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PTCA後再狭窄に対する遺伝子治療の試み

―アンチセンス技術の応用と遺伝子導入―

森下竜一, 檜垣実男, 青木元邦, 中村好男, 荻原俊男, 金田安史

動脈硬化
1995年 23 巻 1-2 号 51-56
発行日: 1995/10/20
公開日: 2011/09/21

DOI https://doi.org/10.5551/jat1973.23.1-2_51

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To develop an effective strategy to prevent neointimal formation after angioplasty injury, we have identified cell cycle regulatory proteins as targets for inhibition using antisense oligonucleotides (
ODN
). In addition, we have developed a novel intraluminal molecular delivery method that employs the protein coat of the Hemagglutinating virus of Japan (HVJ) complexed with liposomes, to enhance the efficiency of cellular uptake and the stability of antisense oligonucleotides while minimizing non-specific toxicity. Phosphorothioate
ODN
are complexed with liposomes and the protein coat of the inactivated HVJ. This method results in a more rapid cellular uptake and a ten-fold higher transfection efficiency of plasmids or
ODN
than lipofection or passive uptake methods. HVJ-liposome complex containing
ODN
was incubated within the rat carotid artery for 10 minutes immediately after balloon injury. First, we examined the effect of antisense cdc (cell division cycle) 2 kinase, proliferating cell nuclear antigen (PCNA), cdk (cyclin dependent kinase) 2 kinase and cyclin B₁
ODN
on neointimal formation after balloon injury in the rat carotid artery model. Reverse transcription PCR in antisense
ODN
(PCNA and cdc 2 kinase) transfected vessels showed a marked decrease in cdc 2 and PCNA gene expression as compared to that in sense control
ODN
transfected vessels. The measurement of DNA synthesis as assessed by bromodeoxyuridine index and DNA content revealed a decrease in antisense PCNA and cdc 2 kinase
ODN
treated vessels on day 4 after injury as compared to sense
ODN
treated vessels. Transfection with either antisense cdc 2, cdk 2 kinase and cyclin B₁
ODN
alone resulted in a partial inhibition of neointimal formation. We then evaluated the strategy of combined antisense
ODN
to achieve complete inhibition. The combination of antisense
ODN
directed against both cdc 2 kinase and PCNA inhibited completely neointimal formation at 2 weeks after angioplasty. Moreover, the inhibitory effect of this antisense
ODN
combination on neointimal formation persisted up to 8 weeks after a single transfection. The combination of antisense cdc 2 kinase and cyclin B₁
ODN
, or antisense cdc 2 and cdk 2 kinase
ODN
, also resulted in near complete inhibition of neointimal formation. The presistent inhibition of neointimal formation after balloon injury was also observed by the combination of antisense cdc 2 kinase and cyclin B₁
ODN
. The present study documents that a single intraluminal molecular in vivo delivery of antisense cell cycle regulatory genes
ODN
results in a inhibition of neointimal formation in the rat carotid balloon injury model as a model of gene therapy. Interestingly, our results reveled that multiple blockade of cell cycle regulatory genes by antisense
ODN
are necessary to achieve the complete inhibition of neointimal formation after angioplasty.
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Time Course of Changes in μ-Opioid Receptor mRNA Levels in the Periaqueductal Gray of Rat Brain by a Single or Repeated Injections of Antisense Oligodeoxynucleotides

Masanobu Yoshikawa, Kouichi Yokogami, Ken Kitamura, Tomomi Kobayashi, Masayuki Kanai, Takao Taniguchi, Kazuhito Akahori, Masaru Nakabayashi, Kayoko Iwao, Masao Hyodo, Tetsuo Oka

The Japanese Journal of Pharmacology
1999年 81 巻 2 号 209-215
発行日: 1999年
公開日: 2001/03/31

DOI https://doi.org/10.1254/jjp.81.209

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The effect of phosphorothioated antisense oligodeoxynucleotide (AS
ODN
) against the μ-opioid receptor (MOR) on MOR mRNA level in the periaqueductal gray (PAG) of rat brain was investigated. The MOR mRNA levels at 3, 6, 12, 24, 48 and 72 h after MOR AS
ODN
microinjection into the PAG were determined by reverse transcriptase-polymerase chain reaction. The MOR mRNA level was significantly decreased only at 12 h after the injection of 10 μg MOR AS
ODN
. When 10 μg MOR AS
ODN
was given three times at the interval of 48 h, MOR mRNA levels were significantly decreased at 6, 12 and 24 h after the last injection of the AS
ODN
. However, MOR mRNA levels were not significantly changed by three injections at 48-h interval of MOR sense
ODN
or AS ODNs against δ- and κ-opioid receptors, although the two latter AS ODNs significantly reduced the respective targeted mRNA levels. In conclusion, the present results show that the selective decrease in MOR mRNA is at least one reason why the reported diminished effects of MOR agonists are produced in animals pretreated with MOR AS
ODN
, although they could be produced through several mechanisms in which MOR mRNA level does not change.
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Development of Photochemical DNA/RNA Manipulation Toward Its Application for Nanotechnology

Kenzo Fujimoto, Hideaki Yoshino, Tomoko Ohtake, Yoshinaga Yoshimura, Isao Saito

Transactions of the Materials Research Society of Japan
2010年 35 巻 1 号 85-89
発行日: 2010/03/01
公開日: 2014/04/18

DOI https://doi.org/10.14723/tmrsj.35.85

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We describe a light-controlled template-directed reversible DNA photoligation via carbazole tethered 5-carboxyvinyluracil. Carbazole tethered 5-carboxyvinyl-2´-deoxyuridine (^CVU)-containing oligodeoxynucleotide (
ODN
) can be ligated by irradiation at 366 nm in the presence of template
ODN
, and the ligated
ODN
can be split by irradiation at 366 nm in the absence of the template
ODN
.
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TLR9アゴニストはFas/FasLを介してマウス肝細胞のミトコンドリア障害に起因するアポトーシス感受性を亢進させる

*宋彬彬, 青木重樹, 劉聡, 伊藤晃成

日本毒性学会学術年会
2019年 46.1 巻 P-45
発行日: 2019年
公開日: 2019/07/10

DOI https://doi.org/10.14869/toxpt.46.1.0_P-45

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Early hepatocyte death occurs in most liver injury cases and triggers liver inflammation, which in combination with other risk factors, leads to the development of liver disease. However, the pathogenesis of early phase hepatocyte death remains poorly understood. Here, C57BL/6J mice were treated with the hepatotoxic drug flucloxacillin (FLUX) and the toll-like receptor 9 agonist CpG oligodeoxynucleotide (
ODN
) to reproduce the early phase of drug-induced hepatotoxicity and investigate its pathogenesis. C57BL/6J mice were treated with FLUX (100 mg/kg, gavage) alone or in combination with
ODN
(40μg/mouse, intraperitoneally). Plasma alanine aminotransferase (ALT) level was measured as a marker of hepatotoxicity. FLUX or
ODN
alone was insufficient to induce ALT elevation, whereas combination treatment with FLUX and
ODN
increased ALT levels 24 h after FLUX treatment and upregulated Fas ligand in natural killer T (NKT) cells and Fas in hepatocytes. FLUX induced mitochondrial permeability transition (MPT), and pretreatment with
ODN
sensitized mitochondria to FLUX-induced MPT. The increase in ALT levels induced by
ODN
and FLUX co-treatment was suppressed in Fas ligand (gld/gld)-deficient mice and in mice deficient in a component of MPT pore opening (cyclophilin D-knockout mice). These results suggested that
ODN
activated the Fas/Fas ligand-mediated pathway in NKT cells and hepatocytes, which may predispose to FLUX-induced mitochondrial dysfunction, and lead to early phase hepatocyte apoptosis. Taken together, these findings elucidate a potentially novel mechanism underlying drug-induced early phase hepatocyte death related to the Fas/Fas ligand death receptor pathway and mitochondrial dysfunction.

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Extracellular Nucleic Acids Secreted by Lactobacillus Regulate TLR9 Expression

Chun-Liang Liu, Shu Wen, Yin-Hui Liu, Hua-Jun Li, Jing Xiao, Li Tang

Journal of Hard Tissue Biology
2011年 20 巻 2 号 153-160
発行日: 2011年
公開日: 2011/10/12

DOI https://doi.org/10.2485/jhtb.20.153

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Objective: The study was designed to determine the immune mechanism of nucleic acid sequences present in the medium filtrate of lactobacillus DM8909 at exponential growth phase. The effect of extracellular nucleic acids on TLR9 on the expression of IL-6 was also investigated.
Methods: Three groups were formed as nucleic acid group, CpG-
ODN
group and control group. Extracellular nucleic acids were examined by TA cloning and sequencing technology. RT-PCR and ELISA were used to detect the expressions of TLR9 and NF-κBp65 in HT-29 cells and the presence of IL-6 in the medium.
Results: The nucleic acids detected in the medium filtrate of lactobacillus DM8909 at exponential growth phase were found to be RNAs. A total of 39 RNA sequences were identified as lactobacillus delbrueckii subsp. Bulgaricus with a size of 49 bp, 23S ribosomal RNA and another RNA with a size of 52 bp identified as lactobacillus rhamnosus, ATCC 53103, tRNA-Leu sequence. TLR9 expressions in CpG-
ODN
and nuclei acid groups were higher compared to the control group (TLR-9 values were 3.2386, 0.4984 and 0.1780 respectively). The same results were obtained for NF-κBp65 where the expressions in CpG-
ODN
and nucleic acid groups were higher than the control group (relative values were 1.6671, 0.6501 and 0.3094 respectively). Furthermore, IL-6 expressions in CpG-
ODN
and nucleic acid groups were significantly higher than the control group (P <0.05, (relative values were 23.6735 ± 1.4515, 20.2041 ± 1.6820 and 15.7143 ± 0.7047 respectively).
Conclusion: The extracellular nucleic acids in the medium of lactobacillus DM8909 are RNAs and they could elevate the level of IL-6 through the activation of NF-κB and TLR9 in vitro. These molecules have similar immune activity with CpG-
ODN
.
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Photochemical
ODN
Manipulation Based on Reversible DNA Photoligation Mediated by Modified Photoresponsive Base

Masayuki Ogino, Shinji Ogasawa, Takehiro Ami, Yoshinaga Yoshimura, Kenzo Fujimoto

Journal of Photopolymer Science and Technology
2005年 18 巻 4 号 507-512
発行日: 2005年
公開日: 2005/08/17

DOI https://doi.org/10.2494/photopolymer.18.507

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We describe an efficient template-directed photoligation of oligodeoxynucleotides (ODNs) using modified photoresponsive base. An efficient photoligation was produced by photoirradiation of an
ODN
containing α-5-cyanovinyldeoxyuridine (α^CU) at the 3' end with an
ODN
containing thymine at the 5' end in the presence of a template
ODN
. This photoligation method is a new, efficient way to synthesize branched ODNs
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Suppression of α-amylase gene expression by antisense oligodeoxynucleotide in barley cultured aleurone layers

Nobuhiko TSUTSUMI, Keiko KANAYAMA, Shigemitsu TANO

遺伝学雑誌
1992年 67 巻 2 号 147-154
発行日: 1992年
公開日: 2005/02/09

DOI https://doi.org/10.1266/jjg.67.147

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Antisense oligodeoxynucleotides (ODNs) have been applied to regulate gene expression using cell-free media or animal cells. Here we demonstrate the specific inhibition of barley α-amylase gene expression by synthetic antisense ODNs. In a cell free system using wheat-germ extracts, 5 μM of a 20-mer antisense
ODN
prevented the synthesis of the polypeptide corresponding to the predetermined length of α-amylase translated in vitro, whereas there was no effect on other protein synthesis. Furthermore, in cultured aleurone cells, a-amylase activity was efficiently decreased by addition of ODNs. At the concentrations higher than 5 μM, antisense
ODN
inhibited α-amylase gene expression almost completely. These results imply that
ODN
could transport into the cultured aleurone cells crossing the cell membrane, and regulate specific gene expression. This simple model system could be applicable not only for the analysis of the α-amylase multigene family in barley but also for studying functions of cryptic genes in higher plant.

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β-1,3-グルカンを用いた薬物送達システムへの応用

Yoshiya Maegawa, Shinichi Mochizuki, Noriko Miyamoto, Yusuke Sanada, Kazuo Sakurai

Trends in Glycoscience and Glycotechnology
2015年 27 巻 153 号 13-29
発行日: 2015/01/25
公開日: 2015/01/23

DOI https://doi.org/10.4052/tigg.27.13

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β-1,3-D-グルカンの一種であるシゾフィラン（SPG）はホモ配列のオリゴデオキシヌクレオチド（
ODN
）と水素結合や疎水性相互作用によって
ODN
/SPG複合体を形成する。また、マクロファージや樹状細胞などの抗原提示細胞上には、β-1,3-D-グルカンの受容体であるデクチン-1が発現している。そのため、この複合体を用いることで、アンチセンス
ODN
（AS-
ODN
）や非メチル化CpG配列を持った
ODN
（CpG-
ODN
）を抗原提示細胞に特異的に送達することが可能になると考えられる。実際、AS-
ODN
/SPG複合体をリポ多糖誘導型マウス肝炎モデルに投与したとき、炎症を抑えることができた。また、カニクイザルにCpG-
ODN
/SPG複合体をインフルエンザワクチンのアジュバントとして投与したとき、高い抗体価を促すことができた。以上より、SPGは特に抗原提示細胞を標的にした薬物送達システムのキャリアとして有用であると考えられる。
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Radioprotection of Bone Marrow Hematopoiesis by CpG-oligodeoxynucleotides Administered to Mice after Total-body Irradiation

Chao ZHANG, Jing LIN, Jianguo CUI, Bailong LI, Cong LIU, Jichao WANG, Fu GAO, Jianming CAI

Journal of Radiation Research
2011年 52 巻 6 号 828-833
発行日: 2011年
公開日: 2011/11/22

DOI https://doi.org/10.1269/jrr.10098

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CpG-oligodeoxynucleotide (
ODN
), a synthetic analog of bacteria DNA, has attracted attention because it activates cells of an adaptive immune system and the innate immune system. In this study, we investigated whether CpG-
ODN
has radioprotective effects, when administered after total-body irradiation (TBI). Mice were treated with 50 μg CpG-
ODN
via intraperitoneal injection (i.p) within 30 min, 24 h and 48 h after TBI. Our results showed that the survival rate was enhanced at various levels of TBI. The calculated dose reduction factor (DRF) was 1.2. Bone marrow cell count and bone marrow histological examination indicated that CpG-
ODN
minimized the bone marrow damage induced by TBI. The data of the white blood cell (WBC) count, exogenous (CFU-S) and endogenous (endoCFU-S) colony forming unit-spleen count demonstrated that CpG-
ODN
reduced primitive hematopoietic stem cells damage and reconstituted hematopoiesis after TBI. Thus, we suggested that CpG-
ODN
had the potential to contribute to the improvement of the survival rate and limitation of myelosuppression induced by TBI.
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Immunotherapeutic Potential of CpG Oligonucleotides in Chickens

Arshud Dar, Brenda Allan, Susantha Gomis, Andrew Potter, George Mutwiri

The Journal of Poultry Science
2009年 46 巻 2 号 69-80
発行日: 2009/03/25
公開日: 2009/04/25

DOI https://doi.org/10.2141/jpsa.46.69

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Synthetic oligodeoxynucleotides (
ODN
) containing CpG motifs activate innate and adaptive immune responses in numerous vertebrate species. The protective effects of CpG
ODN
against viral, bacterial and protozoal pathogens have been well documented in various mouse models of disease. CpG
ODN
are also being evaluated in humans as an immunotherapeutic agent against infectious diseases, cancer, allergy and as a vaccine adjuvant. In species of veterinary importance where the immune activity of CpG
ODN
has been investigated, CpG
ODN
has shown the greatest potential in chickens, as indicated by its protective effects against experimental bacterial infections. Surprisingly, chicken do not appear to express Toll-like receptor 9 (TLR9), the receptor that is involved in CpG-mediated immune activation in humans and many animal species. We will review progress on CpG research with particular emphasis on avian species.
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新規アジュバントを添加した次世代SE36マラリアワクチンの開発：旅行者用ワクチンへのアプローチ

東岸任弘, 石井健, 堀井俊宏

YAKUGAKU ZASSHI
2013年 133 巻 11 号 1153-1157
発行日: 2013/11/01
公開日: 2013/11/01

DOI https://doi.org/10.1248/yakushi.13-00212-2

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The SE36 antigen, derived from serine repeat antigen 5 (SERA5) of Plasmodium falciparum, is a promising blood stage malaria vaccine candidate. Previous clinical trials indicated the protective efficacy of BK-SE36 malaria vaccine that is constituted of SE36 recombinant protein and aluminum hydroxide gel. In this study, we assessed the safety, immunogenicity and protective efficacy of SE36/AHG formulated with TLR9 ligand adjuvants K3 CpG oligodeoxyribonucleotides (CpG ODNs) (K3
ODN
), D3
ODN
or synthetic hemozoin, in two non-human primate models. SE36/AHG with or without each adjuvant was administrated to cynomolgus monkeys. A combination of TLR9 ligand adjuvant with SE36/AHG induced higher humoral and cellular immune response compared with SE36/AHG alone. The most effective TLR9 ligand, K3
ODN
, was chosen for further vaccine trials in squirrel monkeys, in combination with SE36/AHG. All monkeys immunized SE36/AHG with K3
ODN
effectively suppressed parasitemia and symptoms of malaria following challenge infection. Furthermore, no serious adverse events were observed. Our results show that the novel vaccine formulation of K3
ODN
with SE36/AHG is safety, potent immunogenicity and efficacy in nonhuman primates. We are conducting the first in human clinical trials with this formulation.

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Development of Efficient Packaging Method of Oligodeoxynucleotides by a Condensed Nano Particle in Lipid Envelope Structure

Yuma Yamada, Kentaro Kogure, Yoshio Nakamura, Kaori Inoue, Hidetaka Akita, Fumi Nagatsugi, Shigeki Sasaki, Tetsuya Suhara, Hideyoshi Harashima

Biological and Pharmaceutical Bulletin
2005年 28 巻 10 号 1939-1942
発行日: 2005年
公開日: 2005/10/01

DOI https://doi.org/10.1248/bpb.28.1939

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An efficient delivery system is required if antisense oligodeoxynucleotides (
ODN
) are to be utilized for gene therapy. We report herein on the development of a novel
ODN
delivery system,
ODN
-encapsulated nano particles (
ODN
-ENP) using an efficient and simple packaging method. The
ODN
-ENP consists of a condensed
ODN
particle and a lipid envelope, which can be equipped with various functional devices for the efficient delivery of
ODN
with a small diameter (150 nm). The encapsulation efficiency and
ODN
recovery of
ODN
-ENP were significantly higher than those of other packaging methods, such as a stabilized antisense-lipid particles method or a freeze-thaw method. Furthermore, the time required for the preparation of the
ODN
-ENP was shorter than the other methods. The method developed in this study is a simple and efficient packaging method for
ODN
with a condensed nano particle in lipid-envelope structure.
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Enhanced Immunostimulatory Activity of CpG Oligodeoxynucleotide by the Combination of Mannose Modification and Incorporation into Nanostructured DNA

Wenqing Liao, Sakiko Akahira, Rintaro Iwata Hara, Takeshi Wada, Kosuke Kusamori, Yoshinobu Takakura, Makiya Nishikawa

Biological and Pharmaceutical Bulletin
2020年 43 巻 8 号 1188-1195
発行日: 2020/08/01
公開日: 2020/08/01

DOI https://doi.org/10.1248/bpb.b20-00052

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電子付録

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The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG
ODN
) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG
ODN
,
ODN
1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored
ODN
1668 or the mannosylated CpG
ODN
(Man-
ODN
1668) synthesized via modification of the 5′-terminal of
ODN
1668 with a synthesized mannose motif. By mixing
ODN
1668 or Man-
ODN
1668 with the hexapodna,
ODN
1668/hexapodna and Man-
ODN
1668/hexapodna were successfully formed with high yields. However, Man-
ODN
1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-
ODN
1668 or
ODN
1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG
ODN
.

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In vitro Enhancement by CpG
ODN
of Phagocytic Activity of Rainbow Trout Head Kidney Phagocytes against Fish Pathogen Vibrio ordalii, but not against Polystyrene Particles

Masahiro NAKATANI, Tadashi IWASAKI, Shinobu WATARAI, Hiroshi KODAMA

Journal of Veterinary Medical Science
2007年 69 巻 12 号 1287-1290
発行日: 2007年
公開日: 2008/01/05

DOI https://doi.org/10.1292/jvms.69.1287

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Rainbow trout (Oncorhynchus mykiss) head kidney phagocytes precultured with a synthetic cytidine-phosphate-guanosine (CpG) oligodeoxynucleotide (
ODN
) displayed significantly higher phagocytic activity agaist Vibrio ordalii than phagocytes precultured with non-CpG
ODN
. However, head kidney phagocytes precultured with CpG
ODN
did not show enhanced phagocytic activity against polystyrene particles.

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Photochemical Ligation of DNA Probe prepared in Click Chemistry

Yoshinaga Yoshimura, Tomoya Matsuzaki, Kenzo Fujimoto

Journal of Photopolymer Science and Technology
2009年 22 巻 2 号 267-272
発行日: 2009/06/30
公開日: 2009/08/07

DOI https://doi.org/10.2494/photopolymer.22.267

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Template-directed photoligation with 5-carboxyvinyl-2'-deoxyuridine (CVU) can be used for genomic research, biotechnology and biomedical agents. However, in photoligation with CVU, a long photoirradiation time at 366 nm is required to complete photoligation. To overcome the limitation of photoligation with CVU, we synthesized new photosensitive probes in the copper-catalyzed azide-alkyne cycloaddition reaction, as the best example of click chemistry.
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アンチセンスオリゴヌクレオチドによるメサンギウム細胞増殖抑制

柏原直樹, 前島洋平, 守田吉孝, 杉山斉, 関川孝司, 岡本一徳, 金尾浩一郎, 山崎康司, 槇野博史, 太田善介, 保田立二

炎症
1995年 15 巻 5 号 383-388
発行日: 1995/09/30
公開日: 2010/04/12

DOI https://doi.org/10.2492/jsir1981.15.383

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Mesangial cell (MC) proliferation is the hallmark of various forms of renal disease. Many protooncogenes are involved in the signal transduction pathways leading to cell proliferation induced by a wide variety of growth factors. We evaluated the inhibitory effects of antisense oligonucleotides (AS-ODNs) targeting protooncogenes on MC proliferation. The antisense and sense phosphorothioate ODNs of c-myc, c-fos and c-myb were synthesized. Quiescent MCs were stimulated with 20% FCS in the presence of AS-
ODN
or control sense
ODN
, which were mixed with liposome. Cell proliferation was determined by counting the number of trypan blue-viable cells. Inhibition of protooncogene mRNA was monitored by reverse transcriptase polymerase chain reaction (RT-PCR) . AS-
ODN
inhibited MC proliferation in a dose-dependent manner. Inhibitory effects of 10μM of each AS-
ODN
for MC proliferation were : c-myc 56%, c-myb 45% and c-fos 12%. Sense-
ODN
had little effect. Any significant morphological change in MC was not noticed with ASODN treatment.
These results indicate that these protooncogenes are involved in the signal transduction pathways mediating MC proliferation. Furthermore, the results suggest a potential role of antisense strategies designed to inhibit protooncogene expression to prevent MC proliferation.
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アンチセンスオリゴデオキシヌクレオチドによる脳内特定タンパク質の発現抑制

吉川正信, 岡哲雄

日本薬理学雑誌
1997年 109 巻 4 号 187-191
発行日: 1997年
公開日: 2007/01/30

DOI https://doi.org/10.1254/fpj.109.187

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The synthetic oligodeoxynucleotide (
ODN
) complementary to the normal (sense) mRNA, socalled antisense
ODN
, has been used to regulate the gene expression in the brain. It has been reported to interfere with transcription, pre-mRNA splicing and translation through at least two mechanisms; i.e., its competition with transcription and protein synthesis machinery or induction of mRNA cleavage. The unmodified antisense
ODN
was shown to be the RNase activator when it hybridizes with at least four contiguous bases of mRNA. In contrast, the phosphorothioate
ODN
(S-
ODN
) is reported to be a less effective activator of RNase H and more resistant to the nuclease attack than unmodified
ODN
. Because of these properties, S-ODNs are preferentially employed in antisense
ODN
experiments. When the DNA sequence of the target gene is determined, we can design an antisense
ODN
that selectively hybridizes with the bases of a nucleic acid (DNA or RNA) related to the target gene. The initial sites of specific binding of most drugs are known to be proteins such as receptors and enzymes. Therefore, the specific modulation of target protein synthesis by the antisense
ODN
method is quite interesting to the pharmacologist. We have studied the change in the morphine-induced behaviors after the microinjection of antisense S-
ODN
directed against the m-opioid receptor (MOR) into the periaqueductal gray (PAG) or lateral ventricle of rat brain. We could detect the decrease of the MOR mRNA level in PAG by the RT-PCR method and that in whole brain by the Northern blot technique. Although the antisense
ODN
method seems to be quite useful for the modulation of a given gene expression, many problems still remain to be elucidated. These include the mechanism of the regulation of a target gene, pharmacokinetics of antisense
ODN
and toxicity of antisense
ODN
.
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抗HSV活性を有するアンチセンス・オリゴDNAの安定性およびウイルス感染細胞における動態の検討

岩谷若夫, 東海林洋子, 田村信也, 乗松美貴, 嶋田甚五郎, 水島裕

Drug Delivery System
1996年 11 巻 6 号 427-434
発行日: 1996/11/10
公開日: 2009/02/23

DOI https://doi.org/10.2745/dds.11.427

ジャーナルフリー

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We have synthesized antisense phosphorothioate oligodeoxynucleotide (S-
ODN
) tageted 3' splice junction of immediate early (IE) pre-mRNA 4/5 in herpes simplex virus type 1 (HSV-1) and evaluated their antiviral activities as a novel anti-herpetic agent. The antisense S-
ODN
completely protected the permissive Vero cells from a cytopathic effect of HSV-1 at a lower concentration range of 0.78 to 1.56 microM in a serum-free assay system, whereas its sequence including mismatced bases of S-
ODN
and the sense sequence of S-
ODN
exhibited the protective effect at higher concentrations (=> 50 microM). The results indicated that the antisense S-ODNs targeted 3' splice junction inhibit viral growth in a sequence-specific manner. To get more informations for the usage as a therapeutic agent, the biological stability and the intracellular distribution of the antisense S-
ODN
were examined. In the nuclease degradation test, the antisense S-
ODN
had a half life of 5 to 7 hrs in culture medium containing 10% fetal calf serum at 37°C. The similar antisense phosphodiester (D)
ODN
was completely degraded within 60 mins. The S-
ODN
was shown to be considerablely more stable to nuclease degradation than D-
ODN
. In the same condition, the antiviral activity of the antisense S-
ODN
was 8 times less than that in the serum-free culture system. No antivial effect of the antisense D-
ODN
was detected at any concentration even in the serum-free culture system. In HSV-1 infected cells, a uniform distribution of S-
ODN
was observed both in nuclei and in cyto plasm at 12-hr incubation. On the other hand, in non-infected cells, S-
ODN
was localized into lysosome-like vesicles and such localization of S-
ODN
continued on and after 12-hr incubation. This indicates that a critical disadvantage of intracellular delivery to target site might be solved by viral infection. These findings suggest that the antisense S-
ODN
may be sufficiently worth developing as an antiherpetic therapeutic agent, and that some more devices for the improvement of nuclease-resistance and intracellular permeability could make the antisense S-
ODN
more effective against HSV-1 infection.
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Oligodeoxynucleotide-Modified Capillary for Electrophoretic Separation of Single-Stranded DNAs with a Single-Base Difference

Takahisa ANADA, Masako OGAWA, Hisashi YOKOMIZO, Yoshihisa OZAKI, Tohru TAKARADA, Yoshiki KATAYAMA, Mizuo MAEDA

Analytical Sciences
2003年 19 巻 1 号 73-77
発行日: 2003年
公開日: 2003/07/31

DOI https://doi.org/10.2116/analsci.19.73

ジャーナルフリー

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We describe here a method of affinity capillary electrophoresis in which oligodeoxynucleotide(
ODN
) was immobilized onto the inner surface of the capillary. The immobilized
ODN
functioned successfully as an affinity ligand for sequence-based DNA separation. Six-or 12-mer
ODN
with a sequence complementary to one of the c-K-ras gene was used as an immobilized ligand. When the 12-mer
ODN
was used, the detection peak for the complementary
ODN
disappeared selectively, while the single-base mutant was detected as useal. In contrast, when the 6-mer
ODN
was used as the affinity ligand with a mixture of the complementary
ODN
and its single-base mutant, it was possible to detect both as completely separate peaks. That is, the separation mode was dependent on the base number of the immobilized
ODN
used as an affinity ligand.
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Bioimaging Analysis of Cellular Uptake of Cholesterol-Conjugated Oligonucleotides Using a Real-Time Confocal Laser Microscope

Kenichi Hanaki, Tetsuro Ueno, Hiroshi Nakatake, Chikateru Nozaki, Takahide Taniguchi, Eiichi Honda, Masanobu Hayashi, Kenji Yamamoto

bioimages
1997年 5 巻 4 号 127-132
発行日: 1997年
公開日: 2021/09/10

DOI https://doi.org/10.11169/bioimages.5.127

ジャーナルフリー

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The uptake of 3'-cholesterol conjugated phosphorothioate oligonucleotides with 5'-fluorescein (ChPSf-
ODN
) into rat immortalized hepatocytes was visualized with a real-time confocal laser microscope. The images showed that the uptake of ChPSf-
ODN
in the cytosol had started within the first 5 minutes, and it was not until a 40 minute-incubation that the endosomal and/or lysosomal accumulation appeared. Histogram analysis of fluorescence intensity indicated that cytosolic uptake of ChPSf-
ODN
was five times as great as that of phosphorothioate oligonucleotides with 5'-fluorescein (PSf-
ODN
). However, nuclear uptake of ChPSf-
ODN
was only twice as great as that of PSf-
ODN
. The mechanisms of the different amounts of ChPSf-
ODN
distribution in the subcellular compartments are discussed. This study shows that the visual data derived from time-lapse confocal laser microscopy are helpful in understanding the cellular uptake mechanism of fluorescein labeled
ODN
.
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