Biomedical subjects

Y Nishimune

Publications and source records attributed to Y Nishimune.

At least 73 records · Page 4Linked to original sources

PubMed1996

Differentiation-dependent enhanced expression of protein phosphatase 2Cbeta in germ cells of mouse seminiferous tubules.

The presence of five distinct isoforms of protein phosphatase 2Cbeta (PP2Cbeta-1 approximately -5) is known. In this study, we demonstrate that the mRNA levels of PP2Cbeta-3, -4 and -5 and PP2Cbeta protein level increased during the course of the first wave of spermatogenesis in neonatal mouse testis. Northern blot and in situ hybridization analyses revealed that PP2Cbeta-3, -4 and -5 were expressed predominantly in pachytene spermatocytes and in more highly differentiated germ cells. The substrate specificity of PP2Cbeta-4 determined with artificial substrates differed from those of PP2Cbeta-3 and -5, suggesting that the difference in the structure of PP2Cbeta-3, -4 and -5 reflect their unique physiological functions in testicular germ cells.

Animals↗

PubMed1996

Abrogation of c-kit/Steel factor-dependent tumorigenesis by kinase defective mutants of the c-kit receptor: c-kit kinase defective mutants as candidate tools for cancer gene therapy.

The growth and survival of many types of cancer cells are known to be supported by specific growth factor/cytokine systems. Among these, the activation of c-kit receptor and its ligand steel factor participates in several types of human carcinogenesis. W mutations of laboratory mouse strains are loss of functional mutations of the c-kit receptor. To examine the validity of these mutants in investigating c-kit-mediated carcinogenesis and in the treatment of c-kit-dependent tumors, we introduced various W mutations (W, Wv, and W42) into a transgenic mouse strain carrying human papillomavirus oncogenes, in which c-kit/Steel-mediated tumorigenesis occurs with a very high incidence. In all transgenic strains carrying a W mutation, the c-kit deficiency affected the tumorgenic process to various degrees. Tumor development was markedly suppressed in transgenic strains carrying kinase defective mutations (Wv and W42) in a heterozygous condition. In null-type (W) heterozygous transgenic mice, tumorigenesis was suppressed at a lower level. Moreover, minimal focal legions or, in some cases, no focal legions were found in the testes of W/Wv heterozygous transgenic mice, showing a close relationship between tumor cell growth and the degree of c-kit inactivation. These results indicated that c-kit activity is a pivotal determinant of testicular tumor development and that the kinase defective mutants of c-kit are valuable for treating c-kit-dependent cancer, as well as for clarifying the c-kit-mediated carcinogenesis.

Animals↗

PubMed1996

Cell cycle-dependent expression of the mouse Rad51 gene in proliferating cells.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouse Rad51 mRNA was examined in synchronized mouse m5S cells. The Rad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression of Rad51 mRNA. By immunohistochemical analyses, in mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouse Rad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes.

Animals↗

PubMed1996

The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis.

Genetic recombination in meiosis plays an important role in generating diversity of genetic information. In yeast an Escherichia coli RecA-like gene, DMC1, is expressed in meiotic prophase and its product co-localizes with Rad51 protein on zygotene chromosomes. We have cloned the mouse and human homologs of the yeast DMC1 gene. The predicted human and mouse DMC1 proteins showed 54.1% sequence identity with yeast Dmc1 protein. The domain II region, highly conserved in the E.coli RecA-like protein family, was also found in the mammalian DMC1 proteins, including the two ATP binding motifs and DNA binding sites with the region. In situ hybridization analysis revealed expression of the mouse Dmc1 gene in testicular germ cells in meiosis; RT-PCR showed expression in embryonal ovaries. These findings suggest that DMC1 plays an important role in meiotic homologous recombination. From both the man and mouse we have isolated an alternative spliced form of Dmc1 cDNA (Dmc1-d), which is deleted for a region between the two motifs involved in nucleotide binding. Since the alternatively spliced Dmc1-d transcript was detected in both male and female germ cells, the encoded protein DMC1-D may have a novel role in mammalian genetic recombination in meiosis.

Adenosine Triphosphatases↗

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↗

PubMed1995

Isolation of a novel cDNA that encodes a protein localized to the pre-acrosome region of spermatids.

We have identified a novel cDNA clone, named AZ1, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent demethylating reagent. The amount of transcript increased with 5-azacytidine treatment of C3H10T1/2 cells and the transcript was highly expressed in mouse testis. As the mutant mouse jsd/jsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed specifically in spermatocytes. The mRNA was detected at significant levels in the testes from mice aged 16 days after birth, suggesting that its expression started at the pachytene spermatocyte stage. The elucidated nucleotide sequence contained a 2841-nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 107,254 Da. Specific antibodies raised against the fusion protein including glutathione S-transferase revealed an approximately 130-kDa band of a translation product in testis and in cultured cells transfected with AZ1 cDNA in the expression vector on Western-blot analysis. The protein was localized to the pre-acrosome region of round and elongated spermatids. However, it was not detected at a more advanced stage of spermatids, i.e. just before their release from Sertoli cells. This protein may play an important role in spermatogenesis.

Acrosome↗

PubMed1995

A rapid and non-invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP).

Non-invasive selection of transgenic mice was performed at the stage of preimplantation embryos. The morulae collected from wild female mated with hemizygous transgenic male expressing Aequorea victoria green fluorescent protein (GFP) under chicken beta-actin promoter could be classified as green or non-green under a fluorescent microscope. All the green embryos were shown to carry the transgene by PCR analysis. Taking advantage of the detection of GFP expression can be done non-invasively, the selected embryos were demonstrated to be able to developed to term with 100% of accuracy of the selection.

Animals↗

PubMed1995

Regulation of the mouse histone H2A.X gene promoter by the transcription factor E2F and CCAAT binding protein.

We have molecularly cloned the genomic gene encoding the mouse histone variant H2A.X and characterized the promoter. The promoter region of the H2A.X gene was characterized by chloramphenicol acetyltransferase analysis using Balb/c 3T3 cells. Maximal promoter activity was found in the construct containing up to -282 base pairs H2A.X upstream region. Within this region, we found two sequences regulating the promoter activation; one was an E2F site and another was a CCAAT box. These sequences were also required for the DNA/protein binding activities. Thus, these activities corresponded to the promoter activities, implying that the promoter activity H2A.X gene was controlled by both the transcription factor E2F and H1TF2 through the E2F and CCAAT element. The CCAAT box binding activity was constitutive when cell cycle was progressed by release from G1 arrest, but transiently transfected chloramphenicol acetyltransferase activity slightly increased when cells entered S phase. Similarly, the level of the smallest form of E2F (free E2F) became higher when cells reentered the cell cycle, indicating that the free E2F was one capable of inducing the promoter activation. Thus, the free E2F and CCAAT DNA binding activity correlated with regulation of the promoter activity.

3T3 Cells↗

PubMed1995

Characterization of the testis-specific gene 'calmegin' promoter sequence and its activity defined by transgenic mouse experiments.

We have cloned the genomic DNA of calmegin [(1992) J. Biol. Chem. 269, 7744-7749] and analyzed its promoter region. It contained GC-rich sequences and potential binding sites for AP 2 and Sp 1, but lacked the TATA sequence. The 330 bp 5' flanking sequence of calmegin genomic DNA fused with the CAT gene was used for the study of promoter activity in transgenic mice. The CAT gene activity was detected exclusively in testes, indicating that the 330 bp calmegin 5' sequence was sufficient for the testis-specific expression. The existence of testicular nuclear factors specifically bound to the putative promoter sequence was also demonstrated.

Animals↗

PubMed1995

An in vivo model for receptor tyrosine kinase autocrine/paracrine activation: auto-stimulated KIT receptor acts as a tumor promoting factor in papillomavirus-induced tumorigenesis.

Constitutive overactivation of growth factor receptors through autocrine/paracrine mechanisms occurs frequently in cancer cells and are thought to play a critical role in carcinogenesis. In the present report, we propose a refined in vivo model which explains the significance of these mechanisms in tumour development. We have previously established transgenic mouse lines containing human papillomavirus type 16 (HPV16) E6E7 oncogenes, in male mice of which a Leydig cell tumor developes with a very high incidence. Not only HPV transgene but also the c-kit proto-oncogene receptor tyrosine kinase and its ligand Steel Factor (SLF) were coexpressed in all tumors analysed. This coexpression of c-kit/SLF was also found in two other Leydig cell tumor lines. Moreover, the proliferation of transgenic tumor cells was attenuated by treatment with a c-kit neutralizing antibody in vitro, strongly suggesting that tumorigenesis is closely related to stimulation of receptors through ligand induction. To confirm the significance of these findings, a defective mutation of the SLF gene in a laboratory mouse, the Steel-Dickey (Sld) mutation, was introduced into a line of transgenic mice showing 100% incidence of the tumor. In Sld-E6E7 transgenic mice, tumorigenesis was initiated but numbers of tumor cells were markedly reduced compared with transgenic mice carrying both wild type SLF allele, showing that c-kit activation through the induction of SLF is essential for testicular tumorigenesis, especially in tumour promotion. This transgenic mice system should be a useful in vivo model for clarifying the implication of growth factor autostimulation in carcinogenesis.

Animals↗

PubMed1995

Induction of teratocarcinoma F9 cell differentiation with cis-diammine dichloroplatinum(II) (CDDP).

cis-Diammine dichloroplatinum(II) (CDDP) is the salt of a platinum compound which has been noted to have a wide spectrum of activity against malignant disorders. We have studied the effects of CDDP on embryonal carcinoma F9 cell differentiation. In the presence of this agent in vitro, the cells showed rapid morphological changes, a marked increase in the mRNA expression of various differentiation markers accompanied by a loss of tumorigenicity. These results indicate that the differentiation of F9 cells is induced with CDDP.

Animals↗

PubMed1995

Characterization of a novel spermatogenic cell antigen specific for early stages of germ cells in mouse testis.

To study the mechanism of spermatogenesis during the premeiotic phase, a hybridoma producing monoclonal antibody (mAb) specific for early stages of spermatogenic cells was obtained. In immunohistochemical staining of adult testis, this mAb, designated as EE2, was able to react with type A to B spermatogonia and early meiotic cells, but not with Sertoli cells, Leydig cells, and other somatic tissues. Precursor cells of type A spermatogonia (gonocytes) were also positive for EE2 in perinatal mouse testis. The antigenic molecule recognized by mAb EE2 was a novel glycoprotein with molecular weight of 114 kDa, which had affinity with Con A and WGA lectins, and was susceptible to N-glycanase, suggesting the presence of asparagine-linked sugar chains. Furthermore, EE2 antigen was found to localize on the germ cell surface. The specific expression of this antigenic molecule suggests that it may play an important role in early spermatogenesis, of which only a little information is available at present.

Animals↗

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.

Animals↗

PubMed1995

Effects of protein phosphatase inhibition by okadaic acid on the differentiation of F9 embryonal carcinoma cells.

Okadaic acid, a protein phosphatase inhibitor, is a strong tumor promoter which apparently activates protein phosphorylation. To examine the role of protein phosphatases in stem cell growth and differentiation, embryonal carcinoma F9 cells were treated with okadaic acid. In the presence of this agent, the cells showed rapid morphological changes and arrest of proliferation at the M phase of the cell cycle, accompanied by a marked increase in the mRNA expression of various differentiation markers. Okadaic acid induced rapid increase in the mRNA levels of both c-jun and junB and results indicate that the inhibition of phosphatase by okadaic acid induces apparent activation of protein phosphorylation and may cause the expression of differentiation marker genes in F9 cells via the activation of AP-1.

Animals↗

PubMed1995

Protection against hantavirus infection by dam's immunity transferred vertically to neonates.

Antibodies to hantavirus, Seoul type B-1 strain, vertically transferred to rat neonates prevented lethal as well as persistent infection. When relatively high titer viruses were inoculated into neonates, the mother's antibodies protected all the neonates from lethal virus infection. However, the antibodies could not protect all of the neonates from persistent infection but only half of them underwent persistent infection. The other half was completely cured but also became persistently infected when rechallenged with the active viruses after reaching maturity.

Animals↗

PubMed1995

The kinetics of induction of Hox1.6 and C-jun mRNA during three different ways of inducing differentiation in teratocarcinoma F9 cells.

Changes in Hox1.6 and c-jun gene expression were examined upon F9 cell differentiation that was induced by three independent methods: a drug treatment with retinoic acid (RA), that with sodium butyrate (NaB), and a genetic approach using the ts mutant. To obtain further information on the mechanism of teratocarcinoma cell differentiation we have examined the kinetics of the induction of Hox1.6 and c-jun mRNA whose gene products have been demonstrated to have specific roles in gene regulation. Expression of Hox1.6 mRNA was induced more rapidly than c-jun mRNA by all the above three inducing methods. Furthermore, protein synthesis was not required for the induction of Hox1.6 mRNA as well as of c-jun mRNA synthesis in all three methods. The data suggested that the transcriptional increase in the Hox1.6 mRNA was a primary response and could play an important role in F9 cell differentiation.

Butyrates↗

PubMed1995

Characterization of development-specific, cell type-specific mouse testicular antigens using testis-specific polyclonal antibodies.

Antisera were raised by immunizing rabbits with mouse testicular germ cells, and absorbed in vivo by injection into castrated male whole mice to obtain a specific antiserum which reacted with mouse germ cells. The expression of mouse testis-specific antigenic macromolecules was then studied immunochemically with the antiserum. Approximately 20 antigenic macromolecules with molecular weights ranging from 26 to 110 kD were detected in the normal adult testis. At least 12 of these were differentiation-specific antigens appearing during development of germ cells, while others were expressed in testicular germ and/or somatic cells or detected only in mature spermatozoa. This technique for raising testis-specific antisera could be useful for isolation of cDNA clones encoding their antigens, as well as for investigation of the physiological roles of these molecules in germ cell differentiation at the molecular level.

Animals↗

PubMed1995

Insulin-like growth factor-I and transforming growth factor-alpha stimulate differentiation of type A spermatogonia in organ culture of adult mouse cryptorchid testes.

This study assessed the effect of growth factors on testicular germ cell differentiation in vitro. Testicular fragments of experimentally prepared cryptorchid testes of adult mice were cultured for 9 days in serum-free media containing various concentrations of IGF-I, TGF-alpha, FGF, and PDGF. Their histology was then examined under a light microscope. Each type of germ cell and mitotic cell in the seminiferous tubules was counted per 1000 Sertoli cells. IGF-I at a concentration of 10 ng/ml induced maximal differentiation of type A spermatogonia. TGF-alpha at concentrations ranging from 1 to 10 ng/ml also stimulated differentiation, whereas FGF and PDGF did not show any stimulation of spermatogonial differentiation in this experimental system.

Animals↗