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Y Tomooka

Publications and source records attributed to Y Tomooka.

At least 19 recordsLinked to original sources
PubMed2001

Mouse nestin cDNA cloning and protein expression in the cytoskeleton of transfected cells.

Complementary DNA (cDNA) corresponding to mouse nestin intermediate filament protein, a specific marker for neural stem cells, was isolated and characterized. The complete sequence comprised 5983 base pairs encoding 1821 amino acids, and the deduced polypeptide was similar to rat (84%), hamster (73%), and human (62%) nestin. Southern blots showed that mouse nestin was a single-copy gene, and Northern blots detected a 6.0 kilobase mRNA transcript. When the cDNA was overexpressed as an enhanced green fluorescent fusion protein in COS7 cells, nestin immunoreactivity appeared in the filamentous cytoskeletal network. Accordingly, biologically active mouse nestin cDNA may offer an important new tool for stem cell research.

Amino Acid Sequence↗
PubMed2001

Establishment of an androgen-responsive prostatic cell line "PEA5" from a p53-deficient mouse.

BACKGROUND: We demonstrated that p53-deficiency is sufficient for the establishment of clonal cell lines from the uterus and prostate. In the present study, we improved cloning methods to establish androgen-responsive cell lines. METHODS: In our previous study, a prostatic cell line was established from the ventral prostate of a p53-deficient mouse and was maintained in a medium containing heat-inactivated fetal calf serum at 10% supplemented with insulin (10 microg/ml), transferrin (10 microg/ml), cholera toxin (10 ng/ml) and selenium (10(-8) M). In the present study, 5alpha-dihydrotestosterone (10(-8) M) was added to the medium from the beginning of cloning procedures. RESULTS: We succeeded in the establishment of an androgen receptor positive prostatic cell line, designated PEA5. PEA5 cells exhibited a typical epithelial morphology in culture and growth was stimulated by androgens in a dose-dependent manner. In addition, they grew and formed three-dimensional structures in collagen gel, in which growth was also stimulated by androgen. CONCLUSIONS: Although PEA5 lacks p53 gene, it still retains androgen sensitivity. In collagen gel culture, PEA5 cells can grow and form three-dimensional structures similar to those of the primary cultures reported previously. Furthermore, prostates of p53-deficient mice are shown to be useful sources for obtaining androgen-responsive cells lines.

Animals↗
PubMed1999

The adenovirus E1A domains required for induction of DNA rereplication in G2/M arrested cells coincide with those required for apoptosis.

Induction of apoptosis by adenovirus E1A in rodent cells is stimulated by wild type (wt) p53 but completely suppressed by mutated p53. The suppression is overcome by coexpression with Id proteins (Ids). The cells expressing E1A and Ids undergo apoptosis after accumulation in S phase, suggesting that S phase events are perturbed by E1A and Ids. The E1A domains required for induction of apoptosis, analysed by transfection with expression vectors for E1A, Ids and their mutants, followed by flow cytometry, reside in N-terminal (positions 17 - 38), CR1 and CR2 regions. Interaction of E1A with Ids requires the N-terminal and CR1 regions. The cyclin D1 promoter activity in S phase was reduced severely by E1A and this reduction is caused through CR1 and CR2 regions required for interaction with pRB. Analysis of DNA synthesis in G2/M arrested cells indicated that E1A is capable of inducing >4 N cells and this E1A-mediated DNA rereplication is enhanced by coexpression with Id-1H. The E1A domains required for induction of DNA rereplication coincide with those required for apoptosis.

Adenovirus E1A Proteins↗
PubMed1999

Effects of wild-type and mutated p53 and Id proteins on the induction of apoptosis by adenovirus E1A, c-Myc, Bax, and Nip3 in p53 null mouse cerebellum cells.

The sensitivities of apoptosis induced by E1A, c-Myc, Bax, and Nip3 to wild-type (wt) and mutated p53 and Id proteins were analyzed by transient transfection followed by flow cytometry with p53 null mouse cerebellum cell lines W7 and M13 that express wt and mutated p53 in response to dexamethasone, respectively. Apoptosis induced by c-Myc was stimulated weakly by wt p53, strongly by Ids, but suppressed completely by mutated p53 irrespective of coexpression with Ids, while apoptosis induced by E1A was suppressed by mutated p53 but stimulated when coexpressed with Ids. Apoptosis induced by Bax was little affected by wt and mutated p53, but inhibited by Ids, while apoptosis induced by Nip3 was inhibited by both wt and mutated p53 and inhibition was stimulated by Ids. Caspase-1 was activated only by Bax significantly when coexpressed with mutated p53 but not with wt p53. These results indicate that the apoptotic processes elicited by these inducers are different and differently affected by wt and mutated p53 and by Ids.

Adenovirus E1A Proteins↗
PubMed1998

Cerebellar cell lines established from a p53-deficient adult mouse.

p53-Deficient mice are prone to develop spontaneous tumors [L.A. Donehower, M. Harvey, B.L. Slagle, M.J. McArthur, C.A. Montgomery Jr. , J.S. Butel, A. Bradley, Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors, Nature 356 (1992) 215-221], but brain neoplasms are rare and difficult to culture in vitro. Here we describe cloning and long-term culture of heterogeneous neural cell lines from a multifocal cerebellar neoplasm that arose in an adult p53-/- mutant mouse. These lines may be useful for studies of neoplastic transformation and cell lineage in cerebellar development.

Animals↗
PubMed1998

An alternatively spliced fibroblast growth factor (FGF)-5 mRNA is abundant in brain and translates into a partial agonist/antagonist for FGF-5 neurotrophic activity.

We detected in the brain and then cloned two novel, short forms of human and mouse fibroblast growth factor (FGF)-5 mRNA, which were designated human FGF-5S (hFGF-5S) and mouse FGF-5S (mFGF-5S), respectively. Genomic analysis indicated that mFGF-5S and authentic mFGF-5 mRNAs were transcribed from a single gene; hFGF-5S and mFGF-5S mRNAs were generated by excluding the second exon of the respective FGF-5 genes, and the alternatively spliced mRNAs encoded for 123- and 121-amino acid proteins, respectively. Indeed, a neuron-like cell line expressing mFGF-5S mRNA secreted a protein of the expected size and with FGF-5 antigenicity. In PC12 cells, expression of hFGF-5 or exposure to hFGF-5 protein induced differentiation. Neither expression of hFGF-5S, alone, nor co-expression of hFGF-5S with hFGF-5 induced significant differentiation. At high concentrations, hFGF-5S protein partially antagonized FGF-5 activity, whereas by itself, hFGF-5S exerted very weak neurotrophic activity. hFGF-5S protein binds to FGF receptor (FGFR)-1 on PC12 transfectants and partially inhibits hFGF-5-induced tyrosine phosphorylation of FGFR-1 and an FGFR substrate, but it also induces phosphorylation by itself. These results suggest that FGF-5S is a naturally expressed partial agonist/antagonist of FGF-5 neurotrophic activity in the brain and that its effects are exerted in part at the level of the receptor.

Alternative Splicing↗
PubMed1998

Suppression of adenovirus E1A-induced apoptosis by mutated p53 is overcome by coexpression with Id proteins.

The rat 3Y1 derivative cell lines, EId10 and EId23, established by introducing the adenovirus E1A12S, Id-1H, and Id-2H cDNAs linked to the hormone-inducible promoter, express these proteins upon treatment with dexamethasone and elicit apoptosis, although these cell lines express mutated p53. The E1A mutants containing a deletion in either the N terminus or the conserved region 1 were unable to induce apoptosis in cooperation with Ids. Western blot analysis of the immunoprecipitates prepared from the dexamethasone-treated EId10 cell extract showed that Id-2H preferentially binds to E1A and E2A (E12/E47) helix-loop-helix transcription factors in vivo, but scarcely to the retinoblastoma protein. After induction of E1A and Ids, EId10 and EId23 cells began to accumulate in S phase and undergo apoptosis before entering G2 phase, suggesting that abnormal synthesis of DNA induced by coexpression of E1A, Id-1H, and Id-2H results in the induction of apoptosis. Apoptosis also is induced in mouse A40 (p53-/-) cells by E1A alone or E1A plus Ids after transient transfection of the expression vectors. The induction of apoptosis is stimulated by coexpression with wild-type p53; however, apoptosis induced by E1A alone was suppressed completely by coexpression with mutated p53, whereas apoptosis induced by E1A plus Ids was stimulated by the mutated p53 as done by wild-type p53. These results suggest that the suppressive function of mutated p53 is overcome by Ids.

Adenovirus E1A Proteins↗
PubMed1998

Establishment of prostatic cell line "Pro9ad" from a p53-deficient mouse.

BACKGROUND: We demonstrated that p53-deficiency is sufficient for immortalization of fetal uterine cells. In the present study, we further extended our previous observations to prostate tissues from a young p53-deficient adult mouse. METHODS: Cell lines were established from the ventral prostate of a p53-deficient male mouse and maintained in medium containing 10% heat-inactivated fetal calf serum supplemented with insulin (10 microg/ml), transferrin (10 microg/ml), cholera toxin (10 ng/ml), and selenium (10(-8) M). RESULTS: Pro9ad, one of the lines established, exhibits a typical epithelial morphology in culture. Despite the possession of androgen receptors, the growth of Pro9ad was not stimulated by 5alpha-dihydrotestosterone. Hepatocyte growth factor (HGF) slightly stimulated proliferation, whereas fibroblast growth factor-1 (FGF-1), keratinocyte growth factor (KGF), and platelet-derived growth factor AB (PDGF-AB) had no stimulating effect on growth. However, FGF-2, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) accelerated proliferation in a dose-dependent manner. EGF and IGF-1 additively stimulated growth. CONCLUSIONS: These results suggest that Pro9ad shares characteristics in common with primary prostatic epithelial cells despite p53-deficiency, and that p53-deficiency alone allows establishment of clonal cell lines of the prostate epithelium. Furthermore, the prostates of p53-deficient mice are useful sources for obtaining cell lines.

Animals↗
PubMed1998

Morphogenic activity of fibroblast growth factor-2 on primary neural precursor cells in three-dimensional culture.

Mouse neural precursor cells (NPC) were dissociated from fetal heads at the 10th day of gestation. When clumps of NPC were cultured in collagen gel, they grew and reorganized neural tube-like structures in medium containing fetal calf serum at 10% and supplemented with insulin, transferrin, cholera toxin and selenite. However, dissociated NPC died when they were cultured in collagen gel at low density in the same medium. Addition of fibroblast growth factor-2 (FGF-2) to this culture stimulated growth of NPC and formation of neural tube-like structures. The requirement for FGF-2 disappeared in high seeding density culture: they grew and formed neural tube-like structures without FGF-2. The structures formed in collagen gel were immunohistochemically positive against anti-FGF-2 antibody. The results show that the three-dimensional culture system provides a useful tool to study the roles of FGF-2 in morphogenesis of the central nervous system.

Animals↗
PubMed1998

Cell lines established from fetal brains of p53-deficient mice.

Brains from 7 p53-deficient mice on fetal day 13 were divided into 4 regions: cerebral cortex, cerebellum, upper spinal cord, and the rest of brain. They were trypsinized and cultured in medium containing 10% fetal calf serum. By dilution culture, 138 clonal lines were established from every regions. The lines were characterized morphologically and immunocytochemically as neuronal, glial, myogenic, or unidentified. They were cultured for more than a year, indicating that the lines are immortalized. p53-deficiency alone is sufficient for establishing clonal cell lines of the central nervous system.

Animals↗
PubMed1996

A possible role of the nestin protein in the developing central nervous system in rat embryos.

Rat embryos at the head-hold stage (Slc:SD strain; 9.5 days of gestation) were cultured for 48 h in rat serum with the anti-nestin peptide antiserum. The antiserum identified a single band in Western blots of the tissue extracts from rat embryos and stained the cells from the neural tube, migrating neural crest, and somites immunohistochemically. The antiserum-treated embryos appeared to develop normally for the most part. However, histological observation disclosed that the ventral portion of the neural tube was deformed. The cells in the deformed portion did not show the elongated shape but were round. These round cells tended to crowd near the ventricular surface, and a gap was observed between the original pial surface and cells arranged in the most pialward region. The penetration of the anti-nestin peptide antibody into the embryos from the culture medium was confirmed by visualization of the penetrated antibody using biotinylated anti-rabbit IgG antibody raised in goats and Texas red-conjugated streptavidin. These results indicate that the nestin protein plays an important role in the organization or the maintenance of neuroepithelial cells of the elongated shape spanning the neural tube from the luminar to the pial side.

Animals↗
PubMed1994

Extracorporeal urinary bypass for malignant ureteral obstruction.

Extracorporeal urinary bypass was attempted in a patient with malignant ureteral obstruction. A nephrostomy was drained into the bladder by connecting the tube to a cystostomy catheter. This method made the patient free from a collecting bag without any significant complications. This method may improve the quality of life of patients with malignant ureteral obstruction, when their bladder is intact.

Cystostomy↗
PubMed1994

A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption.

We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function.

Amino Acid Sequence↗
PubMed1993

Reconstruction of neural tube-like structures in vitro from primary neural precursor cells.

Vertebrate central nervous system develops from a neural tube derived from the embryonic ectoderm. In mouse, the neural tube around embryonic day 10 primarily consists of neural precursor cells (NPCs). During the development of embryonic central nervous system, NPCs proliferate and migrate outward; thus later stages show NPCs toward the lumen of the neural tube and neurofilament-positive differentiated cells toward the periphery. In conventional liquid culture, NPCs isolated from mouse on embryonic day 10 proliferate and differentiate into neurofilament-positive neurons. In the present communication, we show that fragments of neural tubes and aggregates of NPCs, when placed into collagen gel matrix, form three-dimensional structures which resemble the neural tube formed in vivo in the developing embryos. Even dissociated NPCs form the three-dimensional structures in the collagen gel matrix. Our results indicate that individual NPCs or fragments of neural tubes carry morphogenetic information which allows them to reconstruct neural tube-like structures in vitro.

Animals↗
PubMed1993

Enhanced proliferative potential in culture of cells from p53-deficient mice.

Normal somatic cells are endowed with limited doubling potential in culture, and the process of immortalization is an inevitable step in neoplastic transformation of the cells. To examine the roles of p53 in this process, the cells of p53-deficient mice were examined for doubling potential. Fibroblast-like cells from a variety of tissues of these mice proliferated continuously without showing aging or crisis. The aneuploid cells overcome the population with passage, but cloning experiment indicated that chromosomal changes were not essential to this process. The enhanced proliferative potential in culture of cells from the p53-deficient mice was also observed in epithelial cells of lens, mammary glands and seminal vesicles and in neural precursor cells. Proliferation of bone marrow cells in response to stem cell factor was enhanced in long term culture, but not in in vitro colony assay; no permanent cell lines could be obtained. No effects of p53-deficiency were found in proliferation of cardiac muscle cells or hepatocytes.

Aneuploidy↗
PubMed1992

A novel tyrosine kinase, hyk, expressed in murine embryonic stem cells.

To identify tyrosine kinases which play roles in mammalian early development, the 3' rapid amplification of cDNA ends (RACE) was performed on mouse embryonic stem (ES) cells. Among eight tyrosine kinases thus identified, we report here a novel tyrosine kinase, hyk (adhesion structures linked tyrosine kinase). The sequences of the 4.7 kb cDNA indicated the presence of RGD motif and three epidermal growth factor-like domains put between two immunoglobulin-like domains and three fibronectin type III domains in its extracellular region. It is strongly expressed in ES cells and later stages of embryos, but at low levels in midgestation embryos. It is also expressed at a low level in neural precursor cells from 10-day embryos, but at high levels in embryonic day 15 and neonatal brains. In adult tissues it is expressed ubiquitously.

Amino Acid Sequence↗