SEIBUTSU BUTSURI KAGAKU
Online ISSN : 1349-9785
Print ISSN : 0031-9082
ISSN-L : 0031-9082
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Volume 51, Issue 3
Displaying 1-12 of 12 articles from this issue
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Abstracts of the 58th General Meeting of the Japanese Electrophoresis Society
The 46th Kodama Memorial Prize Lectures
The Hirai Memorial Prize Lectures of 2007
Special Lecture
Symposium 1
Symposium 2
Plenary Session
Full Paper
  • Novel type of phosphate-affinity electrophoresis for protein kinase profiling
    Eiji Kinoshita, Emiko Kinoshita-Kikuta, Yuri Aoki, Tohru Koike
    2007 Volume 51 Issue 3 Pages 199-206
    Published: 2007
    Released on J-STAGE: December 04, 2009
    DOIhttps://doi.org/10.2198/sbk.51.199
    JOURNAL FREE ACCESS
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    We developed a novel type of phosphate-affinity polyacrylamide gel electrophoresis using an alkoxide-bridged dinuclear manganese(II) complex as a phosphate-binding tag, Phos-tag. The phosphate-affinity site is a polyacrylamide-bound Mn2+-Phos-tag that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart in an SDS-PAGE gel and the quantitative analysis of kinase reactions. Herein, we describe three applications of protein kinase profiling using the phosphate-affinity electrophoresis (Mn2+-Phos-tag SDS-PAGE). The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein status. The activity profiles of six kinds of kinases were determined using a substrate, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time-course changes in the EGF-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting analysis. The final application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl, was determined using the substrate Abltide-GST and the approved inhibitor Glivec. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the monophosphorylated and nonphosphorylated Abltide-GST bands.
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Symposium
  • World of antisense RNA
    Tadao Funato, Mayu Takeda, Mami Yoshida
    2007 Volume 51 Issue 3 Pages 207-209
    Published: 2007
    Released on J-STAGE: December 04, 2009
    DOIhttps://doi.org/10.2198/sbk.51.207
    JOURNAL FREE ACCESS
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    In many experimental systems, antisense nucleic acids of various compositions, including antisense oligodeoxynucleotides, ribozymes, and DNAzymes, have been shown to perturb gene expression in a sequence specific manner. Nevertheless, we have employed need to be optimized with regard to intracellular delivery, targeting, chemical composition, and efficiency of mRNA destruction. These sequence-specific inhibitors of gene expression may have therapeutic potential in the treatment of a wide range of diseases. We have addressed these critical issues lead to the development of practical and effective nucleic acid drugs within this review.
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  • Establishment of microRNA detection / expression / inhibition system for functional analysis
    Takayuki Mizutani, Kayoko Yamada
    2007 Volume 51 Issue 3 Pages 211-214
    Published: 2007
    Released on J-STAGE: December 04, 2009
    DOIhttps://doi.org/10.2198/sbk.51.211
    JOURNAL FREE ACCESS
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    MicroRNAs (miRNAs) are relatively short, 22nt～, and endogenous non-coding RNAs. It is known to act as a sequence specific regulator of post-transcriptional gene expression in many eukaryotes, most of the time with some sequence mismatches against target genes. Recently, a couple of miRNAs have been shown to be involved in oncogenic pathway, development, or cell differentiation. miRNAs are now getting attention to its diverse regulatory and catalytic functions.
    Although many of the miRNA sequences have been registered in the public databases and their target genes are predicted by computational analysis, the empirical verification has to be done. One of the difficulties to study the miRNA function will be that its structural and regulatory characteristics.
    In order to overcome this difficulty and utilize miRNA as an experimental tool, we investigated a system to detect, knockdown, and / or overexpress miRNAs. First, we adopted bicyclic nucleic acid, Locked Nucleic Acid (LNA), to improve affinity and single nucleotide discrimination. Next, we established the system to introduce synthetic oligos or RNAs expressed from lentiviral vectors to cells. A novel approach based on these principles on miRNA and its pathway analysis will be discussed.
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  • Epigenetic regulation by DNA methylation during mammalian embryogenesis
    Masaki Okano
    2007 Volume 51 Issue 3 Pages 215-217
    Published: 2007
    Released on J-STAGE: December 04, 2009
    DOIhttps://doi.org/10.2198/sbk.51.215
    JOURNAL FREE ACCESS
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    DNA methylation plays important roles in repressing gene expression and transmitting the silenced state to daughter cells. During embryogenesis, DNA methylation of the mammalian genome is dynamically regulated in stage-, sequence- and lineage-dependent manners. Loss of DNA methylation by inactivation of DNA methyltransferase genes resulted in developmental arrest in mice. These observations suggest that DNA methylation controls developmental gene regulation during embryogenesis, but its regulatory mechanisms remain largely unclear. Here we show that CpG islands associated with the X-linked homeobox gene cluster Rhox, which is highly expressed in the extraembryonic trophectoderm, are differentially methylated in a stage- and lineage-specific manner during the post-implantation development of mice. Inactivation of both Dnmt3a and Dnmt3b, DNA methyltransferases essential for the initiation of de novo DNA methylation, abolished the establishment of DNA methylation and the silencing of Rhox cluster genes in the embryo proper. The Dnmt3-dependent CpG island methylation at the Rhox locus extended for a large genomic region (about 1 Mb) containing the Rhox cluster and its surrounding genes. Complementation experiments using ES cells deficient in the DNA methyltransferases suggested that the regulation of Dnmt3a and Dnmt3b was restricted within this large genomic region, and did not affect the neighboring genes outside it, implicating the existence of region-specific boundaries. Our results suggest that DNA methylation plays important roles in both long-range gene silencing and lineage-specific silencing in embryogenesis.
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