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Biomedical subjects

M Obinata

Publications and source records attributed to M Obinata.

At least 73 records · Page 4Linked to original sources
PubMed1997

A new type-II membrane protein in erythropoietic organs enhances erythropoiesis.

Erythropoiesis is regulated by erythropoietin and microenvironment of erythropoietic organs such as fetal liver and spleen in mice. We developed in vitro erythropoietic microenvironment by established stromal cell lines from erythropoietic organs, in which proliferation and differentiation of erythroid progenitor cells are supported by direct cell-to-cell contact between erythroid progenitor cells and the stroma cells. To identify the functional molecules of the stromal cells, we established monoclonal antibodies which detected specific molecules of the erythropoietic organs. Among those monoclonal antibodies, 11 D exhibited specific immunohistochemical staining of the red pulp of spleen where erythropoiesis dominates. Using this monoclonal antibody, we cloned a new gene (SMAP-1) which codes a type-II membrane protein. Over expression of its antisense cDNA in a stroma cell line caused a decrease in the erythropoietic supporting activity. Thus, SMAP-1 may play an important role in the erythropoietic stimulatory activity of the stromal cells.

Animals↗
PubMed1996

A receptor tyrosine kinase, Sky, and its ligand Gas 6 are expressed in gonads and support primordial germ cell growth or survival in culture.

Although a number of growth factors and their receptors are involved in the proliferation and differentiation of primordial germ cells (PGCs), the only factor that has been shown to be active in vivo is Steel factor, a ligand for c-Kit. To identify new growth factor receptors that may be required for PGCs function in vivo, we used an reverse transcription-polymerase chain reaction-based strategy to screen for protein kinase genes expressed in PGC-derived embryonic germ cells. We report here that one such gene encoding the receptor tyrosine kinase, Sky, is expressed in both PGCs and their supporting cells in male genital ridges after 11.5 dpc. Interestingly, Sky expression was not detected in female genital ridges, although transcripts were detected in supporting cells in the developing ovary at later stages. Gas 6, a ligand for Sky, was also expressed in interstitial cells which surround Sky positive cells in genital ridges, and, in addition, it supported PGC growth or survival in culture. After birth, Sky expression in testis was restricted to Sertoli cells, and Gas 6 was detected around peritubular cells and Leydig cells. These results suggest that Gas 6-Sky signaling plays a role in PGC growth, sexual differentiation, and Sertoli cell functions in vivo. Sky expression in Sertoli cells diminished by 3 weeks of age, when haploid germ cells first appear. On the other hand, the expression in Sertoli cells was markedly upregulated in the testis of germ cell-deficient W/Wv and jsd/jsd mice. The results suggest that signals from differentiated germ cells suppress Sky gene expression in Sertoli cells. High-resolution chromosomal mapping of Sky is also reported.

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PubMed1996

Regulatory function of delta/YY-1 on the locus control region-like sequence of mouse glycophorin gene in erythroleukemia cells.

The far upstream region (-1.2-0.9 kilobase pairs) of the mouse glycophorin gene contains the locus control region (LCR)-like region, which acts as an erythroid-specific enhancer dependent on chromosomal integration in murine erythroleukemia (MEL) cells. In the present study, we demonstrated that this region binds six nuclear factors. The binding of GATA-1 to corresponding sites did not show any change before or after induction with dimethyl sulfoxide, but the binding of Spi-1/PU.l and an unidentified factor called glycophorin regulatory element binding factor (GRBF) showed a change during induction. While binding activity of Spi-l/PU.l dropped soon after induction, the GRBF activity increased after induction when expression of the glycophorin gene began. After identification of the consensus binding site of GRBF, we cloned cDNA for that factor by Southwestern method, and it was identified as a previously reported transcription factor, delta, a murine form of YY-l which is a versatile transcription factor. Mutation analysis in the delta/YY-1 binding site within the LCR-like region indicated that delta/YY-1 acts as a regulatory protein in combination with the E-box-binding protein that binds to the neighboring sequence.

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PubMed1996

Overexpression of c-myc induces apoptosis at the prophase of meiosis of rat primary spermatocytes.

Transgenic rats expressing the rat c-myc gene under the control of the human metallothionein II A promoter were produced. We found that the female transgenic rats were fertile, but that the male transgenic rats were sterile. Atrophy of the seminiferous tubules and depletion of sperm were observed in the sterile male testes. The expression of differential stage-specific mRNAs, including those of the c-kit receptor proto-oncogene, meiotic heat-shock protein 70 gene, acrosin gene, and transition protein 1 gene, was analyzed by the reverse transcriptase-polymerase chain reaction during spermatogenesis. The results suggested that spermatogenesis in these sterile rats were arrested at the prophase of meiosis in the primary spermatocytes. We found that apoptotic DNA fragmentation occurred in primary spermatocytes of the sterile transgenic rats. These results suggest that overexpression of the c-myc gene induces apoptosis at the prophase meiosis of the primary spermatocytes thereby causing male sterility in the c-myc transgenic rats.

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PubMed1996

A novel serine/threonine kinase gene, Gek1, is expressed in meiotic testicular germ cells and primordial germ cells.

We have isolated a novel serine/ threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects.

Amino Acid Sequence↗
PubMed1996

Establishment of a novel chondrocyte-like cell line derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene.

We established a clonal chondrocyte-like cell line (TC6, TC stands for large T immortalized chondrocyte-like cell line) derived from articular cartilage of transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. TC6 cells exhibited spindle-like or polygonal morphology and grew well at 33 degrees C in alpha-minimal essential medium supplemented with 0.5% fetal bovine serum. After confluence, these cells formed nodules that were positive for staining with alcian blue. Northern blot analysis demonstrated that these cells expressed messenger RNAs (mRNA) of the genes encoding cartilage-specific proteins such as type II procollagen, link protein, and aggrecan. Furthermore, the expression of type II procollagen and link protein genes in TC6 cells was regulated by parathyroid hormone and basic fibroblast growth factor, suggesting the presence of the receptors for the hormone and cytokine. The expression of link protein mRNA in TC6 cells was regulated in a time-dependent manner and was enhanced in culture within a week and increased continuously up to 10-fold by the end of 4 weeks. Expression of mRNAs encoding type II procollagen and versican/PG-M also increased moderately during the culture period. TC6 cells expressed type I procollagen mRNA, however, its level declined along with time in culture in contrast to the enhancement of the genes encoding cartilage-specific molecules in these cells. Interestingly, alkaline phosphatase mRNA expression was barely detectable in the TC6 cells in their growing phase while it was enhanced dramatically more than 7-fold by day 14 in culture. These results indicate that the TC6 cells could serve as an excellent model for the studies on chondrocyte physiology.

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PubMed1996

Cisplatin-induced toxicity in immortalized renal cell lines established from transgenic mice harboring temperature sensitive SV40 large T-antigen gene.

We established renal cell lines from definite nephron segments which were microdissected from kidneys of transgenic C57BL/6 mice, harboring the large T-antigen gene of temperature-sensitive mutant simian virus 40, pSVtsA58(ori-). Cell culture was under a humidified atmosphere of 5% CO2 in air, on collagen-coated dishes, and in RITC80-7 medium with 5% fetal bovine serum, 10 micrograms/ml transferrin, 1 microgram/ml insulin, 10 ng/ml recombinant human EGF, penicillin and streptomycin. Cell line which kept contact inhibition character was established from each segment. Cells derived from distal tubule, cortical and outer medullary collecting duct possessed their cyclic AMP response to arginine-vasopressin, like their original nephron segment. On the other hand, cells derived from terminal proximal tubules (S3 segment) formed a cobblestone-like confluent monolayer, and did not respond to arginine-vasopressin like their fresh segments. Since cisplatin, a well-known nephrotoxic substance, damages proximal tubules (especially S3) rather than collecting ducts, we assayed cell number, protein content, and ATP content of cultured S3 cells at various times after addition of 0.2 mM cisplatin. Decrease of cell number, total protein content and total ATP content of culture cells occurred after 10 h incubation with 0.2 mM cisplatin. The 50% lethal dose (LD50) of cisplatin in S3 cells was 4 x 10(-5) M after 20 h incubation and 8.5 x 10(-6) M after 40 h incubation. Outer medullary collecting duct (OMCD) cells were damaged 30% maximally after 20 h incubation with cisplatin, and LD50 in them became 2.5 x 10(-5) M after 40 h incubation. We could show that the LD50 of cisplatin in the OMCD cell line was three times higher than that in the S3 cell line. Thus, these cell lines are the first in the kidney to definite the segmental origin and to maintain some differentiated unique functions. They are valuable for studies on intrarenal site-specific actions and possible mechanisms of action of pharmacological and toxic substances.

Animals↗
PubMed1996

Specific arrest of spermatogenesis caused by apoptotic cell death in transgenic mice.

BACKGROUND: The c-myc protooncogene has been implicated in the control of cell proliferation, differentiation and/or apoptosis in various cellular systems. However, the role of c-myc in germ cell lineage is largely unknown. RESULTS: We have produced transgenic mouse lines carrying the rat c-myc protooncogene under the control of human metallothionein promoter (hMT-c-myc). It was found that the male transgenic mice were sterile. In contrast, all of the female transgenic mice were completely fertile and transmitted the transgene to the next generation. However, male transgenic mice from the female transgenic founders were also found to be sterile. This sterility was due to a defect in spermatogenic cell differentiation, since virtually no sperm were seen within the seminiferous tubules or the cauda epididymis. Histological examination revealed that germ cell death occurred approximately 7 days after birth and, consequently, spermatogenesis was arrested at an early stage in meiotic division in the transgenic mice. Moreover, this germ cell death was found to be caused by apoptosis. CONCLUSION: We conclude that an excess level of c-myc expression in differentiating spermatogenic cells is responsible for the apoptotic death of germ cell, and that a decrease in c-myc level would be an obligatory step for the completion of normal spermatogenesis.

Animals↗
PubMed1996

Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia.

During normal spermatogenesis, more than half of the germ cells undergo apoptosis, but the physiological significance and molecular mechanisms of this programmed cell death are largely unknown. Because Bcl-2 functions as a death repressor, we have investigated the effect of misexpressing Bcl-2 in spermatogonia in transgenic mice using the human bcl-2 cDNA under the control of the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter. In the 2-week-old transgenic testes, exogenous Bcl-2 was expressed in spermatogonia and massive accumulation of spermatogonia was observed in seminiferous tubules by 4 weeks. At this time, only a few spermatocytes were apparent, and the accumulated cells degenerated, leading to vacuolization in some seminiferous tubules by 7 weeks. In older transgenic mice, abnormal accumulation of spermatogonia and degeneration of these germ cells was still observed, but some seminiferous tubules in which the level of Bcl-2 expression was reduced recovered normal spermatogenesis. These observations indicate that spermatogonial apoptosis is part of the normal program of mammalian spermatogenesis and is regulated by a pathway affected by Bcl-2.

Animals↗
PubMed1996

A retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene.

We established a retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 T-antigen gene (tsSV40T) and examined its characteristics. We enucleated both eyes from a 2-month-old transgenic mouse and removed the retinal pigment epithelial (RPE) cells and neuroretinal cells. After cloning the RPE cells, we obtained a cell line (RPET). RPET cells grew well at 33 degrees C but not at 37 degrees C, expressing on the temperature-sensitive character of tsSV40T, and maintained characters of RPE cells such as T1-tyrosinase production, phagocytosis of rod outer segments, and presence of cytokeratin, microvilli on the cell surface and lysosome-like granules around the Golgi apparatus in the cytoplasm. Conditioned medium (CM) from a culture of neuroretinal cells harboring tsSV40T was essentially required for growth. The factor(s) in CM was heat-and acid labile, but was resistant to trypsin digestion. In the presence of 3% CM, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and strong effects on RPET cells, whereas insulin, insulin-like growth factor I (IGF-I), and IGF-II had moderate growth effects. Interestingly, none of these growth factors stimulated the RPET cells in the absence of CM. EHS-Matrix had growth effect, whereas laminin, collagen types I and IV, and fibronectin had no marked growth effects on RPET cells. RPET cells were morphologically changed on a laminin-coated dish. They could not spread on the coated dish, and the majority of the cells floated. But when the floating cells were transferred to non-coated dishes, they immediately attached themselves. These results suggest that RPET cells are a good model for for finding novel growth factor(s) and for investigating the mechanism of cell-laminin attachment.

Animals↗
PubMed1996

v-src interferes with the in vitro erythropoietic stimulatory ability of spleen stromal cells through repression of vascular cell adhesion molecule-1 and stem cell factor.

A mouse spleen stromal cell line, MSS62, can create an in vitro erythropoietic microenvironment in which development of erythropoietin-responsive progentor cells is stimulated by cell-cell contact via stem cell factor (SCF)/c-Kit and vascular cell adhesion molecule-1 (VCAM-1)/very late activation antigen-4 (VLA-4) interactions between stromal and erythroid cells. To find out the effect of src on the erythropoietic microenvironment, MSS62 cells were transfected with v-src oncogene, and its effect on erythropoietic stimulatory activity was measured. Transfectants with high v-Src activity showed reduction in erythropoietic stimulatory activity. A decrease in cell-surface VCAM-1 and SCF mRNA was accompanied by high v-Src activity. These results suggest that v-Src interferes with the erythropoietic stimulatory activity of the stromal cells through repression of VCAM-1 and SCF.

Animals↗
PubMed1995

MIDA1, a protein associated with Id, regulates cell growth.

We have isolated cDNA clone encoding a protein that can associate with Id, a helix-loop-helix (HLH) protein. This protein is named MIDA1 (Mouse Id Associate 1), and its predicted amino acid sequence consists of Zuotin (a putative Z-DNA binding protein in yeast) homology region and tryptophan-mediated repeats similar to c-Myb oncoprotein. MIDA1 associates with the HLH region of Id with the conserved region adjacent to eukaryotic DnaJ conserved motif within the Zuotin homology region, although it does not have any canonical HLH motif. The addition of antisense oligonucleotide of MIDA1 inhibited growth of murine erythroleukemia cells without interfering with erythroid differentiation, indicating that it regulates cell growth.

Amino Acid Sequence↗
PubMed1995

Bone marrow stromal cells induce myeloid and lymphoid development of the sorted hematopoietic stem cells in vitro.

Regulation of development of hematopoietic stem cells was examined by culturing Lin- c-Kit+ Sca1+ stem cells sorted from bone marrow (BM) cells by fluorescence-activated cell sorting on a layer of TBR59, a BM stromal cell line established from simian virus 40 T-antigen gene transgenic mice. The sorted stem cells did not show self-renewal, but two waves (at 7 and 13 days) of a cobblestone formation were induced by the stromal cell layer. The cobblestones were formed by finite cell division (eight divisions on average) of sorted Lin- c-Kit+ Sca1+ stem cells, and divided cells were still immature. The c-Kithigh stem cell population was induced to form the first wave of cobblestone formation committed to myeloid lineage, whereas c-Kitlow population was induced to form the second wave of this formation committed to lymphoid lineage. Both cobblestone formations require c-Kit function, but very late activation antigen-4-vascular cell adhesion molecule-1 interaction plays different parts in the two lineages.

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PubMed1995

Mammalian antioxidant protein complements alkylhydroperoxide reductase (ahpC) mutation in Escherichia coli.

The MER5 [now called the Aop1 (antioxidant protein 1) gene] was cloned as a transiently expressed gene of murine erythroleukaemia (MEL) cell differentiation and its antisense expression inhibited differentiation of MEL cells. We found that the Aop1 gene shows significant nucleotide sequence similarity to the gene coding for the C22 subunit of Salmonella typhimurium alkylhydroperoxide reductase, which is also found in other bacteria, suggesting it functions as an antioxidant protein. Expression of the Aop1 gene product in E. coli deficient in the C22-subunit gene rescued resistance of the bacteria to alkylhydroperoxide. The human and mouse Aop1 genes are highly conserved, and they mapped to the regions syntenic between mouse and human chromosomes. Sequence comparisons with recently cloned mammalian Aop1 homologues suggest that these genes consist of a family that is responsible for regulation of cellular proliferation, differentiation and antioxidant functions.

Animals↗
PubMed1995

Phosphorylation of helix-loop-helix proteins ID1, ID2 and ID3.

Id is a helix-loop-helix protein which forms heterodimer with ubiquitous and/or tissue-specific basic helix-loop-helix proteins and inhibits their DNA binding. It has been noted that putative phosphorylation sites for various protein kinases exist in rat Id1, Id2 and Id3. We show here that Id1 and Id2 can be phosphorylated in vitro by cAMP-dependent protein kinase, Id2 and Id3 by cdc2 kinase, and all three Ids by protein kinase C. The phosphorylated Id1 was actually immunoprecipitated in nerve-growth-factor-stimulated PC12 cells. Gel mobility shift assays, however, demonstrated that neither phosphorylation of Id proteins by cAMP-dependent protein kinase nor phosphorylation of E47 by protein kinase C affected the inhibition of E47 homodimer formation and its DNA binding. Taken together with other observations, phosphorylation of Id proteins may play a role in regulation of cell differentiation but not directly in the dimerization and DNA binding.

Amino Acid Sequence↗
PubMed1995

Bone marrow stromal cells selectively stimulate the rapid expansion of lineage-restricted myeloid progenitors.

Bone marrow stromal cells serve hematopoietic microenvironments where different blood cells are controlled in their growth and differentiation. To characterize functions of stromal cells, 33 bone marrow stromal cells including preadipocytes, endothelial cells, and fibroblasts were established from transgenic mice harboring temperature-sensitive SV40 T-antigen gene and their selective stimulatory abilities to support large colony formation of lineage-specific hematopoietic progenitor cells (erythroid, monocyte/macrophage, granulocyte, and monocyte-granulocyte) were examined. Among established stromal cells, 27 clones showed erythropoietic stimulatory activity in the presence of erythropoietin. On myeloid progenitors, the stromal cells showed lineage-restricted stimulatory activity and a reciprocal relationship was observed between granulocyte formation and macrophage formation, but these activities were not dependent on the amount of produced colony-stimulating factors (CSFs). Our present study with many stromal cells established from bone marrow indicated that each stromal cell in the bone marrow may provide the preferable microenvironment for a rapid expansion of the lineage-restricted progenitor cells in combination with CSFs.

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PubMed1995

Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes.

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding regions shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythro-leukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the Plk1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage.

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