fundic mucosal cells of adult transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. At the permissive temperature (33 degrees C), GSM06 and GSM10 cells grew until confluent monolayers were formed and have now been in culture for more than 9 months with regular passaging. Con A-horseradish peroxidase- and PAS-positive staining indicated that these cells retain the characteristics of gastric surface mucous cells. GSM06 cells showed temperature-sensitive growth in culture and expressed SV40 large T-antigen at the permissive temperature but not at a nonpermissive temperature (39 degrees C). In contrast, GSM10 cells showed temperature-insensitive growth and expressed T-antigen at both permissive and nonpermissive temperatures. To our knowledge, this is the first report of the establishment of gastric surface mucous cell lines from animals. These immortalized cell lines with normal characteristics may serve as good experimental models for the basic and applied biology of gastric surface mucous cells.

Animals↗
PubMed1993

Coordinated expression of messenger RNAs for nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in the rat hippocampus following transient forebrain ischemia.

Changes in nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 messenger RNA expression in the rat hippocampus following 20 min of transient forebrain ischemia were evaluated using Northern blot analysis and in situ hybridization histochemistry. Twelve hours after the insult, the level of nerve growth factor messenger RNA increased markedly in the granular cell layer of the dentate gyrus and by day 2 returned to control levels. The level of brain-derived neurotrophic factor messenger RNA showed a persistent and moderate increase. The highest expression of brain-derived neurotrophic factor messenger RNA was seen in the dentate granule cells on day 2 after the insult, and then the expression returned to the control levels. At 2 days post-ischemia, contents of messenger RNAs for nerve growth factor and brain-derived neurotrophic factor were reduced in the CA1 region, which may represent delayed loss of vulnerable CA1 pyramidal neurons. In contrast to brain-derived neurotrophic factor and nerve growth factor messenger RNA expression, the level of neurotrophin-3 messenger RNA declined in the CA1, the CA2 and the dentate granular layer immediately after ischemic insult. In the CA1 region, the reduced expression persisted for at least seven days, but in the dentate gyrus, neurotrophin-3 messenger RNA expression returned to the control levels after two days of post-ischemic recovery. These results suggest that nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 genes are differentially regulated and that each of their gene products may play different roles in the central nervous system under pathophysiological conditions.

Animals↗
PubMed1993

c-Myc selectively regulates the latent period and erythroid-specific genes in murine erythroleukemia cell differentiation.

During the latent period of murine erythroleukemia (MEL) cell differentiation, c-myc levels showed a significant change and the overexpression of the transferred c-myc gene inhibited the commitment and differentiation of MEL cells, suggesting that c-Myc may be a key molecule for the commitment. Since c-Myc may function as a DNA binding transcription factor, we examined whether c-Myc regulates the latent period genes (hsp and hsc70, MER5, Id and Spi-1 genes) and the erythroid-specific genes [beta-globin, glycophorin, delta-aminolevulinic acid synthase (ALAS-E), GATA-1 and erythropoietin receptor (EpoR)] in the MEL cell transformant having transferred c-myc gene. The overexpression of c-myc gene affected the latent period genes in different ways: hsc and hsp 70 genes and Id gene were positively regulated, while expression of MER5 gene was repressed. While c-myc is thought to be involved in DNA replication, its overexpression showed no effect on the expression of proliferating cell specific nuclear antigen or DNA polymerase a. The overexpression of c-myc repressed the expression of glycophorin, ALAS-E and beta-globin genes, of the five erythroid-specific genes, but had no effect on expression of GATA-1 or EpoR gene. These results suggest that c-Myc differentially regulates the expression of the latent period and erythroid-specific genes.

Animals↗
PubMed1993

Functional domains of c-Myc involved in the commitment and differentiation of murine erythroleukemia cells.

In the early event of the induction of mouse erythroleukemia (MEL) cell differentiation, c-myc mRNA levels show a drastic change. The elevated expression of a transfected c-myc gene inhibits the commitment and differentiation of MEL cell transformants. In the present work, we have introduced human c-myc mutants into MEL cells under the inducible promoter to define the functional domains of c-Myc involved in erythroid differentiation. The c-Myc domains necessary for commitment and differentiation are not co-localized; almost entire regions are required for inhibition of commitment, whereas domains II and IV that are essential for co-transforming activity with ras are required for inhibition of differentiation. Interestingly, mutants that delete domains for c-Myc dimerization motifs enhanced differentiation. These results suggest that c-Myc may regulate commitment and differentiation by interacting with proteins through different domains.

Animals↗