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O Niwa

Publications and source records attributed to O Niwa.

At least 37 records · Page 2Linked to original sources
PubMed2000

Increased frequencies of gene and chromosome mutations after X-irradiation in mouse embryonal carcinoma cells transfected with the bcl-2 gene.

Preimplantation stage mouse embryos are known to be highly sensitive to the killing effect of DNA-damaging agents such as radiation. Interestingly, however, this stage of development is well protected from radiation induction of malformation and carcinogenesis in postnatal life. In recent years, it has become clear that the stem cells of preimplantation stage embryos undergo extensive apoptosis after DNA damage. It has been postulated that this apoptosis is likely to be responsible for the resistance to malformation, by excluding cells carrying deleterious DNA damage. We have tested the possible role of apoptosis in elimination of gene and chromosome mutations in undifferentiated mouse embryonal carcinoma cell line, F9, transfected with human bcl-2 cDNA. The colony radiosensitivity of F9 cells was not affected by overexpression of the bcl-2 gene, but the apoptotic cell death was suppressed, as examined by DNA ladder assay and Hoechst staining. This suppression was accompanied by an increase in the frequencies of hprt mutation and micronucleus formation after X-irradiation. These results support the idea that maintenance of genomic integrity during early development is likely to be executed by apoptotic elimination of cells at risk.

Animals↗
PubMed2000

The effect of caffeine on p53-dependent radioresponses in undifferentiated mouse embryonal carcinoma cells after X-ray and UV-irradiations.

The effect of caffeine was studied on the radioresponses of undifferentiated mouse embryonal carcinoma cells (EC cells) with or without the functional p53. The radioresponses studied included radiosensitivity, the activation of p53, apoptosis with characteristic DNA ladder formation and cell cycle progression. An undifferentiated mouse EC cell line, ECA2, and a newly established p53-deficient EC cell line, p53 delta, were used in the present study. The status of the p53 gene did not significantly affect the colony survivals of undifferentiated EC cells to X-rays and UV. Although a post-irradiation treatment with caffeine sensitized both lines to X-rays marginally, the sensitization was prominent for UV regardless of the p53 status of the cells. The activation of a p53 responsible lacZ reporter construct was observed in stably transfected ECA2 cells after X-ray and UV irradiations. Caffeine suppressed the X-ray induced activation of the lacZ reporter, while it drastically enhanced the activation after UV irradiation. X-rays and UV readily triggered the apoptosis of ECA2 cells with the characteristic DNA ladder. Although UV-induced DNA ladder formation was enhanced by caffeine, that induced by X-rays was unaffected. Therefore, the effects of caffeine on the p53-dependent radioresponses were found to be agent specific: suppression for the X-ray induced and augmentation for the UV induced. In contrast to p53-proficient ECA2 cells, smear-like DNA degradation was observed for irradiated p53 delta cells, suggesting the presence of a mode of cell death without DNA ladder formation. UV induction of the smear-like DNA degradation was enhanced in the presence of caffeine. Regardless of the state of the p53 gene, G1/S arrest was not observed in X-ray and UV irradiated EC cells. X-ray induced G2/M arrest in both lines, which was abrogated by caffeine, while G2/M arrest after UV was unaffected by a caffeine treatment. These results indicate that the radioresponses of undifferentiated EC cells differ considerably from those of somatic cells, and that these radioresponses were modulated by a post-irradiation treatment with caffeine.

Animals↗
PubMed1999

Down-regulation of nuclear factor kappaB is required for p53-dependent apoptosis in X-ray-irradiated mouse lymphoma cells and thymocytes.

Transcription factors p53 and nuclear factor kappaB (NF-kappaB) have been implicated in apoptosis induced by DNA-damaging agents, but the relationship between these two factors at the molecular level is largely unknown. We have isolated apoptosis-resistant mutant sublines from a radiosensitive mouse lymphoma 3SB cell line that undergoes p53-depen-dent apoptosis after X-ray irradiation, and we have analyzed the NF-kappaB activity. Two of these apoptosis-resistant sublines expressed mutant p53 protein and exhibited a defect in the induction of cyclin-dependent kinase inhibitor p21 after X-ray irradiation. A decrease in the DNA binding activity of NF-kappaB was observed in the parental 3SB cells after exposure to X-rays, whereas the same activity was unaffected by radiation in the two mutant sublines. A similar down-regulation of NF-kappaB activity by X-rays was observed in thymocytes derived from p53 wild-type and heterozygous mice, but not in thymocytes from p53 homozygous knock-out mice. These results suggest that NF-kappaB inactivation is p53 dependent and is required for X-ray-induced apoptosis in thymic lymphoma cells and normal thymocytes.

Animals↗
PubMed1999

Homozygous deletions and point mutations of the Ikaros gene in gamma-ray-induced mouse thymic lymphomas.

Our previous genome-wide analysis of allelic loss for thymic lymphomas that were induced by gamma-irradiation in F1 hybrid mice between BALB/c and MSM strains suggested the centromeric region on chromosome 11 as a site harboring a tumor suppressor gene. Interestingly, to this region the mouse Ikaros gene was mapped which was postulated to participate in oncogenic process from the study of Ikaros knockout mice. Here we show fine allelic loss mapping in the vicinity of Ikaros in 191 lymphomas, indicating that the critical region of allelic loss was centered at the Ikaros locus. PCR analysis revealed that nine lymphomas failed to give PCR-amplification for either of two exon primer pairs, indicative of homozygous deletion. Six and five mutations were detected in the N-terminal zinc finger domain and the activation domain of Ikaros, respectively, and six of the eleven were frameshift or nonsense mutations that resulted in truncation of Ikaros protein. The results strongly suggest a direct role for Ikaros in development of mouse thymic lymphomas. This provides the experimental basis for further analysis of Ikaros mutations in human cancer.

Animals↗
PubMed1999

Allelic loss mapping and physical delineation of a region harboring a putative thymic lymphoma suppressor gene on mouse chromosome 12.

Our previous allelic loss analysis of gamma-ray induced thymic lymphomas in F1 hybrid and backcross mice between BALB/c and MSM strains mapped the Tlsr4 region exhibiting a high frequency of allelic loss (62%) to a 2.9 cM interval between the markers D12Mit53 and D12Mit279 on mouse chromosome 12. To narrow further the interval harboring a putative tumor suppressor gene, a high-density scan has been carried out for informative 361 thymic lymphomas. Construction of a physical map of Tlsr4 with 3 YAC and 15 BAC clones and isolation of YAC- and BAC-derived polymorphic probes lead to fine allelic loss mapping. Three successive polymorphic sites within one BAC exhibit the retention of both alleles in seven, one and four lymphomas, suggesting that a common region of allelic loss for Tlsr4 exists within the BAC region. Pulsed-field gel electrophoresis of NotI digests of this and other clones determines that the commonly lost region is a 35 kb interval with a NotI site. NotI sites are frequently associated with coding regions, and our preliminary sequencing has identified ESTs in the region. Thus, the present study facilitates the identification of genes in the Tlsr4 region that would lead to isolation of a novel tumor suppressor gene.

Alleles↗
PubMed1999

Mutations in the p53 and scid genes do not cooperate in lymphomagenesis in doubly heterozygous mice.

Analysis of double mutant mice of the p53 and scid genes, which have a combination of cell cycle checkpoint/apoptosis and DNA repair defects, shows that the latter defect synergistically enhances lymphoma development with loss of the former function. These mice lack the ability to eliminate lymphocytes predisposed to neoplastic transformation resulting from faulty antigen receptor gene rearrangement. Here we examine the cooperativity in double heterozygotes of p53 and scid in which normal development of lymphocytes is not impaired. MSM mice carrying a p53-knockout allele were crossed with BALB/c mice heterozygous for the scid locus and 129 offspring were obtained. They were subjected to gamma-ray irradiation, 84 thymic lymphomas being generated. The tumors and host mice were genotyped of p53 and scid. Among 42 mice developing p53-deficient lymphomas, scid/+ and +/+ genotypes did not provide difference in onset and latency. Besides, allelic loss of the Scid gene occurred at a high frequency in those lymphomas but the loss exhibited no allelic bias. The results suggest that the scid/+ genotype is not a modifier of loss of p53 function in the double heterozygotes.

Alleles↗
PubMed1999

Accumulation of recombinant chromosomes and low fidelity of transmission of chromosome X DNA markers in gamma-ray-induced lymphomas lacking p53.

F1 offspring of male MSM male mice with a p53-deficient (knockout) allele and normal female BALB/c mice were backcrossed with MSM mice to produce N2 mice. Female F1 and N2 mice were irradiated with gamma-radiation, and thymic lymphomas were obtained from 69 F1 and 82 N2 mice heterozygous for X chromosome markers. Of these 151 mice, 91 carried a p53-deficient allele. These lymphomas were analyzed for allelic loss by using four marker loci distributed on X chromosome to assess the stability of the inactive X chromosome, which contributes little to cellular functions. Twenty lymphomas showed allelic loss of all four loci, suggesting loss of a whole inactive X chromosome due to mitotic nondisjunction, whereas 24 lost only a part of an X chromosome, as a result of somatic recombination. The p53 status of the lymphomas was determined by genotyping and allelic loss analysis: 53 had retained two wild-type p53 alleles, suggesting normal function; 69 had lost both alleles, indicating loss of function; and the remaining 29 had at least one wild-type p53 allele, so their p53 status was unclear. Compilation of these two data revealed one nondisjunction-type change and five recombination-type mutations on X chromosome in 53 lymphomas retaining functional p53. In contrast, 14 and 16 of these alterations, respectively, were observed in 69 lymphomas lacking p53 function. These results suggest that p53 loss significantly increases the accumulation of recombinant chromosomes and decreases the fidelity of mitotic chromosome transmission of the X chromosome in gamma-ray-induced lymphomas.

Animals↗
PubMed1999

Analysis of the c-myc, K-ras and p53 genes in methylcholanthrene-induced mouse sarcomas.

We have examined 63 methylcholanthrene (MCA)-induced mouse sarcomas for possible correlations of mutations involving the c-myc, ras and p53 genes. The c-myc gene was found to be amplified in 18 of these sarcomas (29%). Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and subsequent direct sequencing identified 18 cases carrying K-ras mutation at codons 12, 13 and 61 (29%). No mutation was detected in the H-ras and N-ras genes. Mutations of the p53 gene in exons 5 to 8 were found in 45 cases (71%). Comparison of these mutations revealed that out of 18 cases with c-myc gene amplifications, 10 carried K-ras mutations (56%) and 14 carried p53 mutations (78%). In contrast, among 45 cases of sarcomas without c-myc gene amplification, 8 were found to have K-ras mutations (18%). The same 45 cases were found to have 31 p53 mutations (69%). The present study suggests a strong correlation between c-myc gene amplification and K-ras gene mutation (P < 0.01). p53 gene mutation was frequently found among MCA-induced mouse sarcomas, indicating the importance of this mutation in the etiology of these tumors. However, p53 mutations were present in sarcomas regardless of the state of c-myc amplification and K-ras mutation. Therefore, a defect in the p53 gene is independent of amplification of the c-myc gene or point mutation of the K-ras gene.

Animals↗
PubMed1999

Characterization of a fission yeast SUMO-1 homologue, pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation.

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of the pmt3(+) gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. The pmt3Delta cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found that pmt3(+) is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase- and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.

Amino Acid Sequence↗
PubMed1999

Accumulation of aberrant Y chromosomes in gamma-ray-induced thymic lymphomas lacking p53.

Male F(1) hybrids between MSM mice carrying a deficient p53 allele and BALB/c mice were irradiated with gamma-rays, and 80 thymic lymphomas were obtained, 46 of which developed in mice carrying the deficient p53 allele. Because the Y chromosome contributes little to cellular function, the stability of the Y chromosome in the tumors was assessed by polymerase chain reaction by examining three genes: Smcy and Sry on the short arm and Sts in the pseudoautosomal region of the long arm of the Y chromosome. Twenty-one lymphomas had lost one or two genes, probably as a result of mitotic recombination or interstitial deletion, whereas no lymphomas had lost all three genes. The p53 status of the lymphomas was determined by genotyping and allelic loss analysis; 34 had retained two wild-type p53 alleles, suggesting normal function; 34 had lost both alleles, indicating loss of function; and the other 12 had at least one wild-type p53 allele, so their p53 status was unclear. Compilation of these data revealed that changes in the Y chromosome were detected in only two of the 34 lymphomas retaining functional p53 but in 18 of the 34 lymphomas lacking p53 function, suggesting that p53 deficiency leads to an increase in the accumulation of radiation-induced aberrant chromosomes. This is consistent with our previous result from analysis of the inactive X chromosome. In contrast, a decrease in the fidelity of mitotic transmission in p53-deficient lymphomas was not noted for the Y chromosome.

Abnormalities, Radiation-Induced↗
PubMed1998

Allelic loss analysis of gamma-ray-induced mouse thymic lymphomas: two candidate tumor suppressor gene loci on chromosomes 12 and 16.

A total of 429 gamma-ray-induced thymic lymphomas were obtained from F1 and backcross mice between BALB/c and MSM strains, about a half of which carried a p53-deficient allele. A genome-wide allelic loss analysis has revealed two loci exhibiting frequent allelic losses but no allelic preference, one is localized within a 2.9 cM region between D12Mit53 and D12Mit279 loci on chromosome 12, and the other is near the D16Mit122/D16Mit162 loci on chromosome 16. The frequency of allelic loss in the D12Mit279 region is 62% and does not differ in tumors between the presence and absence of the p53-deficient allele. In contrast, the loss frequency of D16Mit122 is raised by the existence of p53-deficient allele: 62% for p63(-/+) and 13% for p53(+/+), suggesting co-operative function of the two losses. The D12Mit279 and D16Mit122 regions probably harbor different types of tumor suppressor gene that play key roles in lymphoma development.

Animals↗
PubMed1998

Plastic film carbon electrodes: enzymatic modification for on-line, continuous, and simultaneous measurement of lactate and glucose using microdialysis sampling.

Ring and split-disk plastic film carbon electrodes (PFCEs) were fabricated for use in thin-layer radial flow cells which were coupled to a microdialysis sampling system. PFCEs, were initially coated with osmium poly(vinylpyridine) redox polymer horseradish peroxidase (Os-gel-HRP). Then a second coat of oxidase enzyme was applied to produce enzyme bilayer (oxidase/Os-gel-HRP) PFCEs which were subsequently over-coated with cellulose acetate for use in the determination of glucose or lactate at 0 mV (vs Ag/AgCl). Split-disk electrode geometry enabled different oxidase enzymes to be immobilized on each half of a split-disk, Os-gel-HRP-coated, PFCE to facilitate the electrochemically independent yet continuous on-line determination of these two analytes from a single dialysate. In continuous-flow experiments, cellulose acetate overcoated oxidase/Os-gel-HRP cast-coated PFCEs were quick to stabilize background current and displayed linear and sensitive responses to substrates. The effect of ascorbic acid was minimal and cross talk between partner split-disk electrodes was demonstrated to be acceptable for in vivo applications. The utility of this analytical system is demonstrated by the quantitative on-line continuous assay of changes in dialysate striatal extracellular glucose and lactate from a conscious rat during (a) local stimulation of neurons by perfusion with the depolarizing agent, Veratridine, and (b) physical restraint.

Animals↗
PubMed1998

On-line electrochemical sensor for selective continuous measurement of acetylcholine in cultured brain tissue.

An on-line acetylcholine (ACh) sensor was developed in order to determine extracellular ACh concentrations without interference from choline (Ch). The sensor is composed of a small-volume enzymatic prereactor (22-microL inner volume) in which choline oxidase and catalase are immobilized in series. Carbon electrodes were modified with an acetylcholine esterase (AChE), choline oxidase (ChOx), and osmium poly(vinylpyridine)-based redox polymer containing horseradish peroxidase (Os-gel-HRP). The sensor sensitivity was 43.7 nA/microM (+/- 0.15, n = 3) for ACh under optimized conditions. Almost no response was seen when 100 microM Ch was continuously injected. The detection limit for ACh with the sensor was comparable to that obtained using liquid chromatography with electrochemical detection combined with an enzymatic reactor. The electrical stimulation of cultured rat hippocampal tissue resulted in an extracellular ACh increase of 20 nM (+/- 11 nM, n = 3). This increase was observed continuously with our online sensor combined with a microcapillary sampling probe located very close to the tissue. The continuous measurement of ACh and Ch using a split disk carbon film dual electrode in which one electrode surface was modified with ChOx/Os-gel-HRP and the other with AChE-ChOx/Os-gel-HRP bilayer film was also demonstrated to improve the response time by eliminating the prereactor.

Acetylcholine↗
PubMed1998

Direct detection of uncaged glutamate and the laser photostimulation of cultured rat cortex.

The photostimulation of nerve cells using a caged compound is very useful because it is non-invasive and non-destructive compared with standard electrophysiological techniques. There are no methods, however, for continuously measuring the photo-uncaged 'free' compound concentration at high temporal and spatial resolutions which can detect how much uncaged compound has been applied to cells. Here, we used an electrochemical detection method for the real-time measurement of photo-uncaged glutamate. In this way, we were able to determine the amount of uncaged glutamate and investigate neural activities by tracing [Ca]i while simultaneously employing photostimulation and on-line glutamate measurement. The combination of an on-line sensor and laser-photostimulation with [Ca]i measurement could be a powerful tool with which to investigate synaptic connections and activities.

Animals↗
PubMed1998

A novel fission yeast gene, tht1+, is required for the fusion of nuclear envelopes during karyogamy.

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1(+) gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1(+) gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.

Amino Acid Sequence↗
PubMed1998

Small-volume on-line sensor for continuous measurement of gamma-aminobutyric acid.

We report the first on-line electrochemical sensor for the continuous measurement of gamma-aminobutyric acid (GABA), which is a well-known inhibitory neurotransmitter in the nervous system. The sensor is composed of a glutamate oxidase (GluOx) and catalase immobilized small-volume enzymatic reactor and a glassy carbon (GC) electrode modified with a top layer film consisting of gabase and GluOx coimmobilized bovine serum albumin and an Ospoly(vinylpyrridine) bottom layer film containing horseradish peroxidase. The response of the sensor depends on the alpha-ketoglutarate concentration and is almost saturated when its concentration is 100 times higher than GABA. The sensor exhibits a sensitivity of 1.56 nA/microM for GABA under optimized conditions and shows almost no response when 10 microM glutamate is continuously injected. A detection limit of 0.1 microM is obtained with a linear range of 0.1-10 microM. GABA can be measured in the absence of alpha-ketoglutarate when there is L-glutamate in the sample solution, which is a typical condition for the extracellular measurement of cultured nerve cells.

Animals↗