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Biomedical subjects

Y Ikawa

Publications and source records attributed to Y Ikawa.

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

Point mutation of p53 tumor suppressor gene in bovine leukemia virus-induced lymphosarcoma.

To elucidate the mechanism of leukemogenesis induced by bovine leukemia virus (BLV), the abnormality of p53 tumor suppressor gene was examined using the sequencing method of polymerase chain reaction (PCR)-amplified DNA from peripheral blood lymphocytes (PBL) and tumors from BLV-infected cattle that showed evidence of different stages during the progression of enzootic bovine leukosis (EBL). Mutations of the p53 gene were found in tumor cells from cattle with EBL, but not in PBL from BLV-free normal cattle or BLV-infected cattle without any evidence of tumor, suggesting mutation of p53 gene occurred specifically at the lymphoma stage of the disease. Twelve of eighteen cattle with EBL had seven missense and five silent mutations. The mutations were mapped to the highly evolutionarily conserved regions of p53 gene, and were involved in the DNA binding of p53. Thus, it appeared that the p53 point mutation is one of the critical events leading from the asymptomatic stage to the lymphoma stage.

Animals↗
PubMed1997

Up-regulation of cell cycle-associated genes by p53 in apoptosis of an erythroleukemic cell line.

A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. When these cells were cultured at 32 degrees C, the growth rate was reduced significantly and DNA fragmentation, a typical character of apoptosis, was observed. In this process, p53 migrated from cytoplasm to nucleus and protein complexes binding to the p53-responsive element were detected in nuclear extracts of the cells cultured at 32 degrees C by gel-shift assay and transactivation from the p53-responsive element was detected. The expression of the p21 (waf1/cip1/sdi1), cyclin G and gadd45 genes was increased (about 3 to 4 fold that at 37 degrees C), when the cells were cultured at 32 degrees C. However, the expression of the bax gene was increased slightly (about 1.5 fold that at 37 degrees C) and no significant change was detected in expression of the mdm2 gene. No change in the amount of Fas antigen was observed by flow cytometric analysis. Transcripts of the bcl-2 and fasl gene were not detected in the cells both at 37 degrees C and 32 degrees C. These results suggest that up-regulation of the genes associated with the cell cycle and/or DNA replication, such as p21, cyclinG and gadd45 rather than bax, fas, fasl and bcl-2 may be important for induction of apoptosis of this erythroleukemic cell line by p53.

Animals↗
PubMed1997

Activation of the JAK1-STAT5 pathway by binding of the Friend virus gp55 glycoprotein to the erythropoietin receptor.

Friend spleen focus forming-virus (F-SFFV) induces acute erythroleukemia in susceptible mice. Initiation of the erythroleukemia is due to binding of the env-related glycoprotein gp55 encoded by F-SFFV to the erythropoietin receptor (EPOR). The gp55/EPOR interaction induces prolonged and growth factor independent proliferation in a factor-dependent cell line. In erythropoietin (EPO) signaling, the JAK2/STAT5 pathway was shown to be activated downstream of the EPOR to transmit the signal to the cells. To determine members of the JAK family and the STAT transcription factor family involved in the gp55/EPOR signaling, we examined tyrosine phosphorylation of JAKs and STATs in F-SFFV-infected erythroid or erythroleukemic cells. JAK1 and STAT5 were constitutively tyrosine-phosphorylated but the DNA binding activity of STAT5 was not induced without EPO stimulation in erythroblastoid cells from spleens of F-SFFV-infected mice and erythroleukemia cell lines derived from gp55-transgenic mice. These results indicate that JAK1 is involved in the gp55/EPOR signaling but STAT5 is not playing an essential role in the growth of those erythroid cells.

Animals↗
PubMed1996

A murine neural-specific homolog corrects cholinergic defects in Caenorhabditis elegans unc-18 mutants.

Caenorhabditis elegans UNC-18 protein, homologous to yeast Sec1p, is important in neurotransmitter release, because the unc-18 mutation leads to severe paralysis and presynaptic acetylcholine (ACh) accumulation. To examine the functional conservation in mammals, we tried to isolate unc-18 isoforms from mouse and human brain cDNA libraries and obtained two classes of isoforms-neural genes and ubiquitous genes. Neural genes were identical to Munc-18 (also known as n-Sec1 or rbSec1), identified in rat and bovine brains as a syntaxin-binding protein. According to "Munc-18" terminology, we call the neural genes Munc-18-1 and the ubiquitous genes Munc-18-3. These mammalian isoforms exhibit 58% (Munc-18-1) and 42-43% (Munc-18-3) amino acid sequence identity with UNC-18. Next, we constructed transgenic unc-18 mutants to test biological activity of mouse Munc-18-1 and Munc-18-3 under the control of C. elegans unc-18 promoter. Munc-18-1 compensates for severe locomotion disability and cholinergic defects, e.g., abnormal sensitivities to cholinesterase inhibitors and cholinergic receptor agonists in unc-18 mutants, but Munc-18-3 fails. These data suggest that Munc-18-1 and C. elegans unc-18 may play positive roles in ACh release and that the molecular mechanism of neuronal regulated secretion has been partially conserved from nematodes to mammals.

Animals↗
PubMed1996

Characterization of the newly constructed domains that replace P5abc within the Tetrahymena ribozyme.

The P5abc domain of the Tetrahymena ribozyme has been shown to function as an activator that enhances core catalytic activity of the ribozyme. We reported previously that several new domains in that their primary sequences are different from that of P5abc are also capable of activating the ribozyme. It was unclear whether the mechanism of activation by the new domains is identical to that by P5abc. We have investigated structural and functional properties of the new domains and obtained evidence that strongly indicates that a particular domain activates the ribozyme in a different manner from that by P5abc.

Adenine↗
PubMed1996

A C3H strain-specific allele (alpha va126) of the murine alpha-globin gene newly detected by UT-PAGE and RT-PCR-SSCP analysis.

An extra band. distinct from the well-characterized globin chains (alpha, beta-maj, beta-min, beta-s), was detected in an adult erythrocyte sample of the C3H strain by urea triton polyacrylamide gel electrophoresis (UT-PAGE) analysis. The extra band was recognized by an antibody against the alpha-globin chain by Western blot analysis. Reverse transcription, polymerase chain reaction and single strand conformation polymorphism (RT-PCR-SSCP) analysis and direct sequencing analysis of cDNA of the alpha-globin gene revealed a nucleotide substitution (GGA to GTA) corresponding to an amino acid substitution (Gly to Val) at codon 26 in the alpha-globin gene only in the erythrocyte sample of the C3H strain. Polypeptides generated by in vitro translation from the alpha-globin gene with the nucleotide substitution at codon 26 (alpha Val26) had the same mobility as that of the extra band of the C3H strain in UT-PAGE. These results suggest that the substitution GGA (Gly) to GTA (Val) at codon 26 of the murine alpha-globin gene may directly affect the mobility of alpha-globin in UT-PAGE and the base substitution may be a C3H strain-specific polymorphism.

Alleles↗
PubMed1996

A follistatin-like gene, mac25, may act as a growth suppressor of osteosarcoma cells.

mac25, a retinoic acid-inducible gene that is expressed at high levels in senescent epithelial cells, was initially cloned as a gene that is differentially expressed in meningioma. Although the homology of its product with members of family of insulin-like growth factor-binding proteins was suggested, the product also exhibits strong homology to follistatin, an activin-binding protein. However, a domain corresponding to the carboxyl terminus of follistatin is not found in mac25. The carboxyl-terminally truncated form of follistatin, generated by alternative splicing, has stronger activin-binding activity than the complete form. This result suggests that mac25 might act as an activated follistatin. Clonal growth of a p53-deficient osteosarcoma cell line was strongly inhibited when the murine mac25 gene, as well as the p53 gene, was introduced. Resembling activins that belong to the transforming growth factor-beta (TGF-beta) superfamily, mac25 and p53 might associate with similar but distinct targets, namely cyclin-dependent kinase inhibitors. However, there is no evidence for compensation of p53 function by mac25 in the development of p53-deficient mice, as judged from the pattern of expression of mac25 in mice. mac25 might act as a tumor suppressor, modulating signaling of the TGF-beta family, as does alpha-inhibin.

Amino Acid Sequence↗
PubMed1996

Water channel protein subtype suggests the origin of renal cell carcinoma.

PURPOSE: Several subtypes of water selective channel protein have been cloned. In normal kidneys, CHIP-28 (channel forming integral protein of 28 kd) is expressed solely in the proximal tubules and descending thin limbs, and aquaporin-CD (AQP-CD) expression is restricted to collecting ducts. We assessed expression of these 2 distinct types of water channels in renal cancer tissues. MATERIALS AND METHODS: Expression of CHIP-28 and AQP-CD was examined in 12 samples of primary renal cell carcinoma by Northern blot, reverse transcriptase-polymerase chain reaction and immunohistochemistry. We also evaluated expression of these 2 molecules immunohistochemically in 4 samples of Bellini duct tumor, which has been thought to arise from renal collecting ducts. RESULTS: Expression of CHIP-28 was detected in all of the samples of primary renal cell carcinoma, whereas none of them expressed AQP-CD. None of 4 samples of Bellini duct tumor expressed CHIP-28, although expression of AQP-CD was recognized in 2. CONCLUSIONS: Our current observation confirms the idea of the proximal or descending tubule origin of renal cell carcinoma.

Aged↗
PubMed1996

Genomic imprinting of the human serotonin-receptor (HTR2) gene involved in development of retinoblastoma.

Epidemiological and genetic studies of retinoblastoma (RB) suggested that imprinted genes might be genetically linked to the RB gene. In this study, we found that the human serotonin-receptor, HTR2, gene, which had been mapped nearby the RB gene on chromosome 13, was expressed only in human fibroblasts with a maternal allele and not in cells without a maternal allele. The 5' genomic region of the human HTR2 gene was cloned by PCR-mediated method. Only the 5' region of the gene was methylated in cells with the maternal gene, and it was not methylated in cells without the maternal gene. A polymorphism of PvuII site of the gene was also found and useful for the segregation analysis in a family of a RB patient and for analysis of loss of heterozygosity on chromosome 13 in tumor and its parental origin. These results suggest that the human HTR2 gene might be affected by genomic imprinting and that exclusive expression of the maternal HTR2 gene may be associated with the delayed occurrence of RB, which had lost the maternal chromosome 13.

Alleles↗
PubMed1995

Molecular cloning of a novel serine/threonine kinase, MRK, possibly involved in cardiac development.

We have isolated a novel member of putative serine/threonine kinase from a rat heart cDNA library using polymerase chain reaction methods. The novel kinase is transcribed as 2.6 kb mRNA encoding for a protein of 629 amino acids with the C-terminal non-catalytic portion. Amino acids analysis revealed that the N-terminal catalytic domain is 87% identical to the male-germ cell associated kinase (MAK), a cdc2-related serine/threonine kinase found to promote meiosis during spermatogenesis. Therefore, we designated this novel kinase as the MAK-related kinase (MRK). MRK protein, with a molecular weight of 66 kD, was shown to phosphorylate itself and the exogenous substrates, histone H1 and myelin basic protein. In addition, phosphoamino acid analysis confirmed the serine/threonine-specific protein kinase activity of MRK. Although MRK was ubiquitous in adult rat tissues, the expression of MRK protein in embryos was restricted primarily to embryonic myocardium during early organogenesis. This finding suggests that MRK may be a participant in cardiac development.

Amino Acid Sequence↗
PubMed1995

Repression of p53-dependent sequence-specific transactivation by MEF2c.

Lambda ZAP cDNA library constructed from spleen of a p53-deficient mouse was screened by South-Western technique using Fragment A, a DNA sequence that p53 specifically binds to, as a probe. One (clone 2) of six clones isolated was identical to MEF2c, a MADS-family transcription factor. Transcripts of the mef2c gene was also detected in spleen where the expression has not been reported so far. Isolated clones except clone 2 had a growth-suppression activity on p53-deficient osteosarcoma cell line (Saos II). Clone 2 repressed the transactivation from Fragment A by p53, suggesting that MEF2c may act as a negative regulator of p53-responsive element.

Animals↗
PubMed1995

K rev-1 protein is abundantly expressed in the rat spinal cord.

The K rev-1 gene, which was originally identified as a dominantly functioning tumor suppressor gene inducing a flat revertant of a v-K-ras-transformed NIH 3T3 cell line, was abundantly expressed in the mammalian brain [Kitayama et al. (1989) Cell 56, 77-84]. To investigate where K rev-1 and its family ras proteins are distributed in the central nervous system, we isolated the membrane fractions from several regions of the brain and spinal cord of rats by subcellular fractionation and analyzed those proteins by immunoblot analysis with the specific monoclonal antibodies, K rev-1 protein was detected at the highest level in the spinal cord among areas of the central nervous system which included cerebral cortex, cerebellum, hippocampus, and olfactory bulb. On the other hand, ras proteins were found at similar levels in these regions. Within the spinal cord, K rev-1 and ras proteins were detected at a comparable level in the ventral and dorsal parts, while they were much less in the dorsal root ganglion than in the spinal cord. They showed the differential expression during early postnatal development: K rev-1 protein increased and ras proteins were at relatively high levels. When K rev-1 and ras proteins were examined in synaptosomes from the lumbar spinal cord of newborn rats, most of them were detected not in the synaptic vesicles but in the synaptic plasma membranes. K rev-1 protein as well as ras proteins might be involved in neuronal functions in the spinal cord such as sensory processing and motor control.

Animals↗
PubMed1995

Focal adhesion kinase is not essential for in vitro and in vivo differentiation of ES cells.

Focal adhesion kinase, FAK, is a unique protein tyrosine kinase found in cellular focal adhesions. It is widely expressed and highly phosphorylated during embryogenesis. To examine the function of FAK in cell differentiation, we made FAK-deficient embryonic stem (ES) cells by homologous recombination. However, FAK-deficiency did not interfere with differentiation of the ES cells into cells of three germ layers when implanted subcutaneously into nude mice or when treated with retinoic acid in vitro, nor was there any evidence of defects in hematopoiesis in vitro.

Animals↗
PubMed1995

A new strategy of gene trapping in ES cells using 3'RACE.

"Gene trapping" in embryonic stem (ES) cells is a novel approach to identify a series of genes in mammals concomitant with the production of the corresponding mutant mice. However, this approach is currently unable to identify genes that are not expressed in ES cells. Here we describe a strategy to identify gene trapping clones which is not based on expression of a reporter gene. It uses the neor gene which lacks a polyadenylation signal and has a splice donor signal. Expression of the neor gene as fusion transcripts with the 3' end containing the polyadenylation signal of tagged genes allows the identification of these clones by 3' rapid amplification of the cDNA end in undifferentiated ES cells, even if the genes are not expressed in ES cells. Amplification was observed in about 25% of G418-resistant clones. Sequence analyses suggested the amplifications represent gene trapping events. The feasibility of this approach was further assessed by analysing one clone, PAT-12, in detail.

Amino Acid Sequence↗
PubMed1995

Ovarian teratomas in mice lacking the protooncogene c-mos.

Parthenogenesis has been suggested to be tightly coupled with development of ovarian teratomas. Indeed, ovarian tumors developed in c-mos-deficient female mice, which are characterized by the parthenogenetic activation of oocytes. The tumors appeared at a frequency of 30% between 4 and 8 months of age, and did not develop in younger or older mice. Most of the tumors were benign and consisted of multi-focal cysts most notably with mature ectodermal components, but also with mesodermal and endodermal components. One among 17 tumors observed consisted of extra-embryonic tissues alone, and two bore malignant components with metastasis to peritoneal organs. The results strongly suggest the involvement of c-mos mutations in human germ cell tumors.

Age Factors↗
PubMed1995

A mouse erythroleukemia cell line possessing friend spleen focus-forming virus gp55 transgene and temperature-sensitive mutant p53 gene.

Two different erythroleukemia cell lines have been established from the splenic lesions of transgenic mice possessing the Friend spleen focus-forming virus (F-SFFV) gp55 gene. One showed a near-diploid karyotype and a temperature-sensitive (ts) p53 mutation, and the other, a hyper-triploid karyotype with double p53 mutations found by single-strand conformation polymorphism (SSCP) analysis. The cell lines both retained No.11 chromosomes on which p53 genes are localized. Another p53 allele in the cell line with the ts-p53 mutation appeared intact in the SSCP analysis of the genomic exon 5. The cells with the ts-mutant p53 gene showed no apparent change with temperature shift in their growth or dimethylsulfoxide-induced differentiation, although the wild-type p53 gene on the other allele was not expressing. This ts-p53Val-135 gene made p53-deficient fibroblasts anchorage-independent at 37 degrees C but not at 32 degrees C. This non-virus-producing, mouse erythroleukemia cell line will be useful for the study of mutated p53 function during the induction of erythrodifferentiation or apoptotic change.

Animals↗
PubMed1995

Both the changes of six amino acids and the C-terminal truncation caused by a one-base insertion in the defective env gene of Friend spleen focus-forming virus significantly affect the pathogenic activity of the encoded leukemogenic membrane glycoprotein (gp55).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in mice and encodes in its defective env gene an Env-like membrane glycoprotein (gp55). The F-SFFV env gene has three characteristic structures compared with that of ecotropic murine leukemia viruses (MuLVs): substitution by the polytropic MuLV env sequence, a 585-bp deletion, and a 1-bp insertion. All of these characteristic structures are essential for the leukemogenic potential of gp55 of polycythemia-inducing isolates of F-SFFV (F-SFFVp). The 1-bp insertion causes changes of six amino acids and truncation by 34 amino acids at the C terminus. In this study, we constructed 12 mutant F-SFFV genomes starting from the wild-type F-SFFVp and examined the effect of the C-terminal truncation and the six altered amino acids on the pathogenic activity of gp55. The results indicated that at least 18 to 24 amino acids must be deleted from the C terminus for the env product to be pathogenically active. We also found that the six altered amino acids contributed significantly to the pathogenic activity of gp55. Analyses of the cellular processing of these mutant gp55s supported a correlation between the pathogenic activity of gp55 and its efficiency in overall cellular processing.

3T3 Cells↗