Yoshiko Shimizu

Keio University, Edo, Tokyo, Japan

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Publications (37)151.12 Total impact

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    ABSTRACT: We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.
    No preview · Article · May 2015 · Chromosome Research
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    ABSTRACT: Resveratrol was glucosylated to its 3- and 4'-β-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4'-β-glucoside. P. americana cells converted piceatannol into its 4'-β-glucoside. The 3- and 4'-β-glucosides of resveratrol were further glucosylated to 3- and 4'-β-maltosides of resveratrol, 4'-β-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-β-glucoside and 3-β-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4'-β-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4'-β-glucoside showed high phosphodiesterase inhibitory activity.
    No preview · Article · Jul 2014 · Bioscience Biotechnology and Biochemistry

  • No preview · Article · Jan 2014
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    ABSTRACT: Parkin mutations are responsible for the pathogenesis of autosomal-recessive juvenile parkinsonism (AR-JP). On initial screening of Japanese patients with AR-JP, we had found that approximately half of the parkin mutations are deletions occurring between exons 2 and 5, forming a deletion hot spot. In this study, we investigated the deletion breakpoints of the parkin mutations in 22 families with AR-JP and examined the possible association between these deletion events and meiotic recombinations. We identified 18 deletion breakpoints at the DNA nucleotide sequence level. Almost all these deletions were different, indicating that the deletion hot spot was generated by recurrent but independent events. We found no association between the deletions and specific DNA elements. Recent copy number variation (CNV) data from various ethnic groups showed that the deletion hot spot is overlapped by a highly polymorphic CNV region, indicating that the recurrent deletion mutation or CNV is observable worldwide. By comparing Marshfield and deCODE linkage maps, we found that the parkin deletion hot spot may be associated with a meiotic recombination hot spot, although such association was not found on comparison with recent high-resolution genetic maps generated from the International HapMap project. Here, we discuss the possible mechanisms for deletion hot spot formation and its effects on human genomes.
    No preview · Article · Sep 2009 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Among the salivary gland carcinomas, carcinoma in pleomorphic adenoma has been regarded as a representative carcinoma type which arises secondarily in the background of a pre-existent benign pleomorphic adenoma. It is still poorly understood how and which benign pleomorphic adenoma cells transform into its malignant form, carcinoma ex pleomorphic adenoma. We have established five cell systems from a benign pleomorphic adenoma of the parotid gland of a 61-year-old woman. They were characterized by immunofluorescence, classical cytogenetics, p53 gene mutational analysis, fluorescence in-situ hybridization, and histopathological and immunohistochemical examinations of their xenografts, to demonstrate their potency of secondary transformation. We established and characterized five cell systems (designated as SM-AP1 to SM-AP5) from a benign pleomorphic adenoma of the parotid gland. SM-AP1 to SM-AP3 showed polygonal cell shapes while SM-AP4 and SM-AP5 were spindle-shaped. SM-AP1-3 cells were immunopositive for keratin only, indicating their duct-epithelial or squamous cell differentiation, while SM-AP4/5 cells were positive for both keratin and S-100 protein, indicating their myoepithelial cell differentiation. Chromosome analyses showed numeral abnormalities such as 5n ploidies and various kinds of structural abnormalities, such as deletions, translocations, derivatives and isodicentric chromosomes. Among them, der(9)t(9;13)(p13.3;q12.3) was shared by all five of the cell systems. In addition, they all had a common deletion of the last base G of codon 249 (AGG to AG_) of the p53 gene, which resulted in generation of its nonsense gene product. Transplanted cells in nude mice formed subcutaneous tumors, which had histological features of squamous cell carcinoma with apparent keratinizing tendencies. In addition, they had ductal arrangements or plasmacytoid appearances of tumor cells and myxoid or hyaline stromata, indicating some characteristics of pleomorphic adenoma. This study demonstrates in vitro that certain cell types from pleomorphic adenoma are able to clone and survive over a long term and develop subcutaneous tumors in nude mice. The histological features of squamous cell carcinoma from the transplanted cell systems in nude mice might suggest a secondary onset of malignancy from a pre-existing benign adenoma.
    Full-text · Article · Feb 2009 · BMC Cancer
  • Satoko Asai · Akiko Yamaki · Jun Kudoh · Nobuyoshi Shimizu · Yoshiko Shimizu
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    ABSTRACT: The gene DSCR4 locates in the band q22.2 of human chromosome 21 and encodes a protein of 118 amino acids. Expression of DSCR4 is restricted to human placenta and placental choriocarcinoma cell lines BeWo and JEG3. The 5'-RACE method using RNA from human placenta indicated the major transcription start site at 93 nt upstream (nt -93) of the initiation codon. Transfection assay using a series of deletion constructs of the 5'-flanking region fused to the luciferase reporter gene identified three positive regions nt -2200 to -2088, nt -2064 to -1924, nt -810 to -632 and two negative regions nt -1923 to -1740, nt -631 to -425. The computer analysis predicted the presence of several cis-elements in these regions and the promoter assay using various mutants of consensus sequence identified two distinct cis-elements for OLF-1 and E47. The electrophoretic mobility shift assay (EMSA) using the extracts of DSCR4-expressing cells confirmed the binding of certain protein factors to these cis-elements. In fact, OLF-1-like transcription factor, EBF-3 and EBF-4 were detected in the DSCR4-expressing cell lines and human placenta. Based on these data, we postulated that transcription of DSCR4 gene is regulated positively by binding of OLF-1-like transcription factor and negatively by binding of E47-like transcription factor.
    No preview · Article · Feb 2008 · Biochimica et Biophysica Acta
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    Satoko Asai · Akiko Yamaki · Jun Kudoh · Nobuyoshi Shimizu · Yoshiko Shimizu
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    ABSTRACT: The Publisher regrets that this article is an accidental duplication of an article that has already been published in Biochem. Biophys. Acta, doi:10.1016/j.bbagrm.2007.09.005. The duplicate article has therefore been withdrawn.
    Preview · Article · Nov 2007 · Biochimica et Biophysica Acta
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    ABSTRACT: Human single-minded 2 (SIM2) is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors and is associated with the etiology of Down syndrome phenotype. Here, we examined a possibility of the post-translational modification of SIM2 protein by transfecting various expression constructs followed by the analysis with immunoprecipitation and Western blotting. In fact, transient expression of SIM2 cDNA in HEK293 cells revealed poly-ubiquitination of SIM2 protein. In the stable transfectants, a proteasome inhibitor MG132 protected the poly-ubiquitinated SIM2 protein from degradation. Furthermore, in the cells co-transfected with SIM2 and each of four different E3 ubiquitin ligases, SIM2 was immunoprecipitated with the RING-IBR-RING-type E3 ubiquitin ligases, Parkin and HHARI, but it was not immunoprecipitated with other E3 ligases, such as one RING-type Siah-1 and the PHD type AIRE. A series of deletion constructs revealed that Parkin actually binds to SIM2 with the IBR (294-377)-RING2 (378-465) domains and that the sites for poly-ubiquitination of SIM2 reside within the PAS1-PAS2 region (aa 141-289). We postulated that transcription factor SIM2 and E3 ubiquitin ligase Parkin may interact each other to play an important physiological role in the brain development which is controlled by ubiquitination.
    No preview · Article · Oct 2005 · Experimental Cell Research
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    ABSTRACT: The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers approximately 99% of the euchromatic genome and is accurate to an error rate of approximately 1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human genome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead.
    Full-text · Article · Oct 2004 · Nature
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    Full-text · Article · Oct 2004 · Nature
  • Akiko Yamaki · Jun Kudoh · Nobuyoshi Shimizu · Yoshiko Shimizu
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    ABSTRACT: Human Single-minded 1 (SIM1) and SIM2 genes were found as homologs of Drosophila sim gene which plays a key role in the midline cell lineage of the central nervous system. SIM proteins belong to a family of transcription factors, called bHLH/PAS. Here we examined the intracellular localization of SIM proteins using the expression constructs of whole SIM2 or SIM1 protein fused with enhanced green fluorescent protein (EGFP). The transient expression analysis revealed the nuclear localization of SIM proteins in the cultured cells. To identify the nuclear localization signal, we made expression constructs of EGFP-fusion protein consisting of various portions of SIM proteins. Transfection assay showed the presence of NLS activity in the small region of 23 and 21 amino acid residues at the central part of SIM2 and SIM1 proteins, respectively. Further analysis with amino acid substitution of this small region of SIM2 protein revealed the critical role of five amino acid residues (Arg367, Lys373, Pro385, Tyr386, and Gln389) in NLS activity. The consensus sequence of RKxxKx[K/R]xxxxKxKxRxxPY was estimated as a presumptive NLS in SIM proteins from various species. Thus, the NLS consisting of a cluster of basic amino acids with Pro and Tyr at the C-terminal end is novel and well conserved in the SIM proteins during evolution.
    No preview · Article · Feb 2004 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary disorders. The prevalence of the ADPKD genotype in the Caucasian and Latin populations has been reported. Here, we used linkage analysis to demonstrate the prevalence of the genotype and the correlation between phenotypes and genotypes among 21 Japanese ADPKD families consisting of 96 individuals and including 57 affected members. Six polymorphic markers, each linked to either the polycystic kidney disease 1 (PKD1) or polycystic kidney disease 2 (PKD2) gene, were used for polymerase chain reaction analysis. Seventeen families (81%) showed linkage to PKD1, two families (10%) showed linkage to PKD2, and two families did not show linkage to either PKD1 or PKD2. One of the PKD1-linked families was indicated to have different mutations of PKD1 gene in the same family. PKD2-linked families did not have milder symptoms than PKD1-linked families.
    Preview · Article · Feb 2002 · Journal of Human Genetics
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    ABSTRACT: We previously postulated that the single-minded 2 (SIM2) gene identified on the human chromosome 21q22.2 is a good candidate gene for the pathogenesis of mental retardation in Down syndrome because its mouse homolog exhibits preferential expression in the mouse diencephalon during early embryogenesis. We analyzed the genomic sequence of the entire SIM2 gene which consists of 11 exons and spans over 50 kb. As a step toward understanding the molecular mechanisms of SIM2 gene expression, we have analyzed the human SIM2 gene expression in nine established human cell lines. Three transcripts of 3.6, 4.4, and 6.0 kb were detected in the glioblastoma cell line, T98G, neuroblastoma cell line, TGW, and transformed embryonic kidney cell line, 293. The RACE analysis using SIM2-expressing human cell line T98G provided evidence for the transcription start site at approximately 1.2 kb upstream of the translation initiation site. The transfection assay using various deletion constructs with reporter gene suggested the presence of a presumptive promoter region. Transient transfection assay in T98G cell line revealed a significant promoter activity located in the 60 bp sequence between nt -1385 and -1325 upstream region of the translation initiation site. This 60 bp sequence contains cis-elements for c-myb, E47 and E2F transcription factors. Moreover, the gel retardation assay using oligo-DNA of various cis-element sequences indicated the presence of protein factor(s) which bind to the cis-element for c-myb. These results suggested that binding of a protein transcription factor(s) such as c-myb or that alike regulates transcription of the SIM2 gene by binding to a small upstream region.
    No preview · Article · Jun 2001 · Gene
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    ABSTRACT: More than 80 mutations of the PKD1 gene have been reported, mostly in patients from Western Europe. New techniques are being used to detect an increasing number of mutations, even in the homologous region of the PKD1 gene. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) or denaturing high-performance liquid chromatography (DHPLC) analyses were performed in the present study to screen mutations from exon 23 to exon 46 in the PKD1 gene and in the entire PKD2 gene. When an abnormal pattern was found in PCR-SSCP or DHPLC, the PCR products were directly sequenced. Four mutations were identified in the PKD1 gene: a missense mutation (C47413T causing T3509M in exon 35), a splicing mutation (del 20bp in 75 bp of intron 43), and two nonsense mutations (C48566A causing C3693X in exon 38, and C51237T causing Q4124X in exon 45). The nonsense mutation Q4124X existed in only two of three affected sib members in family K68. The pattern of the restriction enzyme digest and the haplotype analysis confirmed the presence of a heterozygous mutation in the family. Fifteen single nucleotide polymorphisms were identified in this study. Two of them (C50439A and C51659T) can be used as intragenic polymorphic markers.
    Preview · Article · Feb 2001 · Journal of Human Genetics
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    ABSTRACT: Cat eye syndrome (CES) is a developmental disorder with multiple organ involvement, associated with the duplication of a 2-Mb region of 22q11.2. Using exon trapping and genomic sequence analysis, we have isolated and characterized a gene, CECR1, that maps to this critical region. The protein encoded by CECR1 is similar to previously identified novel growth factors: IDGF from Sarcophaga peregrina (flesh fly) and MDGF from Aplysia californica (sea hare). The CECR1 gene is alternatively spliced and expressed in numerous tissues, with most abundant expression in human adult heart, lung, lymphoblasts, and placenta as well as fetal lung, liver, and kidney. In situ hybridization of a human embryo shows specific expression in the outflow tract and atrium of the developing heart, the VII/VIII cranial nerve ganglion, and the notochord. The location of this gene in the CES critical region and its embryonic expression suggest that the overexpression of CECR1 may be responsible for at least some features of CES, particularly the heart defects.
    No preview · Article · Apr 2000 · Genomics
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    ABSTRACT: The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned between the two DNA-binding zinc fingers that is similar to well-characterized nuclear localization sequences in other proteins. When residues within this region are mutated to nonbasic amino acids, or when this domain is deleted, the receptor is still well expressed, but it no longer associates with the vitamin D-responsive element in DNA, in vitro, and hVDR-mediated transcriptional activation is abolished in transfected cells. Concomitantly, the mutated hVDRs exhibit a significant shift in hVDR cellular distribution favoring cytoplasmic over nuclear retention as assessed by subcellular fractionation and immunoblotting. Independent immunocytochemical studies employing a VDR-specific monoclonal antibody demonstrate that mutation or deletion of this basic domain dramatically attenuates hVDR nuclear localization in transfected COS-7 cells. Although wild-type hVDR is partitioned predominantly to the nucleus in the absence of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone, treatment with ligand further enhances nuclear translocation, as it does to some degree in receptors with the basic region altered. The role of 1,25(OH)2D3may be to facilitate hVDR heterodimerization with retinoid X receptors, stimulating subsequent DNA binding and ultimately enhancing nuclear retention. Taken together, these data reveal that the region of hVDR between Arg-49 and Lys-55 contains a novel constitutive nuclear localization signal, RRSMKRK. J. Cell. Biochem. 70:94-109, 1998. © 1998 Wiley-Liss, Inc.
    No preview · Article · Jul 1998 · Journal of Cellular Biochemistry
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    ABSTRACT: We previously described the cDNA sequence of HPC-1/syntaxin 1A (HGMW-approved symbol STX1A) from rat and bovine brains. HPC-1/syntaxin 1A belongs to the syntaxin family and is apparently involved in intracellular membrane transport and the exocytosis of neurotransmitters. In this study, we isolated the cDNA and the genomic DNA clone for human HPC-1/syntaxin 1A and carried out gene mapping. Polymerase chain reaction analysis of human/rodent somatic cell hybrid panels and fluorescence in situ hybridization analysis using a genomic DNA clone provided evidence that the gene for human HPC-1/syntaxin 1A maps to chromosome region 7q11.2.
    No preview · Article · Jun 1997 · Genomics
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    ABSTRACT: To isolate genes responsible for some features of Down syndrome, we performed exon trapping experiments using a series of cosmid clones derived from "the Down syndrome critical region" of chromosome 21 and isolated six exons which are highly homologous to the sequence of Drosophila minibrain (mnb) gene. The Drosophila mnb gene encodes a serine/threonine protein kinase that is required in distinct neuroblast proliferation centers during postembryonic neurogenesis. Using one of these six exons as a probe, we isolated cDNA clones for human homolog of Drosophila mnb gene (MNB) from a fetal brain cDNA library. Human MNB cDNA encodes a protein of 754 amino acids with a nuclear targeting sequence and a catalytic domain common to the serine/threonine-specific protein kinase. The human MNB protein strikingly resembles the recently discovered rat Dyrk protein kinase with a dual specificity. The MNB mRNA is expressed in various tissues including fetal and adult brains. The remarkable similarity of human MNB protein to Drosophila mnb and rat Dyrk proteins implies that human MNB protein may play a significant role in a signaling pathway regulating nuclear functions of neuronal cell proliferation, contributing to certain features of Down syndrome.
    No preview · Article · Sep 1996 · Biochemical and Biophysical Research Communications
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    ABSTRACT: We have recently isolated a human homolog (hSIM) of the Drosophila single-minded (sim) gene from the Down syndrome critical region of chromosome 21 using the exon trapping method. The Drosophila sim gene encodes a transcription factor that regulates the development of the central nervous system midline cell lineage. To elucidate the structure of the mammalian SIM protein, we have isolated cDNA clones from a mouse embryo cDNA library. The cDNA clones encode a polypeptide of 657 amino acids with a bHLH (basic-helix-loop-helix) domain, characteristic of a large family of transcription factors, and a PAS (Per-Arnt-Sim) domain in the amino-terminal half region. Both of these domains have striking sequence homology with human SIM and Drosophila SIM proteins. In contrast, the carboxy-terminal half of the mouse SIM protein consists of a proline-rich region with no sequence homology to the Drosophila SIM protein. A similar proline-rich domain is known for the activator domain of a number of transcription factors. Whole-mount embryo in situ hybridization experiments revealed that the SIM mRNA is expressed prominently in the diencephalon of mouse embryos at 8-9.5 days postcoitum. The structural characteristics of the mouse SIM protein and its expression in the diencephalon during embryogenesis strongly suggest that the newly isolated mammalian SIM homolog may play a critical role in the development of the mammalian central nervous system. We propose that the human SIM gene may be one of the pathogenic genes of Down syndrome.
    No preview · Article · Aug 1996 · Genomics
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    ABSTRACT: DNA topoisomerase I is phosphorylated after mitogenic stimulation of 3T3-L1 mouse fibroblasts by 12-O-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester tumor promoter. In vivo labeling with [32P]orthophosphate and immunoprecipitation with an anti-DNA topoisomerase I antibody has demonstrated an increase in the phosphorylation of DNA topoisomerase I in Swiss/3T3 mouse fibroblasts treated with epidermal growth factor (EGF) and H35 rat hepatoma cells treated with insulin. The only phosphorylated form of DNA topoisomerase I observed was the 100-kDa protein Digestion of DNA topoisomerase I with trypsin revealed two phosphopeptides. In addition, VT-1, a non-responsive genetic variant of 3T3-L1, and the DNA topoisomerase I inhibitor camptothecin were used to further study TPA-induced DNA topoisomerase I phosphorylation. Our results indicate that the phosphorylation of DNA topoisomerase I may be an ubiquitous response of cultured mammalian cells to mitogenic agents, even in the absence of DNA replication.
    No preview · Article · Sep 1994 · Biochimica et Biophysica Acta

Publication Stats

2k Citations
151.12 Total Impact Points

Institutions

  • 2009-2015
    • Keio University
      • Department of Molecular Biology
      Edo, Tokyo, Japan
  • 1996-2009
    • Kyorin University
      • School of Health Sciences
      Edo, Tōkyō, Japan
  • 1981-1994
    • The University of Arizona
      • Department of Molecular and Cellular Biology
      Tucson, Arizona, United States