Development of real-time intracellular pH sensor using a membrane-permeable peptide nucleic acid.

• Project/Area Number: 16500200
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Neuroscience in general
• Research Institution: Okayama University
• Principal Investigator: TOMIZAWA Kazuhito Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Associate Prof., 大学院・医歯薬学総合研究科, 助教授 (40274287)
• Co-Investigator(Kenkyū-buntansha): MATSUSHITA Masayuki Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Assistant Prof., 大学院・医歯薬学総合研究科, 講師 (30273965) ; MATSUI Hideki Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Professor, 大学院・医歯薬学総合研究科, 教授 (30157234)
• Project Period (FY): 2004 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥3,700,000 (Direct Cost: ¥3,700,000); Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000); Fiscal Year 2004: ¥2,300,000 (Direct Cost: ¥2,300,000)
• Keywords: Peptide nucleic acid / Polyarginine / Nucleic acid / Bio-molecule / Bio-imaging / Cell / 蛋白質導入法 / TAT / センサー / リアルタイム / イメージング / 蛋白導入法 / FRET

Research Abstract

Membrane-permeable peptide nucleic acid (PNA) developed by us is thought to be a powerful tool for the regulation of cell function. The purpose of this project is that development of the tools that are useful for the regulation of protein expression and the function and for the monitoring of nano-ecology in living cells using the PNA. In the present project, we show that membrane-permeable PNA is a powerful tool for the regulation of gene expression and for anti-cancer therapy as follows.
1.Regulation of gene expression.
We synthesized a PNA, which effectively hybridized with plasmid DNA, and the PNA was fused a membrane-permeable poly-arginine to have the ability of membrane permeability. After mixing the PNA with plasmid DNAs, cells were incubated with the complex and the efficiency of the transfection was investigated. The plasmid DNAs were effectively transfected in both cells and the efficiency is better than that of cationic transfection method.
2.Antigene therapy using membrane-permeable PNA.
We synthesized PNAs that are able to bind with p53 mRNA and the double-strand DNA. Cancer cells were incubated with the PNAs. The PNAs were delivered into cells and inhibited the expression of p53 mRNA and the protein. The combination treatment of the PNAs with p53 protein therapy was capable to replace endogenous mutated p53 proteins with wild-type p53 proteins in the cancer cells.

Research Products

• [Journal Article] p53 protein transduction therapy : successful targeting and inhibition of the growth of the bladder cancer cells. (2006)
  • Author(s): Inoue, M., Tomizawa, K.et al.
  • Journal Title: Eur. Urol. 49 ・ 1 Pages: 161-168
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] p53 protein transduction therapy : Successful targeting and inhibition of the growth of the bladder cancer cells. (2006)
  • Author(s): Inoue, M., Tomizawa, K., Matsushita, M., Lu, Y.-F., Yokoyama, T., Yanai, H., Takashima, A., Kumon, H., Matsui, H.
  • Journal Title: Eur.Urol. 49・1 Pages: 161-168
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The NH-2-terminal of influenza virus hemagglutinin-2 subunit peptides enhance the anti-tumor potency of poly-arginine-mediated p53 protein transduction. (2005)
  • Author(s): Michiue, H., Tomizawa, K.et al.
  • Journal Title: J. Bio1. Chem. 280 ・ 9 Pages: 8285-8289
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Ubiquitination-resistant p53 protein transduction therapy facilitates anti-cancer effect on the growth of human malignant glioma cells. (2005)
  • Author(s): Michiue, H., Tomizawa, K.et al.
  • Journal Title: FEBS Lett. 579 ・ 18 Pages: 3965-3969
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Cdk5-dependent regulation of glucose-stimulated insulin secretion. (2005)
  • Author(s): Wei, F.-Y., Tomizawa, K.et al.
  • Journal Title: Nature Med. 11 ・ 10 Pages: 1104-1108
  • NAID: 130005447816
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability. (2005)
  • Author(s): Wei, F.-Y., Tomizawa, K.et al.
  • Journal Title: J. Neurochem. 93 ・ 2 Pages: 502-512
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Truncation and activation of calcineurin A by calpain I in Alzheimer disease brain. (2005)
  • Author(s): Liu, F., Tomizawa, K.et a1.
  • Journal Title: J. Bio1. Chem. 280 ・ 45 Pages: 37755-37762
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The NH-2-terminal of influenza virus hemagglutinin-2 subunit peptides enhance the anti-tumor potency of poly-arginine-mediated p53 protein transduction. (2005)
  • Author(s): Michiue, H., Tomizawa, K., Wei, F.-Y., Matsushita, M., Lu, Y.-F., Ichikawa, T., Tamiya, T., Date, I., Matsui, H.
  • Journal Title: J.Biol.Chem. 280・9 Pages: 8285-8289
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Ubiquitination-resistant p53 protein transduction therapy facilitates anti-cancer effect on the growth of human malignant glioma cells. (2005)
  • Author(s): Michiue, H., Tomizawa, K., Matsushita, M., Tamiya, T., Lu, Y.-F., Ichikawa, T., Date, I., Matsui, H.
  • Journal Title: FEBS Lett. 579・18 Pages: 3965-3969
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Cdk5-dependent regulation of glucose-stimulated insulin secretion. (2005)
  • Author(s): Wei, F.-Y., Nagashima, K., Ohshima, T., Saheki, Y., Lu, Y.-F., Matsushita, M., Yamada, Y., Mikoshiba, K., Seino, Y., Matsui, H., Tomizawa, K.
  • Journal Title: Nature Med. 11・10 Pages: 1104-1108
  • NAID: 130005447816
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Control of Cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability. (2005)
  • Author(s): Wei, F.-Y., Tomizawa, K., Ohshima, T., Asada, A., Saito, T., Nguyen C., Bibb, JA., Ishiguro, K., Kulkami, A.B., Pant, H.C., Mikoshiba, K., Matsui, H., Hisanaga, S.
  • Journal Title: J.Neurochem. 93・2 Pages: 502-512
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Truncation and activation of calcineurin A by calpain I in Alzheimer disease brain. (2005)
  • Author(s): Liu, F., Grundke-Iqbal, I., Iqbal, K., Oda, Y., Tomizawa, K., Gong C.-X.
  • Journal Title: J.Biol.Chem. 280・45 Pages: 37755-37762
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The NH2-terminal of influenza virus hemagglutinin-2 subunit peptides enhance the anti-tumor potency of poly-arginine-mediated p53 protein transduction. (2005)
  • Author(s): Michiue H, Tomizawa K, Wei FY, Matsushita M, Lu YF, Ichikawa T, Tamiya T, Date I, Matsui H.
  • Journal Title: J.Biol.Chem. 280(9) Pages: 8285-8289
• [Journal Article] Calpain inhibitors prevent neuronal cell death and ameliorate motor disturbances after compression-induced spinal cord injury in rats. (2005)
  • Author(s): Arataki S, Tomizawa K, Moriwaki A, Nishida K, Matsushita M, Ozaki T, Kunisada T, Yoshida A, Inoue H, Matsui H.
  • Journal Title: J.Neurotrauma 22(3) Pages: 398-406
• [Journal Article] Regulation of synaptic vesicle recycling by calcineurin in different vesicle pools. (2005)
  • Author(s): Kumashiro S, Lu YF, tomizawa K, Matsushita M, Wei FY, Matsui H.
  • Journal Title: Nerosci.Res. 51(4) Pages: 435-443
  • NAID: 130005447570
• [Journal Article] Channel function is dissociated from the intrinsic kinase acitivty and autophosphorylation of TRPM7/ChaK1. (2005)
  • Author(s): Matsushita M, Kozak JA, Shimizu Y, McLachlin DT, Yamaguchi H, Wei FY, Tomizawa K, Matsui H, Chait BT, Cahalan MD, Nairn AC.
  • Journal Title: J.Biol.Chem. 280(21) Pages: 20793-20803
• [Journal Article] Regulation of N-methyl-D-asparate receptors by calpain in cortical neurons. (2005)
  • Author(s): Wu HY, Yuen EY, Lu YF, Matsushita M, Matsui H, Yan Z, Tomizawa K
  • Journal Title: J.Biol.Chem. 280(22) Pages: 21588-21593
• [Journal Article] Ubiquitination-resistant p53 protein transduction therapy facilitates anti-cancer effect on the growth of human malignant glioma cells. (2005)
  • Author(s): Michiue H, Tomizawa K, Matsushita M, Tamiya T, Lu YF, Ichikawa T, Date I, Matsui H.
  • Journal Title: FEBS Lett. 579(18) Pages: 3965-3969
• [Journal Article] A new approach to inhibiting astrocytic IP3-induced intracellular calcium increase in an astrocyte-neuron co-culture system. (2005)
  • Author(s): Saheki Y, Li ST, Matsushita M, Wu YM, Cai WH, Wei FY, Lu YF, Moriwaki A, tomizawa K, Matsui H.
  • Journal Title: Brain Res. 1055(1-2) Pages: 196-201
• [Journal Article] Cdk5-dependent regulation of glucose-stimulated insulin secretion. (2005)
  • Author(s): Wei FY, Nagashima K, Ohshima T, Saheki Y, Lu YF, Matsushita M, Yamada Y, Mikoshiba K, Seino Y, Matsui H, Tomizawa K.
  • Journal Title: Nat.Med. 11(10) Pages: 1104-1108
  • NAID: 130005447816
• [Journal Article] p53 protein transduction therapy : successful targeting and inhibition of the growth of the bladder cancer cells. (2005)
  • Author(s): Inoue M, Tomizawa K, Matsushita M, Lu YF, Yokoyama T, Yanai H, Takashima A, Kumon H, Matsui H.
  • Journal Title: Eur.Urol. 49(1) Pages: 161-168
• [Journal Article] Truncation and activation of calcineurin A by calpain I in Alzheimer disease brain. (2005)
  • Author(s): Liu F, Grundke-Iqbal I, Iqbal K, Oda Y, tomizawa K, Gong CX.
  • Journal Title: J.Biol.Chem. 280(45) Pages: 37755-37762
• [Journal Article] カルシニューリン(PP2B)の恒常的活性化機構と神経細胞死制御-カルシニューリンとカルパインのクロストーク- (2004)
  • Author(s): 松井秀樹, 呉 海燕, 富澤一仁
  • Journal Title: 細胞工学 Vol.23・No.5 Pages: 533-537
• [Journal Article] タンパク質セラピー法 (2004)
  • Author(s): 松下正之, 富澤一仁, 松井秀樹
  • Journal Title: 実験医学 Vol.22・No.18
• [Journal Article] Critical role of calpain-mediated cleavage of calcineurin in excitotox neurodegeneration. (2004)
  • Author(s): Wu, H.-Y., Tomizawa, K., Matsushita, M., Matsui, H.
  • Journal Title: J.Biol.Chem. 279 Pages: 4929-4940
• [Journal Article] HIV-1 tat inhibits long-term potentiation and attenuates spatial learning. (2004)
  • Author(s): Li, S.-T., Matsushita, M., Tomizawa, K., Matsui, H.
  • Journal Title: Ann.Neurol 55 Pages: 362-371
• [Journal Article] A new cell-permeable peptide allows successful allogeneic islet transplantation in mice. (2004)
  • Author(s): Noguchi, H., Matsushita, M., Tomizawa, K., Matsui, H.
  • Journal Title: Nature Med. 10 Pages: 305-309
• [Journal Article] Photo-acceleration of protein release from endosome in the protein transduction system. (2004)
  • Author(s): Matsushita, M., Tomizawa, K., Matsui, H.
  • Journal Title: FEBS Lett. 572(1-3) Pages: 221-226