Biomedical subjects

M Obinata

Publications and source records attributed to M Obinata.

At least 127 records · Page 7Linked to original sources

PubMed1989

Isolation of the cDNA clone for mouse glycophorin, erythroid-specific membrane protein.

The cDNA clone for a major mouse glycophorin, transmembrane glycoprotein of erythrocytes has been isolated from a mouse spleen erythroblast cDNA library. The primary structure of a major glycophorin indicates that the protein is a single polypeptide chain of 168 amino acids (aa) clearly organized in three domains distinct in the glycophorin of other species. A strong homology of the mouse major glycophorin with human glycophorin A or B, but not with human glycophorin C is observed only in the hydrophobic stretch of 23 nonpolar aa, indicating that the major mouse glycophorin species cloned is similar to human glycophorin A. The glycophorin mRNA is absent in all non-erythroid organs or cell lines examined. The glycophorin mRNA is induced during the differentiation of murine erythroleukemia cells with dimethyl sulfoxide.

Amino Acid Sequence↗

PubMed1989

Selective suppression of endogenous beta-globin gene expression by transferred beta-globin/TK chimeric gene in murine erythroleukemia cells.

To examine the effect of gene transfer on expression of the endogenous beta-globin gene in murine erythroleukemia (MEL) cells, a beta-globin/TK chimeric gene was introduced into MEL cells. In some of the transformants, expression of the endogenous beta-globin gene was only weakly induced with the addition of DMSO, while expression of the introduced beta-globin/TK chimeric gene was well induced. Suppression of the endogenous beta-globin gene is selective for beta-globin gene, since expression of alpha-globin gene was only weakly affected in its induction in the transformants. Analysis of nine individual transformants indicated that suppression of the endogenous gene correlates more closely with the transcriptional activity than the copy number of the exogenous gene. Thus, the selective suppression can be explained by competition of positive trans-acting factor(s), present in limiting amounts, with high copy number of the exogenous gene the conformation of which is active.

Animals↗

PubMed1989

A balance between self-renewal and commitment in the murine erythroleukemia cells with the transferred c-myc gene; an in vitro stochastic model.

When murine erythroleukemia (MEL) cells, having the transferred rat c-myc gene under the control of human metallothionein II gene promoter, are induced to differentiate with dimethyl sulfoxide (DMSO), the level of differentiation is dependent on the c-myc levels which are modulated by the Zn++ ion. The clonal transformant cell line (38-2) can continuously grow in the presence of both DMSO and Zn++ ion. The proportion of differentiated cells in a population of the continuous culture is strongly affected by the concentration of Zn++ ions. These results suggested that a balance between self-renewal and commitment to differentiation of MEL cells is determined by the c-myc level, and that this cell line may be suitable for studying the stochastic process of growth and differentiation of hemopoietic stem cells.

Animals↗

PubMed1989

Mechanism of erythropoietin action on the erythroid progenitor cells induced from murine erythroleukemia cells (TSA8).

Erythropoietin is a well-known erythroid differentiation and growth factor, but the mechanism of its action is not well understood. In this work, we have examined its mechanism of action on the erythropoietin-responsive murine erythroleukemia cells (TSA8). TSA8 cells become responsive to erythropoietin after induction with DMSO. Stimulatory effects on erythropoietin response are observed with the addition of compounds affecting the cAMP level such as forskolin, phosphodiesterase inhibitor and cholera toxin only in the presence of erythropoietin. cAMP analogues themselves show no stimulatory effect on TSA8 cells, nor does erythropoietin increase cAMP level in the cells. Thus, it is suggested that cAMP does not act as a direct second messenger for signal transduction through erythropoietin receptors, but as a stimulator of the erythropoietin receptor pathway and/or as a second messenger in combination with the receptor pathway. The mechanism for acquisition of responsiveness to growth and differentiation factors of progenitor cells is discussed.

Adenylyl Cyclases↗

PubMed1989

Different signalling pathways are used in the commitment of murine erythroleukemia cells (TSA8) to differentiate, and in the erythropoietin action on progenitor cells.

The murine erythroleukemia (MEL) cell line, TSA8, becomes responsive to erythropoietin after induction with dimethyl sulfoxide (DMSO). We examined the signalling pathways involved in the commitment of TSA8 cells to become the erythroid progenitor cells responsive to erythropoietin, comparing them with the pathway used in an erythropoietin-induced change of the progenitor cells. Amiloride, an inhibitor of the Na+/H+ antiporter, completely blocked the commitment of TSA8 cells to become responsive to erythropoietin at a concentration that did not affect cell proliferation, while it showed no effect on the differentiation or proliferation of the erythroid progenitor cells derived from TSA8 cells by erythropoietin. Ethyleneglycol-bis (beta-aminoethyl ether) N,N,N',N'-tetra acetic acid (EGTA) inhibited the commitment of TSA8 cells to CFU-E-like cells without affecting colony formation. In contrast, EGTA did not inhibit erythropoietin-induced differentiation of the progenitor cells, but did inhibit their proliferation. These results indicate that erythropoietin uses different signalling pathways from those used in the induction of the commitment of TSA8 cells.

Amiloride↗

PubMed1989

Characterization of a new endothelial cell growth factor (f-ECGF) partially purified from the supernatant of human fibroblast cells.

A new endothelial cell growth factor (f-ECGF) was partially purified from the cultured medium of human fibroblast cells of embryonic lungs. The partially purified f-ECGF induced neovascularization in rabbit cornea. It showed a selective growth stimulatory activity on the endothelial cells in vitro, whereas acidic- and basic-fibroblast growth factors (a- and b-FGFs) showed a broad spectrum of growth stimulation among tissues or cells. f-ECGF did not compete with the binding of a-FGF to the cell surface receptor in HEP-G2 hepatoblastoma cell lines. These results indicated that f-ECGF is a new endothelial cell growth factor distinct from a- and b-FGFs which are known to be potent endothelial cell growth factors.

Animals↗

PubMed1989

Analysis of the expression of two phosphoglycerate kinase genes in a mouse cultured cell line during activation and inactivation of the c-myc gene.

The effect of a change in the concentration of c-myc protein on the expression of genes for two phosphoglycerate kinase isozymes was investigated. The steady state levels of messenger ribonucleic acids (mRNAs) for sperm-type and non-sperm-type proteins were determined by blot hybridization using the RNA of the mouse cell line 38-2 containing the inducible rat c-myc gene cultured under various conditions. Without induction the c-myc gene. mRNA for non-sperm-type protein was detected at a level that remained essentially constant during both activation and inactivation of the c-myc gene. mRNA for sperm-type protein was not detected in 38-2 cells cultured under any conditions used. Change in the amount of c-myc protein alone does not appear to bring about a switch of the expression of the two phosphoglycerate kinase genes during spermatogenesis in mouse testis.

Cell Line↗

PubMed1988

Probability that the commitment of murine erythroleukemia cell differentiation is determined by the c-myc level.

During the commitment of mouse erythroleukemia cell differentiation, c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the commitment of these cells, we have introduced the rat c-myc gene driven by inducible, heterologous (human metallothionein IIA) gene promoter into murine erythroleukemia cells and we have examined the ability of the transformed cells to undergo commitment to terminal differentiation. The induction of the exogenous c-myc gene expression inhibited the commitment of these cells. Time-dependent inhibition of the commitment was observed with the addition of zinc at an appropriate time after the induction with dimethyl sulfoxide. The result clearly indicated that late decline, not early decline, is required for the commitment. By examining the transformants expressing the exogenous c-myc mRNA at different levels, and the induction of the exogenous c-myc mRNA by varying the concentration of zinc, we demonstrated that the commitment may be determined by a stoichiometric amount of c-myc in the defined period. The data also suggest that the probability value for the commitment process occurring in a stochastic manner is well-correlated with the amount of c-myc mRNA.

Animals↗

PubMed1988

Stage-specific gene expression in erythroid progenitor cells (CFU-E).

In erythropoietic differentiation, mature red blood cells are generated from specific progenitor cells through the action of specific growth regulatory molecules. To know the mechanism of differentiation, it is important to examine the control of gene expression in these progenitor cells in combination with growth regulatory molecules. We have cloned two genes expressing at a maximal level in the CFU-E (colony forming unit-erythroid), one of the erythroid progenitor cells from novel murine erythroleukemia (MEL) cell line (TSA8) which can be induced to CFU-E in vitro. The expression of these genes is well correlated with the appearance of CFU-E during induction of TSA8 cells, and is higher in the CFU-E-cells enriched from mouse fetal livers than in the more differentiated erythroid cells. Combining these with our previous results, it is suggested that in the erythropoiesis the progenitor cells have distinct patterns of gene expression. This expression is replaced through each progenitor cell rather than by the continuous increase in the expression of a set of genes specific to the mature erythroid cell following the commitment process.

Animals↗

PubMed1988

Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.

Animals↗

PubMed1988

Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells.

Animals↗

PubMed1987

Molecular cloning and nucleotide sequence of cDNA encoding hydroxyindole O-methyltransferase of bovine pineal glands.

Messenger RNA for hydroxyindole O-methyltransferase (EC 2.1.1.4) was partially purified from poly(A)+ RNA isolated from bovine pineal glands by sucrose density gradient centrifugation. The enriched mRNA was used to prepare a cDNA library by use of expression vector lambda gt11. The library was screened with monoclonal antibodies to the enzyme, and three cDNA clones were isolated. These cloned cDNAs cross-hybridized with one another, and their fusion proteins reacted to the monoclonal antibodies with different binding properties. Hydroxyindole O-methyltransferase enzymatic activity was demonstrated in the bacteria lysate infected with lambda HIOMT-A16, the clone that contained the longest insert. An almost full-length cDNA clone was isolated from lambda gt10 cDNA library by use of the lambda HIOMT-A16 cDNA as a probe. The primary structure of hydroxyindole O-methyltransferase was determined by analyzing the nucleotide sequence of the cDNAs. It consisted of 1939 nucleotides including a 1050-nucleotide region coding for 350 amino acids. RNA transfer blot analysis indicated that mRNA encoding hydroxyindole O-methyltransferase was present only in the pineal gland and not in the brain, retina, and liver of cow.

Acetylserotonin O-Methyltransferase↗

PubMed1987

Characterization of trans-acting factor(s) regulating beta-globin gene expression by in vivo competition.

A beta-globin/TK fusion gene was microinjected into non-erythroid cells (Ltk- cells) and erythroid cells (murine erythroleukemia (MEL) cells), and the interactions of the regulatory cellular factors with the beta-globin sequences were investigated by the in vivo competition experiment. The fusion gene was expressed efficiently in Ltk- cells. This expression was inhibited by a co-injection with a three-fold molar excess of the 5'-flanking sequence of the beta-globin gene or with a nine-fold molar excess of the mammary tumor virus LTR, but not with the alpha-globin gene. The fusion gene was expressed very poorly in the uninduced MEL cells and highly in the induced MEL cells. The co-injection of the beta-globin gene did not affect expression in the MEL cells in either uninduced or induced conditions.

Animals↗

PubMed1987

Modulation of the transferred mouse 26K casein gene in mouse L cells by glucocorticoid hormone.

The cloned 26K casein gene was transferred to mouse L-cells and its expression was measured by Northern blot hybridization. When the lambda clone with 5'- and 3'-flanking sequences was transferred, transcripts were detected without glucocorticoid, but in the presence of glucocorticoid, the level of the transcripts of heterogeneous sizes increased and their pattern was similar to that observed in the mammary glands of non-lactating mice. When the 6.7 kb EcoRI fragment containing most of the coding region was transferred, putative precursor and mature mRNAs were detected without glucocorticoid. Surprisingly, with the addition of glucocorticoid, the level of the transcripts greatly decreased. The presence of multiple sequences responsible for glucocorticoid receptor binding was detected in the 5'-flanking region of the gene in a competition assay using the subfragments of casein gene and on sequence analysis. These results suggest that the 26K casein gene has multiple regulatory domains which interact with glucocorticoid receptors, and these domains may play different roles in the regulation of the casein gene.

Animals↗

PubMed1987

Factors controlling induction of commitment of murine erythroleukemia (TSA8) cells to CFU-E (colony forming unit-erythroid).

On addition of DMSO, the MEL cell line TSA8 becomes committed into erythroid progenitor cells (CFU-E) which can form differentiated colonies in the presence of erythropoietin. To understand the mechanism of cellular commitment, the number and the affinity of the receptors for erythropoietin were estimated. The affinity of the receptors did not change before or after induction. The number of receptors changed depending on the growth phase, but was not dependent on the addition of the inducer. Thus, the presence of the receptors for erythropoietin may be required, but are not essential for responsiveness to erythropoietin. Further examination of the optimum conditions for commitment suggests that the concomitant actions of induced factor(s) with the receptors may control commitment of TSA8 cells to CFU-E.

Animals↗

PubMed1987

[Factors controlling the differentiation and cancerization of murine erythroleukemia cells].

Cultured murine erythroleukemia (MEL) cells can be induced to differentiate into erythrocytes. During this induced differentiation, a certain type of sequentially programmed gene expression and repression appears to occur in addition to the induction of globin mRNA. This system provides a commitment model for erythrodifferentiation and decancerization. By transfection of a beta-globin/TK chimeric gene into a B8/3 cell line, we examined the regulatory factors controlling beta-globin gene expression. Our results suggested that the timing of the appearance of inducible, positive trans-acting factor (s) and activation of chromatin conformation occur during induction. We demonstrated that a novel MEL cell line, TSA 8, could be induced to be committed to CFU-E, an erythropoietin-mediated progenitor cell, with the addition of DMSO. In the commitment process, we observed an asymmetric cell division, which could explain the self-renewal and the commitment of multipotential hemopoietic stem cells. For this commitment, the receptor for erythropoietin is required, but is insufficient and the other factor (s) are induced in the earlier phase of induction. Finally, we found that erythropoietin induced two signals for proliferation and differentiation of the progenitor cells and that these two signals are not coupled.

Animals↗

PubMed1986

Replication timing: histone genes replicate during early S phase in cleavage-stage embryos of sea urchin.

Newly synthesized DNA was separated from the bulk of the DNA by pulse-labeling with BUdR and centrifugation in an alkaline CsCl buoyant density gradient. The content of histone gene in the newly synthesized DNA was determined by DNA dot hybridization. The gene contents in DNA replicated during the early half of S phase and during the whole S phase were compared. Results showed that histone genes were replicated during the first half of the S phase in embryos in the early cleavage stage.

Animals↗

PubMed1986

Induction of commitment of murine erythroleukemia cells (TSA8) to CFU-E with DMSO.

The commitment of novel mouse erythroleukemic (MEL) cells (TSA8) to colony-forming units of erythroid (CFU-E) by dimethylsulfoxide (DMSO) was investigated. After exposure to the inducer in liquid culture, the cells were transferred to a semi-solid culture to examine their ability to form erythroid colonies which were dependent on erythropoietin. Exposure to DMSO for 2 days is optimum for CFU-E type colony formation and colonies induced in this manner are equivalent to CFU-E. The induction occurred in a synchronous manner. Partly stained colonies appeared prior to CFU-E formation and are thought to be a result of asymmetric cell division. Appearance of these partly stained colonies suggested that the number of erythropoietin receptors is important in the complete responsiveness to erythropoietin. TSA8 cells constitute a suitable model system in which to analyse the mechanism of commitment in early erythropoiesis.

Animals↗