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Biomedical subjects

M Obinata

Publications and source records attributed to M Obinata.

At least 37 records · Page 2Linked to original sources
PubMed2000

Multipotency of a bone marrow stromal cell line, TBR31-2, established from ts-SV40 T antigen gene transgenic mice.

Bone marrow is believed to contain multipotential stromal stem cells which can differentiate into osteoblasts, chondrocytes, adipocytes, and myoblasts (Prockop, D. J. Science 276, 71-74, 1997). Therefore, characterization and identification of the stem-like cell within the stromal cells are important to understand bone marrow function in relation to the hematopoietic microenvironment, and repair/regeneration of tissue defects. TBR31-2 cell, a bone marrow stromal cell line established from bone marrow of transgenic mice harboring temperature-sensitive (ts) simian virus (SV) 40T-antigen gene for immortality, is induced toward both adipocytic and osteogenic cells under conditions of the inactivation of T-antigen (Okuyama, R., Yanai, N., Obinata, M. Exp. Cell Res. 218, 424-429, 1995). In this work, using a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, mRNA expressions of tissue-specific differentiation markers for adipocyte (lipoprotein lipase), osteoblast (type I collagen and osteocalcin), chondrocyte (type II and X collagen), and muscle cell (desmin) were examined during a long-term culture of the cell. In addition, histochemical studies showed the appearance of adipocytic, osteoblastic, chondrocytic, and muscle cells during this long-term culture. Thus, TBR31-2, which has characteristics of an undifferentiated cell, has the potential to express the multipotential cell lineages. These results indicated that a multipotential progenitor cell including potential to differentiate into a muscle cell and which is situated in the mesenchymal cell lineage was first obtained.

Animals↗
PubMed2000

Apoptosis of fetal testicular cells is regulated by both p53-dependent and independent mechanisms.

A portion of fetal germ cells undergoes apoptosis in the physiological context, but the molecular mechanisms of their apoptosis are largely unknown. Because p53 tumor suppressor gene product promotes apoptosis in various types of cells, we have investigated the expression of p53 in fetal gonads and examined the influence of loss of p53 function in fetal gonad cells using mice deficient in the p53 gene. We found that the expression of p53 protein in fetal testis was induced after 15.5 dpc (days post coitum), while the expression was not detected in fetal ovary. The number of apoptotic cells found in the seminiferous tubules of fetal testes was not significantly different between p53-deficient and wild-type mice until 16.5 dpc. At 17.5 dpc, however, more apoptotic cells were observed in wild-type testes than in the p53-deficient mice. In contrast, a similar number of apoptotic cells was found in fetal ovaries throughout these developmental stages. These observations indicate that p53 promotes apoptosis of fetal testicular cells after 16.5 dpc.

Animals↗
PubMed2000

Self-renewal and differentiation of a novel bipotent myeloid progenitor clone in the stroma-dependent culture.

To understand regulation of myeloid development, it is necessary to obtain the myeloid progenitor cell lines with self-renewal and differentiation capacities. Because prolonged hematopoiesis occurs with the production of myeloid cells at all stages of differentiation in the Dexter-type long-term bone marrow cultures, we tried to obtain stroma-dependent myeloid progenitor cells starting from the long-term bone marrow culture. Murine cobblestone areas generated in long-term bone marrow cultures were serially passaged every 10 days. After 4 months, the resultant hematopoietic cells, designated as DFC, were passaged on a monolayer of established spleen stromal cell line, MSS62. After 10-12 passages of DFC cells on MSS62, several clones were obtained by colony formation on MSS62 cell layer. Among these clones, DFC-a cells could be maintained for a long period by coculturing with the established stromal cell line, MSS62.DFC-a cells proliferated by forming cobblestones and contained blast cells, granulocytes, and macrophages. Cell sorting and coculture experiments indicated that the blast type cells exhibiting c-Kit(+) Gr-1(-) Mac-1(-), stroma-dependently self-renewed, and spontaneously differentiated toward granulocytes (c-Kit(+) Gr-1(+) Mac-1(+)) and macrophages (c-Kit(low/+) Gr-1(-) Mac-1(high)). Although most of DFC-a cells expressed c-Kit, SCF-c-Kit interaction was not always necessary for their growth. In the presence of stromal cells, growth and differentiation of DFC-a cells were stimulated by GM-CSF or IL-3. Without stromal cells, DFC-a was transiently expanded by GM-CSF or IL-3 but could not be maintained constantly by these cytokines. The present study demonstrated that DFC-a is a novel bipotent myeloid progenitor cell clone as a simple model system of stroma-dependent myeloid development. It may reflect distinct properties for the earliest myeloid progenitor cells in vivo. It is of interest to know what signals are provided by MSS62 stromal cells to maintain the myeloid progenitor cells.

Animals↗
PubMed2000

MIDA1 is a sequence specific DNA binding protein with novel DNA binding properties.

BACKGROUND: Id proteins not only regulate cell differentiation negatively, but they also promote growth and apoptosis. To know the mechanism of how Id regulates cell fate, we previously isolated an Id-associating protein, MIDA1, which positively regulates cell growth. Its predicted amino acid sequence contains tryptophan-mediated repeats (Tryp-med repeats) similar to the DNA binding region of the c-Myb oncoprotein. We determined whether MIDA1 can bind to DNA in a sequence specific manner by PCR-assisted binding site selection. RESULTS: We identified a 7-base sequence (GTCAAGC) surrounded by a 1-3 bp palindromic sequence as the DNA sequence recognized by the Tryp-med repeats of MIDA1. This motif is located within the 5'-flanking sequence of several growth regulating genes. Gel shift assays revealed that this sequence and a certain length of flanking DNA are necessary for MIDA1 to bind DNA in a stable manner. Methylation interference and DNase I footprint analysis suggested that the DNA binding of MIDA1 is resistant to DNA methylation and that MIDA1 does not specifically localize on this particular motif. CONCLUSIONS: We concluded that MIDA1 is a novel sequence-specific DNA binding protein with some different properties from the usual transcription factors and that MIDA1 may act as a mediator of Id-mediated growth-promoting function through its DNA binding activity.

Base Sequence↗
PubMed2000

Effect of human granulocyte-macrophage colony stimulating factor (hGM-CSF) on lymphoid and myeloid differentiation of sorted hematopoietic stem cells from hGM-CSF receptor gene transgenic mice.

Bone marrow lineage-negative (Lin(-)) c-Kit(+) Sca-1(+) hematopoietic cells from human GM-CSF receptor gene transgenic mice were cultured on established bone marrow stromal cell (TBR59) layers and on semisolid medium. In the semisolid assay, an increasing number of larger colonies were observed in the presence of hGM-CSF. By coculture with the stromal cells, cobblestones containing myeloid and lymphoid lineages of cells were formed from the stem cell enriched fraction, and addition of hGM-CSF strongly stimulated formation of the cobblestones containing both lineages. Repeating passages of the cobblestones on TBR59 stromal cells in the presence of hGM-CSF gradually decreased cobblestone formation and inversely increased macrophages and granulocytes, while mast cells were generated when the cells derived from the semisolid assay were cultured in a liquid medium containing hGM-CSF. These results consistently suggest that cytokines such as GM-CSF may costimulate the immature hematopoietic cells at their stroma-dependent phase before lineage commitment, and after commitment that occurs by an intrinsic program of the cells, they may stimulate maintenance and maturation of progenitor cells.

Animals↗
PubMed2000

Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene.

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37 degrees C but not at 39 degrees C. However, growth was restored when the temperature of the culture was lowered to 33 degrees C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33 degrees C, there was less of this complex at 37 degrees C and 39 degrees C. TM-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. The alkaline phosphatase and gamma-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 +/- 1.16 mmol/l. Moreover, P-glycoprotein (P-gp) with a molecular weight of -170 kDa was expressed in all TM-BBBs. Both mdr1a and mdr1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 micromol/l verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.

3-O-Methylglucose↗
PubMed2000

Stroma-dependent maintenance of cytokine responsive hematopoietic progenitor cells derived from long-term bone marrow culture.

Hematopoietic cells maintained for long periods on primary cultures of bone marrow stromal cells formed cobblestone colonies (Dexter's long-term bone marrow culture, LTBC). These stably maintained hematopoietic cells (for 4 months) were transferred to a coculture on an established spleen stromal cell line (MSS62), and maintained under stromal cell layer, where they retained their invasive ability in the restricted space between the stromal cell layer and culture substratum (DFC culture). DFC contained lineage-negative (Lin-), c-Kit+, Sca-1- cells and spontaneously produced Mac-1+, Gr-1+ cells. DFC could not grow in the absence of MSS62 stromal cells, although, GM-CSF, IL-3, or IL-7 stimulated its growth. Production of granulocyte and monocytic cells was maintained by GM-CSF or IL-3 while it was decreased by IL-7. RT-PCR analysis showed that the IL-7 responsive cell population expressed early lymphoid markers (Ikaros, Pax-5, Oct-2, Rag-1, TdT, IL-7R and Imu), while lacking expression of receptors for G-CSF (G-CSFR) and for M-CSF (M-CSFR), or myeloperoxidase (MPO). These results suggested that DFC simultaneously contained lymphoid-committed progenitors and myeloid-committed progenitors, and that cytokines may expand their responding progenitor cells under the influence of signals provided by the stromal cells. Such a stromal cell-dependent culture system may be useful to analyze the switching mechanism from constitutive to inducible hematopoiesis in vitro.

Animals↗
PubMed2000

Establishment and characterization of a colonic epithelial cell line MCE301 from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

We produced an immortalized colonic epithelial cell line, MCE301, using fetal mice transgenic for the temperature-sensitive simian virus 40 large T-antigen gene. MCE301 cells showed epithelial-like morphology and maintained tight connections with neighboring cells. The cells grew at a permissive temperature (33 degrees C), but the growth of the cells was significantly prevented at the nonpermissive temperature (39 degrees C). The cells expressed large T-antigen at 33 degrees C but not at 39 degrees C. MCE301 cells were not transformed, as judged by the absence of anchorage-independent growth in soft agar gel and lack of tumor formation in nude mice. Electron microscopic studies showed that the cells formed microvilli-like structures on the cell surface and junctional complexes such as tight junctions and desmosomes between the cells. The cells expressed cytosketal (acidic cytokeratins and actin), basement membrane (laminin and collagen type IV) and junctional complex proteins (ZO-1 and desmoplakin I + II), as judged by specific antibodies. Fetal bovine serum, epidermal growth factor, insulin-like growth factor and insulin significantly increased the cell growth at 33 degrees C. Moreover, MCE301 cells expressed colonic mucin Muc2 mRNA as demonstrated by reverse transcriptase-polymerase chain reaction, indicating that the cells originate from mucus-secreting cells. Alkaline phosphatase, a brush border-associated enzyme, was detected in the cells. Sodium butyrate (2 mM), an inducer of cellular differentiation, markedly elevated alkaline phosphatase activity. Thus, the present mouse colonic epithelial cell line MCE301 possessing these unique characteristics should provide a useful in vitro model of colonic epithelium.

Alkaline Phosphatase↗
PubMed2000

mRna expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting.

Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling-time of about 22-31 hr, but did not grow at 39 degrees C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the gamma-glutamyltranspeptidase activity in TR-BBBs was approximately 13% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB13. Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr 1a, mdr 1b, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-methyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 +/- 1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx and efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

MIDA1, an Id-associating protein, has two distinct DNA binding activities that are converted by the association with Id1: a novel function of Id protein.

Id proteins not only regulate cell differentiation negatively, but they also promote growth, immortalization, and apoptosis. To know the mechanism of how Id regulates cell fate, we previously isolated an Id-associating protein, MIDA1, which positively regulates cell growth (1). Its predicted amino acid sequence consists of a Zuotin (a Z-DNA binding protein in yeast) homology region and tryptophan-mediated repeats (Tryp-med repeats). MIDA1 exhibits a sequence-specific DNA binding activity through the Tryp-med repeats (manuscript in preparation). In this study, we revealed that, like Zuotin, MIDA1 can specifically bind to Z-DNA. This suggested that MIDA is a novel DNA binding protein that has two different DNA binding activities. Furthermore, association of Id1 with MIDA1 stimulated the sequence-specific DNA binding activity, while it inhibited the Z-DNA binding activity. Therefore, we concluded that MIDA1 may act as a mediator of the growth-promoting function of Id, by switching the two DNA binding activities of MIDA1.

Animals↗
PubMed1999

Effects of p51/p63 missense mutations on transcriptional activities of p53 downstream gene promoters.

The p51/p63 gene is a homologue of p53, the product of which acts as a transcriptional activator by binding to p53-responsive elements in the promoter regions of several p53 downstream genes. Recently, we identified four distinct mutations in the p51/p63 gene after screening >200 human tumors and cell lines. Because all of the detected p51/p63 mutations were missense mutations, the pathogenic effect of these mutations is difficult to determine without performing a functional analysis. In this study, we examined the transcriptional activity of tumor-derived p51/p63 missense mutations using a yeast-based assay and compared the data with that of artificial p51/p63 missense mutations at residues corresponding to the positions and substituted residues of p53 mutation "hotspots." Although most of the p51/p63 missense mutations at the p53 hotspot residues were unable to transactivate the promoters used in this study, the tumor-derived p51/p63 missense mutations retained their ability to transactivate the MDM2 and/or the BAX promoter but not the p21/WAF1 promoter. These results suggest that the p51/p63 mutation might be involved in an unknown tumor suppression pathway distinct from that of p53.

Amino Acid Sequence↗
PubMed1999

Members of the ErbB receptor tyrosine kinases are involved in germ cell development in fetal mouse gonads.

To isolate the genes involved in mouse primordial germ cell (PGC) development, we carried out subtraction cDNA cloning between PGC-derived embryonic germ (EG) cells and inner cell mass-derived embryonic stem cells. Among the genes preferentially expressed in EG cells, we found a gene encoding a receptor tyrosine kinase ErbB3. By in situ hybridization and immunohistochemical staining, the expression of ErbB3 as well as that of ErbB2, a coreceptor for ErbB3, was detected in PGCs in genital ridges at 12.5 dpc (days postcoitum). The expression was, however, downregulated at 14.5 dpc when the PGCs underwent growth cessation. Neuregulin-beta, a ligand for ErbB2 and ErbB3, was also expressed in genital ridges. In addition, a recombinant Neuregulin-beta enhanced the number of PGCs in 12.5-dpc embryos in culture. Taken together, these observations suggest that ErbB signaling controls the growth or survival of PGCs in genital ridges.

Animals↗
PubMed1999

Alteration of the increase in intracellular [Ca(2+)] in proliferating smooth muscle cells.

Proliferation of smooth muscle cells (SMC) has a role in the development of cardiovascular diseases. We investigated the alteration of contractile signals in proliferating SMC by measuring the increase in intracellular [Ca(2+)] to endothelin-1 (ET-1), noradrenaline (NA), or angiotensin II (AgII). We found that the increase in intracellular [Ca(2+)] by NA or ET-1 decreased in proliferating SMC in comparison to growth-arrested SMC. The increase in intracellular [Ca(2+)] by AgII was stable between the cells. Immunoblotting of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) which are responsible for the mobilization of Ca(2+) by those vasoactive substances revealed that expression of IP(3)R type 1 and type 2 was decreased. Expression of IP(3)R type 3 was increased. The altered Ca(2+) signaling by the cell growth might involve the expression of IP(3)R subtypes.

Animals↗
PubMed1999

Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture.

Induction of hematopoiesis in an embryonic germ (EG) cell line derived from mouse primordial germ cells (PGCs) was examined. When single undifferentiated EG-1 cells were inoculated directly into the methylcellulose medium, both primitive and definitive erythropoiesis were seen in embryoid bodies derived from the EG cells as observed in ES cells, and production of myeloid cell lineages was stimulated by IL-3. These results indicate that EG cells acquired in vitro potency to differentiate toward hematopoietic cells, although they were derived from PGC and are distinct from inner cell mass-derived ES cells with regard to gene expression and patterns of DNA methylation corresponding to genomic imprinting. It turns out that they are useful for study of cell differentiation in the animals whose ES cells are not available.

Animals↗
PubMed1999

The transcriptional activities of p53 and its homologue p51/p63: similarities and differences.

p51/p63 is a novel p53 homologue that has been shown to act as a transcriptional activator through the p53-binding sequence of the p21/WAF1 promoter and to induce apoptosis when it is expressed transiently in a human tumor cell line. We developed transcription assay systems for these two related genes in both Saccharomyces cerevisiae and mammalian cells and used them to investigate the functional similarities and differences of these genes. We found that p51/p63 trans-activated the previously identified p53 target genes, but the degree of the transactivation by p51/p63 differed from that by p53. These results suggest that the cellular signal on p51/p63 cross-talks partially but not completely with that of the p53 pathway.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1999

A novel stromal cell-dependent hematopoietic cell line established from temperature-sensitive SV40 T-antigen transgenic mice.

A novel primitive hematopoietic cell line, THS119, was established from lineage marker negative (Lin-)/Sca-1+ cells from bone marrow of temperature-sensitive (ts) SV40 T-antigen transgenic mice after lengthy passaging by coculture with TBR59 bone marrow stromal cells. THS119 cells exhibited immature primitive hematopoietic cells such as forming cobblestones underneath the stromal cell layers. They retained properties of hematopoietic stem cells as shown by expression of c-Kit, Sca-1 and CD34low, but lacked hematopoietic lineage surface markers of differentiated hematopoietic cells (Gr-1, TER119, Mac-1, CD3, B220). RT-PCR analysis showed that THS119 cells exhibited multiple expression of both earlier developmental markers of myeloid, lymphoid and the hematopoietic cell specific transcription factors. THS119 cells showed temperature-dependent growth reflecting ts T-antigen, and their maintenance was TBR59 stromal cell-dependent. The requirement of stromal cells could not be replaced by cytokines, however, an IL-3 or IL-7 dependent cell line was generated after prolonged culture of THS119 cells on the stromal cells in the presence of these cytokines, and these cytokine-dependent cell lines exhibited phenotypes similar to the parental cells in their gene expression. SCF/c-Kit interaction is one factor required for their maintenance, but involvement of other factor(s) in the conditioned medium of TBR59 stromal cells was suggested. A novel immature hematopoietic cell line, THS119, may provide an appropriate experimental system to resolve how hematopoietic cells are kept in a primitive phase within a hematopoietic microenvironment.

Animals↗
PubMed1999

Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice.

The Pic-1, Oct-1,2, Unc-86 (POU) transcription factor Oct-4 is specifically expressed in the germ cell line, and a previous study has indicated that the expression of the lacZ gene inserted into an 18 kb genomic fragment encompassing the Oct-4 gene can come close to mimicking the endogenous embryonic expression pattern of Oct-4 in transgenic mice. In the present study transgenic mice expressing green fluorescent protein (GFP) in the germ cell line were generated using the same Oct-4 genomic fragments and the expression pattern was analyzed in detail through all stages of germ cell development. The GFP expressing primordial germ cells were first detected as early as 8.0 days post-coitum (d.p.c.; early head fold stage) at the base of the allantois in living embryos. The GFP expression was thereafter found in both male and female germ cells at all developmental stages except in male germ cells after differentiating into type A spermatogonia in the postnatal testis. There was also a lower level of expression in female germ cells in the prophase of the first meiotic division. These transgenic mice therefore proved to be powerful tools for isolating living germ cells at various developmental stages to study their nature and to isolate new genes.

Animals↗