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Toshikazu Ushijima - One of the best experts on this subject based on the ideXlab platform.
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a rat genetic map constructed by Representational Difference Analysis markers with suitability for large scale typing polymorphic markers dot blot hybridization quantitative trait loci
2016Co-Authors: Federico Canzian, Toshikazu Ushijima, Takashi SugimuraAbstract:Representational Difference Analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA (restriction enzymes, BamHI and HindlIl; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa) yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the DiNccl locus. Twenty-five of 27 RDA markers were informative regarding the dot blot Analysis of amplicons, hybridizing only with tester amplicons. Dot blot Analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.
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silencing of peroxiredoxin 2 and aberrant methylation of 33 cpg islands in putative promoter regions in human malignant melanomas
Cancer Research, 2006Co-Authors: Junichi Furuta, Yoshihiro Umebayashi, Kanako Kikuchi, Yoshimasa Nobeyama, Fujio Otsuka, Toshikazu UshijimaAbstract:Aberrant methylation of promoter CpG islands (CGI) is involved in silencing of tumor suppressor genes and is also a potential cancer biomarker. Here, to identify CGIs aberrantly methylated in human melanomas, we did a genome-wide search using methylation-sensitive Representational Difference Analysis. CGIs in putative promoter regions of 34 genes ( ABHD9, BARHL1, CLIC5, CNNM1, COL2A1, CPT1C, DDIT4L, DERL3, DHRS3, DPYS, EFEMP2, FAM62C, FAM78A, FLJ33790, GBX2, GPR10, GPRASP1, HOXA9, HOXD11, HOXD12, HOXD13, p14ARF, PAX6, PRDX2, PTPRG, RASD1, RAX, REC8L1, SLC27A3, TGFB2, TLX2, TMEM22, TMEM30B , and UNC5C ) were found to be methylated in at least 1 of 13 melanoma cell lines but not in two cultured normal melanocytes. Among these genes, Peroxiredoxin 2 ( PRDX2 ) was expressed in normal melanocytes, and its expression was lost in melanomas with methylation. The loss of expression was restored by treatment of melanomas with a demethylating agent 5-aza-2′-deoxycytidine. In surgical melanoma specimens, methylation of PRDX2 was detected in 3 of 36 (8%). Furthermore, immunohistochemical Analysis of PRDX2 showed that disappearance of immunoreactivity tends to associate with its methylation. PRDX2 was recently reported to be a negative regulator of platelet-derived growth factor signaling, and its silencing was suggested to be involved in melanomas. On the other hand, 12 CGIs were methylated in ≥9 of the 13 melanoma cell lines and are considered as candidate melanoma biomarkers. (Cancer Res 2006; 66(12): 6080-6)
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silencing of peroxiredoxin 2 and aberrant methylation of 33 cpg islands in putative promoter regions in human malignant melanomas
Cancer Research, 2006Co-Authors: Junichi Furuta, Yoshihiro Umebayashi, Kanako Kikuchi, Yoshimasa Nobeyama, Fujio Otsuka, Toshikazu UshijimaAbstract:Aberrant methylation of promoter CpG islands (CGI) is involved in silencing of tumor suppressor genes and is also a potential cancer biomarker. Here, to identify CGIs aberrantly methylated in human melanomas, we did a genome-wide search using methylation-sensitive Representational Difference Analysis. CGIs in putative promoter regions of 34 genes ( ABHD9, BARHL1, CLIC5, CNNM1, COL2A1, CPT1C, DDIT4L, DERL3, DHRS3, DPYS, EFEMP2, FAM62C, FAM78A, FLJ33790, GBX2, GPR10, GPRASP1, HOXA9, HOXD11, HOXD12, HOXD13, p14ARF, PAX6, PRDX2, PTPRG, RASD1, RAX, REC8L1, SLC27A3, TGFB2, TLX2, TMEM22, TMEM30B , and UNC5C ) were found to be methylated in at least 1 of 13 melanoma cell lines but not in two cultured normal melanocytes. Among these genes, Peroxiredoxin 2 ( PRDX2 ) was expressed in normal melanocytes, and its expression was lost in melanomas with methylation. The loss of expression was restored by treatment of melanomas with a demethylating agent 5-aza-2′-deoxycytidine. In surgical melanoma specimens, methylation of PRDX2 was detected in 3 of 36 (8%). Furthermore, immunohistochemical Analysis of PRDX2 showed that disappearance of immunoreactivity tends to associate with its methylation. PRDX2 was recently reported to be a negative regulator of platelet-derived growth factor signaling, and its silencing was suggested to be involved in melanomas. On the other hand, 12 CGIs were methylated in ≥9 of the 13 melanoma cell lines and are considered as candidate melanoma biomarkers. (Cancer Res 2006; 66(12): 6080-6)
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construction of a high throughput rat genetic mapping system with 466 arbitrarily primed Representational Difference Analysis markers
Mammalian Genome, 2000Co-Authors: Satoshi Yamashita, Takashi Sugimura, Yukinari Yoshida, Ayako Kurahashi, Toshikazu UshijimaAbstract:Linkage mapping of quantitative trait loci (QTLs) requires genetic markers that can be efficiently genotyped for a large number of individuals. To isolate genetic markers suitable for this purpose, we previously established the arbitrarily primed RDA (AP-RDA) method. Dot-blotting AP-PCR products (AP-amplicons) onto filters at a high density and hybridization of the filters with the AP-RDA markers made it possible to genotype a large number of individuals simultaneously for multiple loci. In this study, by using 25 primers or primer combinations, we isolated a total of 419 AP-RDA markers by subtracting the AP-amplicon of BUF rats from that of ACI rats, and vice versa. By combining 47 previously isolated markers, a rat genetic map was drawn with 466 AP-RDA markers. Between two given strains of rats other than ACI and BUF, the average informativeness of the markers was 38%. As for the intercross of ACI and BUF rats, 12 selected primers served to genotype 259 loci. In addition, the amounts and quality of genomic DNA to be used for AP-PCR were examined to guarantee reliable genotyping. Now, initial genome scanning of the rat for linkage Analysis can be performed efficiently using this mapping system with AP-RDA markers.
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isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by b1 repetitive sequence Representational Difference Analysis
Mammalian Genome, 1998Co-Authors: Toshikazu Ushijima, Takashi Sugimura, Tomoko Nomoto, David E Housman, Minako NagaoAbstract:Mapping of genetic suppressors, modifiers, and quantitative trait loci (QTLs) requires genetic markers that can be efficiently and inexpensively genotyped for a large number of individuals. To isolate rat genetic markers suitable for this purpose, Representational Difference Analysis (RDA) was performed with amplicons prepared by PCR with the B1 repetitive sequence used as the primer (B1-amplicons). In total, 48 polymorphic DNA fragments were isolated by five series of RDA, subtracting the B1-amplicons prepared from an ACI/N (ACI) rat from those prepared from BUF/Nac (BUF), and vice versa. All the polymorphic fragments detected ``presence-or-absence'' polymorphisms with B1-amplicons prepared from ACI, BUF, and their F2 progeny, and each fragment was linkage mapped. Dot-blotting amplicons onto filters at a high density and hybridization of the filters with these B1-RDA markers made it possible to genotype a large number of rats simultaneously for multiple loci. These B1-RDA markers were polymorphic between two given inbred strains of rat at frequencies between 30% and 70%. This is the first report on the isolation of B1-RDA markers among inbred strains of rats.
Atso Raasmaja - One of the best experts on this subject based on the ideXlab platform.
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cellular stress and p53 associated apoptosis by juniperus communis l berry extract treatment in the human sh sy5y neuroblastoma cells
International Journal of Molecular Sciences, 2016Co-Authors: Tiina A Lantto, H Damien J Dorman, Into Laakso, Sulev Koks, Timo Mauriala, Raimo Hiltunen, Atso RaasmajaAbstract:Plant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (Representational Difference Analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition Analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds.
Minako Nagao - One of the best experts on this subject based on the ideXlab platform.
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isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by b1 repetitive sequence Representational Difference Analysis
Mammalian Genome, 1998Co-Authors: Toshikazu Ushijima, Takashi Sugimura, Tomoko Nomoto, David E Housman, Minako NagaoAbstract:Mapping of genetic suppressors, modifiers, and quantitative trait loci (QTLs) requires genetic markers that can be efficiently and inexpensively genotyped for a large number of individuals. To isolate rat genetic markers suitable for this purpose, Representational Difference Analysis (RDA) was performed with amplicons prepared by PCR with the B1 repetitive sequence used as the primer (B1-amplicons). In total, 48 polymorphic DNA fragments were isolated by five series of RDA, subtracting the B1-amplicons prepared from an ACI/N (ACI) rat from those prepared from BUF/Nac (BUF), and vice versa. All the polymorphic fragments detected ``presence-or-absence'' polymorphisms with B1-amplicons prepared from ACI, BUF, and their F2 progeny, and each fragment was linkage mapped. Dot-blotting amplicons onto filters at a high density and hybridization of the filters with these B1-RDA markers made it possible to genotype a large number of rats simultaneously for multiple loci. These B1-RDA markers were polymorphic between two given inbred strains of rat at frequencies between 30% and 70%. This is the first report on the isolation of B1-RDA markers among inbred strains of rats.
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a rat genetic map constructed by Representational Difference Analysis markers with suitability for large scale typing
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Minoru Toyota, Toshikazu Ushijima, Federico Canzian, Takashi Sugimura, Yoko Hosoya, Takashi Kuramoto, Tadao Serikawa, Kohzoh Imai, Minako NagaoAbstract:Abstract Representational Difference Analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot Analysis of amplicons, hybridizing only with tester amplicons. Dot blot Analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.
Minoru Toyota - One of the best experts on this subject based on the ideXlab platform.
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epigenetic inactivation of the candidate tumor suppressor gene hoxb13 in human renal cell carcinoma
Oncogene, 2006Co-Authors: Heiwa Okuda, W Ishida, Mutsumi Tsuchiya, Minoru Toyota, Masayuki Kamada, Mutsuo Furihata, Takashi Tokino, Taro ShuinAbstract:Epigenetic alterations like DNA methylation and the resulting inactivation of cancer-related genes often contribute to the development of various cancers. To identify the genes that are silenced by aberrant methylation in renal cell carcinoma (RCC), we subjected two RCC lines to methylated CpG island amplification/Representational Difference Analysis. This identified 27 CpG islands. Combined bisulfite restriction Analysis of these CpG islands in primary RCC cases revealed that four were methylated in a tumor-specific manner. One of these was identified as the human homeo-box gene B13 (HOXB13) gene, but the remaining three CpG islands were not associated with known genes. The methylation frequencies of HOXB13 in primary RCC samples and lines were 30 and 73%, respectively. The methylation status of HOXB13 correlated with the loss of its expression both in RCC lines and primary tumors, and methyltransferase inhibitor treatment induced the recovery of its expression. Exogenous expression of HOXB13 in RCC cells that lacked endogenous HOXB13 expression suppressed colony formation and induced apoptotic features. Furthermore, HOXB13 methylation correlated positively with tumor grade and microvessel invasion. These results suggest that HOXB13 is a novel candidate tumor suppressor gene in RCC and that its inactivation may play an important role in both RCC tumorigenesis and progression.
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identification of scn3b as a novel p53 inducible proapoptotic gene
Oncogene, 2004Co-Authors: Katsuya Adachi, Minoru Toyota, Yasushi Sasaki, Toshiharu Yamashita, Setsuko Ishida, Mutsumi Ohetoyota, Reo Maruyama, Yuji Hinoda, Tsuyoshi SaitoAbstract:Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. To identify novel p53-inducible genes, we compared the expression of genes in normal mouse embryo fibroblasts (MEFs) to p53-null cells by cDNA Representational Difference Analysis. We report here that expression of endogenous sodium channel subunit beta 3 (SCN3B) is upregulated in mouse embryonic fibroblasts by DNA damage in a p53-dependent manner. In addition, we found that SCN3B levels are upregulated in human cancer cell lines by DNA damaging agents, as well as by overexpression of p53, but not significantly by p63 or p73. Furthermore, we identified two putative p53-binding sites upstream of the first exon (RE1) and in the third intron (RE2). The p53 protein can directly interact with the putative p53-binding sites in vivo, as assessed by chromatin immunoprecipitation. A reporter gene assay revealed that these two p53-binding sites are functional response elements. The SCN3B protein appears to be localized to the endoplasmic reticulum (ER). Introduction of the SCN3B gene into T98G and Saos2 cells potently suppressed colony formation. Furthermore, we found that adenovirus-mediated transfer of SCN3B induced apoptosis when combined with anticancer agents. The results presented here suggest that SCN3B mediates a p53-dependent apoptotic pathway and may be a candidate for gene therapy combined with anticancer drugs.
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a rat genetic map constructed by Representational Difference Analysis markers with suitability for large scale typing
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Minoru Toyota, Toshikazu Ushijima, Federico Canzian, Takashi Sugimura, Yoko Hosoya, Takashi Kuramoto, Tadao Serikawa, Kohzoh Imai, Minako NagaoAbstract:Abstract Representational Difference Analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot Analysis of amplicons, hybridizing only with tester amplicons. Dot blot Analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.
Takashi Sugimura - One of the best experts on this subject based on the ideXlab platform.
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a rat genetic map constructed by Representational Difference Analysis markers with suitability for large scale typing polymorphic markers dot blot hybridization quantitative trait loci
2016Co-Authors: Federico Canzian, Toshikazu Ushijima, Takashi SugimuraAbstract:Representational Difference Analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA (restriction enzymes, BamHI and HindlIl; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa) yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the DiNccl locus. Twenty-five of 27 RDA markers were informative regarding the dot blot Analysis of amplicons, hybridizing only with tester amplicons. Dot blot Analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.
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construction of a high throughput rat genetic mapping system with 466 arbitrarily primed Representational Difference Analysis markers
Mammalian Genome, 2000Co-Authors: Satoshi Yamashita, Takashi Sugimura, Yukinari Yoshida, Ayako Kurahashi, Toshikazu UshijimaAbstract:Linkage mapping of quantitative trait loci (QTLs) requires genetic markers that can be efficiently genotyped for a large number of individuals. To isolate genetic markers suitable for this purpose, we previously established the arbitrarily primed RDA (AP-RDA) method. Dot-blotting AP-PCR products (AP-amplicons) onto filters at a high density and hybridization of the filters with the AP-RDA markers made it possible to genotype a large number of individuals simultaneously for multiple loci. In this study, by using 25 primers or primer combinations, we isolated a total of 419 AP-RDA markers by subtracting the AP-amplicon of BUF rats from that of ACI rats, and vice versa. By combining 47 previously isolated markers, a rat genetic map was drawn with 466 AP-RDA markers. Between two given strains of rats other than ACI and BUF, the average informativeness of the markers was 38%. As for the intercross of ACI and BUF rats, 12 selected primers served to genotype 259 loci. In addition, the amounts and quality of genomic DNA to be used for AP-PCR were examined to guarantee reliable genotyping. Now, initial genome scanning of the rat for linkage Analysis can be performed efficiently using this mapping system with AP-RDA markers.
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isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by b1 repetitive sequence Representational Difference Analysis
Mammalian Genome, 1998Co-Authors: Toshikazu Ushijima, Takashi Sugimura, Tomoko Nomoto, David E Housman, Minako NagaoAbstract:Mapping of genetic suppressors, modifiers, and quantitative trait loci (QTLs) requires genetic markers that can be efficiently and inexpensively genotyped for a large number of individuals. To isolate rat genetic markers suitable for this purpose, Representational Difference Analysis (RDA) was performed with amplicons prepared by PCR with the B1 repetitive sequence used as the primer (B1-amplicons). In total, 48 polymorphic DNA fragments were isolated by five series of RDA, subtracting the B1-amplicons prepared from an ACI/N (ACI) rat from those prepared from BUF/Nac (BUF), and vice versa. All the polymorphic fragments detected ``presence-or-absence'' polymorphisms with B1-amplicons prepared from ACI, BUF, and their F2 progeny, and each fragment was linkage mapped. Dot-blotting amplicons onto filters at a high density and hybridization of the filters with these B1-RDA markers made it possible to genotype a large number of rats simultaneously for multiple loci. These B1-RDA markers were polymorphic between two given inbred strains of rat at frequencies between 30% and 70%. This is the first report on the isolation of B1-RDA markers among inbred strains of rats.
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a rat genetic map constructed by Representational Difference Analysis markers with suitability for large scale typing
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Minoru Toyota, Toshikazu Ushijima, Federico Canzian, Takashi Sugimura, Yoko Hosoya, Takashi Kuramoto, Tadao Serikawa, Kohzoh Imai, Minako NagaoAbstract:Abstract Representational Difference Analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot Analysis of amplicons, hybridizing only with tester amplicons. Dot blot Analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.
