記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI  

A connection of a functional peptide with a cell-penetrating peptide (CPP) used a heterodimeric coiled-coil as a molecular zipper can improve the intracellular delivery and activity of the functional peptide. However, the chain length of the coiled coil required for functioning as the molecular zipper is unknown at present. To solve the problem, we prepared an autophagy-inducing peptide (AIP) that conjugates with the CPP via heterodimeric coiled-coils consisting of 1 to 4 repeating units (K/E zipper; AIP-Kn and En-CPP), and we investigated the optimum length of the K/E zipper for effective intracellular delivery and autophagy induction. Fluorescence spectroscopy showed that K/E zippers with n = 3 and 4 formed a stable 1:1 hybrid (AIP-K3/E3-CPP and AIP-K4/E4-CPP, respectively). Both AIP-K3 and AIP-K4 were successfully delivered into cells by the corresponding hybrid formation with K3-CPP and K4-CPP, respectively. Interestingly, autophagy was also induced by the K/E zippers with n = 3 and 4, more intensively by the former than by the latter. The peptides and K/E zippers used in this study did not show significant cytotoxicity. These results indicate that the effective induction of autophagy occurs via an exquisite balance of the association and dissociation of the K/E zipper in this system.

DOI： 10.3390/pharmaceutics15041048

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI  

BNCT is a non-invasive cancer therapy that allows for cancer cell death without harming adjacent cells. However, the application is limited, owing to the challenges of working with clinically approved boron (B) compounds and drug delivery systems (DDS). To address the issues, we developed self-forming nanoparticles consisting of a biodegradable polymer, namely, "AB-type Lactosome (AB-Lac)" loaded with B compounds. Three carborane isomers (o-, m-, and p-carborane) and three related alkylated derivatives, i.e., 1,2-dimethy-o-carborane (diC1-Carb), 1,2-dihexyl-o-carborane (diC6-Carb), and 1,2-didodecyl-o-carborane (diC12-Carb), were separately loaded. diC6-Carb was highly loaded with AB-Lac particles, and their stability indicated the "molecular glue" effect. The efficiency of in vitro B uptake of diC6-Carb for BNCT was confirmed at non-cytotoxic concentration in several cancer cell lines. In vivo/ex vivo biodistribution studies indicated that the AB-Lac particles were remarkably accumulated within 72 h post-injection in the tumor lesions of mice bearing syngeneic breast cancer (4T1) cells, but the maximum accumulation was reached at 12 h. In ex vivo B biodistribution, the ratios of tumor/normal tissue (T/N) and tumor/blood (T/Bl) of the diC6-Carb-loaded particles remained stably high up to 72 h. Therefore, we propose the diC6-Carb-loaded AB-Lac particles as a promising candidate medicine for BNCT.

DOI： 10.3390/cells11203307

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

We developed a method for detecting DNA by excimer fluorescence from two peptide nucleic acids (PNAs) modified with a pyrene (Pyr). The two PNA-Pyr probes were prepared by solid-phase peptide synthesis, and we assessed fluorescence from the mixture of probes with DNA. From the results, excimer fluorescence derived from the two PNA-Pyr probes forming hybrids with the complementary DNA was observed, and the two probes showed the maximum excimer/monomer ratio when the probes and DNA were hybridized at a 1:1:1 ratio, indicating that the PNA-Pyr probes can detect target DNA. Furthermore, we adjusted the spatial arrangement between the two PNA-Pyr hybrids formed on the DNA to promote optimal excimer formation. As a result, optimal excimer formation was achieved by spacing the two nucleobases between the formed two hybrids and further inserting a hexamethylene linker (C6) between the PNA and Pyr of the PNA-Pyr probe on one side.

DOI： 10.1016/j.bmcl.2022.128838

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics.

DOI： 10.1016/j.bmcl.2022.128767

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI  

In this study, we performed an elaborate adjustment of the structure of peptide nucleic acid (PNA) molecular beacons as probes for detecting nucleic acids. We synthesized the PNA beacons with various numbers of Glu, Lys, and dabcyl (Dab) quenchers in them, and we investigated their fluorescence changes (F-1/1/F-0) with and without full-match DNA. As the numbers of Glu/Lys or Dab increased, the F-1/1/F-0 tended to decrease. Among the different beacons, the PNA beacon with one Glu and one Lys (P1Q1) showed the largest F-1/1/F-0. On the other hand, a relatively large F-1/1/F-0 was obtained when the number of Glu/Lys and the number of Dab were the same, and the balance between the numbers of Glu/Lys and Dab seemed to affect the F-1/1/F-0. We also investigated the DNA detection by the prehybrid of P1Q1, which consists of the T790M base sequence, [P1Q1(T790M)], with quencher-modified DNA (Q-DNA). We examined the DNA detection with single-base mismatch by P1Q1(T790M), and we clarified that there was difficulty in detecting the sequence with P1Q1 alone, but that the sequence was successfully detected by the prehybrid of P1Q1 with the Q-DNA.

DOI： 10.3390/pr10040722

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bmcl.2021.128359

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-021-94249-7.

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記述言語：英語   出版者・発行元：MDPI  

DOI： 10.3390/app11083594

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.xphs.2021.01.026

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bmcl.2021.127811

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms22094982.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

"Theranostics," a new concept of medical advances featuring a fusion of therapeutic and diagnostic systems, provides promising prospects in personalized medicine, especially cancer. The theranostics system comprises a novel 89Zr-labeled drug delivery system (DDS), derived from the novel biodegradable polymeric micelle, "Lactosome" nanoparticles conjugated with specific shortened IgG variant, and aims to successfully deliver therapeutically effective molecules, such as the apoptosis-inducing small interfering RNA (siRNA) intracellularly while offering simultaneous tumor visualization via PET imaging. A 27 kDa-human single chain variable fragment (scFv) of IgG to establish clinically applicable PET imaging and theranostics in cancer medicine was fabricated to target mesothelin (MSLN), a 40 kDa-differentiation-related cell surface glycoprotein antigen, which is frequently and highly expressed by malignant tumors. This system coupled with the cell penetrating peptide (CPP)-modified and photosensitizer (e.g., 5, 10, 15, 20-tetrakis (4-aminophenyl) porphyrin (TPP))-loaded Lactosome particles for photochemical internalized (PCI) driven intracellular siRNA delivery and the combination of 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) offers a promising nano-theranostic-based cancer therapy via its targeted apoptosis-inducing feature. This review focuses on the combined advances in nanotechnology and material sciences utilizing the "89Zr-labeled CPP and TPP-loaded Lactosome particles" and future directions based on important milestones and recent developments in this platform.

DOI： 10.3390/life11020158

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bmc.2021.116036

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/d0an01230k

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI  

Endosomal escape in cell-penetrating peptide (CPP)-based drug/macromolecule delivery systems is frequently insufficient. The CPP-fused molecules tend to remain trapped inside endosomes and end up being degraded rather than delivered into the cytosol. One of the methods for endosomal escape of CPP-fused molecules is photochemical internalization (PCI), which is based on the use of light and a photosensitizer and relies on photoinduced endosomal membrane destabilization to release the cargo molecule. Currently, it remains unclear how this delivery strategy behaves after photostimulation. Recent findings, including our studies using CPP-cargo-photosensitizer conjugates, have shed light on the photoinduced endosomal escape mechanism. In this review, we discuss the structural design of CPP-photosensitizer and CPP-cargo-photosensitizer conjugates, and the PCI mechanism underlying their application.

DOI： 10.3390/molecules26010036

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-020-76100-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/mi11070628

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.bioconjchem.0c00046

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/c9ob02559f

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The inefficient endosomal escape of drugs or macromolecules is a major obstacle to achieving successful delivery to therapeutic targets. An efficient approach to circumvent this barrier is photochemical internalization (PCI), which uses light and photosensitizers for endosomal escape of the delivered macromolecules. The PCI mechanism is related to photogenerated singlet oxygen, but the mechanism is still unclear. In this study, we examined the relation of PCI to heat, pH and Ca2+ ions using cell penetrating peptide (CPP)-cargo-photosensitizer (Alexa546 or Alexa633) conjugates. A cell temperature changing experiment demonstrated that heat (thermal mechanism) does not significantly contribute to the photoinduced endosomal escape. Inhibition of V-ATPase proton pump activity and endosomal pH upregulation indicated that PCI-mediated endosomal escape needs endosomal acidification prior to photoirradiation. Imaging of the CPP-cargo-photosensitizer and Ca2+ ions during photostimulation showed that intracellular calcium increase is not the cause of the endosomal escape of the complex. The increment is mainly due to Ca2+ influx. These findings show the importance of extra- and intracellular milieu conditions in the PCI mechanism and enrich our understanding of PCI-related changes in cell.

DOI： 10.1111/php.13146

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/c9an00725c

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport.

DOI： 10.1016/j.bmcl.2019.02.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jbiosc.2019.01.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0211505

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記述言語：英語   出版者・発行元：WILEY  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.bioconjchem.8b00487

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Blackwell Publishing Ltd  

MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH-SY5Y cells. We identified 15 up-regulated and eight down-regulated miRNAs in SH-SY5Y cells treated with all-trans retinoic acid to induce differentiation. We further showed that one of the up-regulated miRNAs, miR-664a-5p, promoted neuronal differentiation of SH-SY5Y cells. These findings enhance our understanding of the miRNAs involved in the process of neurogenesis and, in particular, highlight an important role of miR-664a-5p in SH-SY5Y cell neuronal differentiation. Further studies will be required to confirm the function of miR-664-5p in neuronal development and disease and to identify its relevant target genes.

DOI： 10.1111/gtc.12559

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier Ltd  

Multivalent cell-penetrating peptides (CPPs) have been reported to show enhancement in cellular uptake and endosomolytic activity. However, its application was limited to trans-delivery of cargo which is lower in cellular uptake efficiency of cargo than cis-delivery. Here, we tried the cis-delivery of cargo with multivalent CPP by preparing bioreducible dimeric CPP–cargo with apoptotic activity using TatBim peptide, a fusion of Tat CPP and Bim peptide derived from Bim apoptosis-inducing protein. Dimeric TatBim was almost twice as highly internalized by cells and significantly induced apoptosis compared to monomeric TatBim. Contribution of bioreducible linkage of dimeric TatBim towards apoptotic activity was also confirmed.

DOI： 10.1016/j.bmcl.2017.12.035

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

A method to induce cytoplasmic peptide delivery, using ultrasound, was demonstrated using a molecular conjugate of a cell-penetrating peptide (CPP), a functional peptide, and a sonosensitizer. As a model of such molecular conjugates, TatBim-RB, consisting of the Tat CPP, the Bim apoptosis inducing peptide, and the sonosensitizer rose bengal was synthesized. CPPs have been widely used for intracellular delivery of various cargos; however, CPP-fused molecules tend to become entrapped in endosomes, as was observed for TatBim-RB molecules applied to cells. To promote escape of the entrapped TatBim-RB molecules, cells were irradiated with ultrasound, which successfully induced endosomal escape and cytoplasmic dispersion of TatBim-RB, and subsequently apoptosis. Our results suggest that this peptidesonosensitizer conjugate strategy may facilitate numerous kinds of medicinal chemistry studies, and furthermore, this specific conjugate may exhibit potential as a novel therapeutic agent for the promotion of apoptosis. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2017.06.024

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

The use of molecular beacons is a versatile method to detect RNAs. Typically, a single-stranded region of RNA is selected as a target sequence for molecular beacons. Therefore, the detection of highly structured short RNAs, such as tRNAs, seems to be difficult. In this study, as an example of highly structured target RNA, we used human tRNA(Lys3), which is known to have functions in protein synthesis and the reverse transcription of human immunodeficiency virus (HIV)-1. No long single-stranded region more than 8 nt is present in this tRNA, which is much shorter than the standard target length of molecular beacons (similar to 20 nt). This study showed that sensitive detection of highly structured RNAs using molecular beacons was much more difficult than that of unstructured or less structured RNAs. However, efficient detection of the tRNA(Lys3) was achieved by selecting the best target region, i.e., the region around the D arm, probably due to the ease of unfolding of this arm. Accordingly, our findings suggested that molecular beacons may have applications in the detection of highly structured RNAs related to various biological functions and diseases.

DOI： 10.1039/c7ay00341b

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PORTLAND PRESS LTD  

Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor-and T-stems of aa-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. We previously reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively, recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode Caenorhabditis elegans. C. elegans EF-Tu2 specifically recognizes the seryl moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e. g. C. elegans) EF-Tu species. We report here that two EF-Tu species also participate in Drosophila melanogaster mitochondria. Both D. melanogaster EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. D. melanogaster EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in D. melanogaster mitochondria, but do exist in related arthropod species. In addition, D. melanogaster EF-Tu2 preferentially bound to aatRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mt translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems.

DOI： 10.1042/BCJ20160929

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl) methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at similar to 405 nm at 125mWcm(-2), DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells.

DOI： 10.1038/ncomms12501

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2016.04.091

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/14686996.2016.1178056

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/bs.pmbts.2015.10.013

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated O-1(2) molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of O-1(2) was confirmed using O-1(2) quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than O-1(2) lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule.

DOI： 10.1038/srep18577

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

A hybrid comprising an autophagy-inducing peptide (AIP) and a cell-penetrating peptide (CPP) connected via heterodimeric leucine zippers was generated and delivered into cells. The hybrid successfully induced autophagy without significant cell death, while the same AIP directly connected to a CPP caused both autophagy and significant cell death.

DOI： 10.1039/c4cc07459a

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Translation systems with four-base codons provide a powerful strategy for protein engineering and protein studies because they enable site-specific incorporation of non-natural amino acids into proteins. In this study, a caged aminoacyl-tRNA with a four-base anticodon was synthesized. The caged aminoacyl-tRNA contains a photocleavable nitroveratryloxycarbonyl (NVOC) group. This study showed that the caged aminoacyl-tRNA was not deacylated, did not bind to EF-Tu, and was activated by light. Photo-dependent translation of an mRNA containing the four-base codon was demonstrated using the caged aminoacyl-tRNA. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2014.10.053

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNA(Met) (iMet) through 5'-3' exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2014.08.003

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記述言語：英語   出版者・発行元：FRONTIERS MEDIA SA  

Conventional tRNAs have highly conserved sequences, four-armed cloverleaf secondary structures, and L-shaped tertiary structures. However, metazoan mitochondrial tRNAs contain several exceptional structures. Almost all tRNA(Ser) for AGY/N codons lack the D-arm. Furthermore, in some nematodes, no four-armed cloverleaf-type tRNAs are present: two tRNAs(Ser) without the D-arm and 20 tRNAs without the T-arm are found. Previously, we showed that in nematode mitochondria, an extra elongation factor Tu (EF-Tu) has evolved to support interaction with tRNAs lacking the T-arm, which interact with C-terminal domain 3 in conventional EF-Tu. Recent mitochondrial genome analyses have suggested that in metazoan lineages other than nematodes, tRNAs without the T-arm are present. Furthermore, even more simplified tRNAs are predicted in some lineages. In this review, we discuss mitochondrial tRNAs with divergent structures, as well as protein factors, including EF-Tu, that support the function of truncated metazoan mitochondrial tRNAs.

DOI： 10.3389/fgene.2014.00109

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掲載種別：論文集(書籍)内論文   出版者・発行元：Springer Netherlands  

DOI： 10.1007/978-94-017-8896-0_19

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Controlled activation of small RNAs, such as small interfering RNA, in cells is very useful for various biological applications. Light is an effective inducer of controlled activation
in particular, near-infrared light is favorable because it can penetrate deeper into tissues than UV or visible light. In this study, near-infrared light control of RNA interference (RNAi) was demonstrated in mammalian cells using a photosensitive RNA carrier molecule, consisting of an RNA carrier protein and a fluorochrome. The photosensitive carrier molecule was identified from six candidates, each with a different fluorochrome. Using this carrier molecule, cytosolic RNA delivery and RNAi can be triggered by near-infrared light. Cytotoxicity was not observed after photoinduction of RNAi. © 2013 American Chemical Society.

DOI： 10.1021/bc4001195

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

In this report, we describe dendritic peptides possessing central fluorescent amino acids with adjacent branched amino acids. These fluorescent-peptide dendrimers were prepared using (9-fluorenyl)methoxycarbonyl (Fmoc)-based solid-phase peptide synthesis and Fmoc-derivative fluorescent and branched amino acids. The branched amino acids featured multiple carboxylic acids in their side chains, making the fluorescent-peptide dendrimers highly water-soluble compared with the analogous peptides containing the fluorescent amino acids only. The branched amino acid units also improved the fluorescence intensity of the dendrimers. Based on high-pressure liquid chromatography and fluorescence spectroscopy results, we determined that the fluorescent groups were located in the core and that the carboxylic acids were located on the surface of the dendrimers. Fluorescence resonance energy transfer was achieved among the three proximal fluorescent groups in one of the fabricated fluorescent-peptide dendrimers. (C) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/bip.22175

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記述言語：英語   出版者・発行元：AMER CHEMICAL SOC  

Because RNA interference (RNAi) can be applied to any gene, this technique has been widely used for studying gene functions. In addition, many researchers are attempting to use RNAi technology in RNAi-based therapies. However, several challenging and controversial issues have arisen during the widespread application of RNAi including target gene specificity, target cell specificity, and spatiotemporal control of gene silencing. To address these issues, several groups have utilized photochemistry to control the RNA release, both spatially and temporally.
In this Account, we foam on recent studies using photocleavable protecting groups, photosensitizes, Hand gold nanoparticles for photoinduced RNAi. In 2005 the first report of photoinduced RNAi used a caged short interfering RNA (siRNA), an siRNA carrying a photocleavable protecting group. Caging groups block the bioactivities of target molecules, but allow for complete recovery of these functions via photoactivation. However, some RNAi activity can occur in these caged siRNAs, so it will be necessary to decrease this "leakage" and raise the RNAi activity restored after irradiation. This technique also uses UV light around 350 nm, which is cytotoxic, but in the near future we expect that it will be possible to use visible and near-infrared light
We also examine the application of photochemical internalization (PCI) to RNAi technology, which involves a combination of photosensitizers and light Instead of inducing RNAi using light, the strategy behind this method was to enhance RNAi using RNA carriers. Many wellknown RNA carriers deliver siRNAs into cells by endocytosis. The siRNAs are trapped in endocytic vesicles and have to be released into the cytoplasm in order to express their activity. To achieve the endosomal escape of siRNAs, PCI technology employed photosensitizers to generate light-dependent reactive oxygen species (ROS) that disrupted the endocytic vesicles. In most studies, RNAi-mediated knockdown of the target gene was detected even without PCI. Recently, a polymer capable of trapping the siRNA in endocytic vesicles controlled RNAi almost entirely by light. CLIP-RNAi uses photosensitizing carrier proteins that can be activated over a wide range of visible light wavelengths. With this method RNA carrier/siRNA complexes are completely trapped within endosomes, and RNAi is controlled strictly by light. Such precise, light-dependent control will open up new possibilities for cellular and molecular biology and therapy.
Most recently, gold nanoparticles (AuNPs) conjugated to siRNA have provided temporal and spatial control of RNAL The light-dependent melting of AuNPs accompanied by a shape transformation induces the release of thiolated siRNAs from AuNPs. In this method, the unique optical properties of the AuNP enable deep penetration of the excitation light into tissues at nearinfrared wavelengths.
The development of photoinduced RNAi technology will lead to novel insights into gene functions and selective drug delivery, and many other scientific fields will continue to influence its progress.

DOI： 10.1021/ar200227n

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2011.06.026

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked REP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.

DOI： 10.1021/bc200095a

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Arginine analogs were incorporated site-specifically into proteins using an in vitro translation system. In this system, mRNAs containing a CGGG codon were translated by an aminoacyl-tRNA(CCCG), which was charged with arginine analogs using yeast arginyl-tRNA synthetase. N(G)-monomethyl-L-arginine, L-citrulline and L-homoarginine were incorporated successfully into proteins using this method. The influence of arginine monomethylation in histone H3 on the acetylation of lysine residues by histone acetyltransferase hGCN5 was investigated, and the results demonstrated that K9 acetylation was suppressed by the methylation of R8 and R17 but not by R26 methylation. K18 acetylation was not affected by the methylation of R8. R17 and R26. This site-specific modification strategy provides a way to explore the roles of post-translational modifications in the absence of heterogeneity due to other modifications. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.09.137

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

To investigate the antisense effect of a pyrrolidine-based oxy-peptide nucleic acid (POPNA), we carried out the LacZ reporter assay using a 12-mer trans-L-POPNA conjugated with a cell-penetrating peptide (antisense reagent). The antisense effect of the conjugated POPNA (inhibition of LacZ activity) was comparable to that shown by a Nielsen-type peptide nucleic acid. Furthermore, the conjugated POPNA could switch the LacZ activity over a wide range of ambient temperatures. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2010.11.034

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

By using the NEXT-A reaction, we introduced a non-natural amino acid at the N-terminus of a peptide/protein that contained a cysteine unit. The side chain of the introduced amino acid spontaneously reacted with the cysteine to afford a cyclic peptide/protein.

DOI： 10.1039/c1cc12196k

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

We synthesized 16 pyrrolidine-based oxy-peptide nucleic acid (POPNA) monomers carrying four different nucleobases onto four different stereoisomers of pyrrolidine rings. Using these monomers, we prepared POPNA oligomers, which formed sequence-specific hybrids with DNAs. The oligomer configurations influenced the hybrid stability. The oligomers were not taken into CHO cells. However, they could enter the cell cytoplasm when mixed with the influenza virus hemagglutinin peptide-arginine heptamer conjugate. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2010.10.056

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

We present three methods relating to tRNA aminoacylation with non-natural amino acids using an Escherichia coli EF-Tu E215A/D216A mutant that can bind tightly to aa-tRNAs carrying either non-natural or natural amino acids: (i) a method for improving aminoacylation efficiency, (ii) a rapid method for analysing aminoacylation efficiency without the use of radioisotope labelling and (iii) a method for purifying aminoacyl-tRNAs. Although the EF-Tu mutant may be incompatible with some kinds of non-natural amino acids, we confirmed that the EF-Tu mutant could efficiently bind to aa-tRNAs carrying various amino acids (Arg, Ser, O-methyltyrosine, Bodipy FL-aminophenylalanine and 2-acrydonylalanine). These methods may be used for the efficient in vitro synthesis of proteins containing various non-natural amino acids.

DOI： 10.1093/jb/mvq053

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN  

We engineered Lactobacillus paracasei to produce a dsRNA that would trigger RNAi-induced silencing of an essential gene in the nematode Caenorhabditis elegans. The dsRNA-expressing L paracasei can be used in experiments conducted on culture plates and may also be used as an orally administrable dsRNA carrier for humans and other mammals. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.08.002

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掲載種別：論文集(書籍)内論文   出版者・発行元：Humana Press  

DOI： 10.1007/978-1-60761-588-0_17

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Bioactive small molecules such as metabolites and drugs play important roles in regulating biological functions. A technique for Visualizing such small molecules is very useful to understand their molecular mechanisms. In this study, an RNA-protein Conjugate, which consists of in RRE-RNA sensor protein (EYFP-Rev-ECFP) and an altered RRE-RNA, was constructed to detect bioactive small molecules by fluorescent resonance energy transfer (FRET). We designed a theophylline-aptamer-inserted RRE-RNA (Theo-RRE) to detect theophylline as a model target molecule. Theo-RRE formed an RNA-protein conjugate with EYFP-Rev-ECFP in the presence of theophylline. As a result, theophylline was specifically detected down to 10 mu M by the FRET increase in distinction from theophylline analogue, caffeine, in cell lysates.

DOI： 10.1021/bc9002184

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

In this study we describe the spatial regulation of RNA interference (RNAi) using an RNA-carrier protein labeled with a fluorescent dye and a light source to trigger the RNAi. We demonstrate photo-dependent gene silencing using several dyes with different excitation wavelengths. Additionally, we use light from a halogen lamp and a photomask to produce photopatterned RNAi, and laser light to trigger single-cell RNAi on cell culture plates. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jconrel.2009.04.015

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE BV  

RNAi-mediated silencing of specific genes is a promising strategy for gene therapy. To utilize RNAi for therapy, an efficient and safe method for delivery of RNA into the cell cytosol is necessary. The plasma membrane is the primary, and most difficult, barrier for RNA to cross, because negatively charged RNA is strongly repulsed by the negatively charged membrane. A variety of cationic polymers can be used as RNA carriers by interacting with RNA and covering its negative charges to form a cell-penetrating complex Among the emerging candidates for RNA carriers are cationic cell-penetrating peptides (CPPs), which can cross the plasma membrane and internalize into cells together with RNA. This review focuses on CPP-based RNA delivery strategies. In using CPP-based RNA delivery, most of the RNA internalized by the cell is entrapped in endosomes. Strategies for endosomal escape of RNAs are also reviewed. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.addr.2009.04.005

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as 'bridgebuilder' to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2009.05.031

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We developed novel Substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide Solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the Suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein Substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2009.03.049

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CHEMICAL SOC JAPAN  

The profiling of microRNAs (miRNAs) using microarray technology has been used in many recent biological and medical studies. In this study, arrays of peptide nucleic acids (PNAs) were prepared and their miRNA capture abilities were evaluated. We found that the sensitivity of 10-mer PNA probes in targeting miRNAs was much higher than that of the corresponding DNA probes and also of longer (20-mer) DNA probes.

DOI： 10.1246/cl.2009.438

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記述言語：英語   掲載種別：論文集(書籍)内論文   出版者・発行元：John Wiley & Sons, Ltd  

DOI： 10.1002/9780470741191.ch1

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Unique taurine-containing Uridine derivatives, 5-taurinomethyluridine (tau m(5)U) and 5-taurinomethyl-2-thiouridine (tau m(5)s(2)U), which were discovered in mammalian mitochondrial tRNAs, exist at the first position of the anticodon. In this paper, we report the first efficient synthesis of tau m(5)U and tau m(5)s(2)U and describe their physicochemical properties. These modified ribonucleosides were synthesized by the reaction of 5-substituted uridine derivatives with a tetrabutylammonium salt of taurine that is highly reactive and well-soluble in common organic solvents. UV and (1)H NMR spectrometric studies revealed the structural properties of the taurine-containing base moieties and the Sugar conformations of these modified ribonucleosides.

DOI： 10.1021/jo802697r

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

In this study, an RNA isolation method was developed using a biotinylated peptide nucleic acid (PNA) that is complementary to the target RNA. Using the biotinylated PNA method, we successfully isolated several RNAs from Escherichia coli and from human total RNA in pure form. Damage to the RNA appears to be negligible by this method because the method is rapid and does not require a high temperature treatment to facilitate RNA-PNA binding.

DOI： 10.1093/jb/mvn107

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The detection of amino acids using aminoacyl-tRNA synthetases (ARSs) as the molecular recognition element was proposed, and the binding activity and specificity of ARSs were evaluated. Using this rapid and easy method, from 15 to 50 mu M tyrosine could be measured specifically. The method suggested in this article could be realized without an amino acid labeling process or a large volume of organic solvents, and the time for measurement was reasonable. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2008.03.037

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Post-transcriptional modifications at the first ( wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS ( mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (tau m(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (Delta mnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U center dot G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing tau m(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the m(5)U center dot G base pair does not have classical U center dot G wobble geometry. These structures provide help to explain how the tau m(5)U modification enables efficient decoding of UUG codons.

DOI： 10.1074/jbc.M800233200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the UIA RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed. to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

DOI： 10.1021/bc800020n

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids. Our previous studies suggested that tRNAs carrying large nonnatural amino acids are bound less tightly to EF-Tu than natural amino acids. To expand the availability of nonnatural mutagenesis, EF-Tu from the E coli translation system was improved to accept such large amino acids. We synthesized EF-Tu mutants, in which the binding pocket of the aminoacyl moiety of aminoacyl-tRNA was enlarged. L-1-Pyrenylalanine, L-2-pyrenylalanine, and DL-2anthraquinonylalanine, which are hardly or only slightly incorporated with the wild-type EF-Tu, were successfully incorporated into a protein using these EF-Tu mutants.

DOI： 10.1021/ja075557u

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記述言語：英語   出版者・発行元：WILEY  

Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins.

DOI： 10.1080/15216540701218722

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically amino-acylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.

DOI： 10.1263/jbb.102.511

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/nar/gkl526

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PORTLAND PRESS LTD  

Nematode mitochondria possess extremely truncated tRNAs. Of 22 tRNAs, 20 lack the entire T-arm. The T-arm is necessary for the binding of canonical tRNAs and EF (elongation factor)-Tu (thermo-unstable). The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm-binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D-arm. This is the first example of an EF-Tu recogpizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.

DOI： 10.1042/BJ20060781

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.

DOI： 10.1021/bi060536i

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記述言語：英語   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.ab.2005.08.008

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：IEEE  

Nonnatural amino acids, such as fluorescent amino acids, can be introduced into proteins by using an expanded protein biosynthesis system. This approach requires orthogonal tRNAs that deliver nonnatural amino acids to a ribosome and expanded codon/anticodon pairs, like four-base codon/anticodon pairs. However, in this system, some of nonnatural amino acids are not efficiently incorporated into proteins. To expand the availability of the nonnatural mugatenesis, translation factors and tRNAs must be improved. In this study, we designed and synthesized an elongation factor Tu (EF-Tu) mutant that efficiently binds to tRNAs charged with nonnatural amino acids. We also devised new tRNAs that decode four-base codons and efficiently deliver nonnatural amino acids into ribosome.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.11.010

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ymeth.2005.04.009

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

In the present study, mitochondrial ribosomes of the nematode Ascaris suum were isolated and their physiochernical properties were compared to ribosomes of Escherichia coli. The sedimentation coefficient and buoyant density of A. suum mitochondrial ribosomes were determined. The sedimentation coefficient of the intact monosome was about 55 S. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.40 g/cm(3), which suggests that the nematode mitoribosomes have a much higher protein composition than other mitoribosomes. The diffusion coefficients obtained from dynamic light scattering measurements were (1.48 +/- 0.04) x 10(-7) cm(2) s(-1) for 55 S mitoribosomes and (1.74 +/- 0.04) X 10(-7) cm(2) S-1 for the 70 S E. coli monosome. The diameter of mitoribosomes was measured by dynamic light-scattering analysis and electron microscopy. Though the nematode mitoribosome has a larger size than the bacterial ribosome, it does not differ significantly in size from mammalian mitoribosomes.

DOI： 10.1021/bi047833c

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

To understand the decoding property of nematode mitochondrial tRNAs with unusual secondary structures, post-transcriptional modifications at wobble positions of Ascaris suum mitochondrial tRNAs corresponding to two-codon families ending with a purine were analyzed. 5-Carboxymethylaminomethyluridine (cmnm(5)U) was identified at the wobble positions of tRNA(Lys), tRNA(Glu) and tRNA(Gln), while 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U) was present in tRNA(UAA)(Leu) and tRNA(Trp). In most bacterial and mitochondrial tRNAs, the 2-thiouridine derivative is present in tRNAs for Lys, Gin and Gln. These is no report that cmnm(5)s(2)U is used in tRNALeu and tRNA(Trp). The unusual usage of wobble modifications might assist decoding of nematode mitochondrial mRNAs. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.04.009

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA(Ser) that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D-T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.

DOI： 10.1093/nar/gki784

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

The mitochondria of the nematode Ascaris suum have tRNAs with unusual secondary structures that lack either the T-arm or D-arm found in most other organisms. Of the twenty-two tRNA species present in the mitochondria of A. suum, twenty lack the entire T-arm and two serine tRNAs lack the D-arm. To understand how such unusual tRNAs work in the nematode mitochondrial translation system, we analyzed post-transcriptional modifications of 11 mitochondrial tRNA species purified from A. suum, 10 of which lacked a T-arm and one of which lacked a D-arm. The most characteristic feature of nematode mitochondrial tRNAs lacking a T-arm was the presence of 1-methyladenosine at position 9 (m(1)A(9)). Synthesis of T-armless tRNAs with or without the modified nucleoside showed that T-armless tRNAs without the modification had much lower aminoacylation and EF-Tu-binding activities than native tRNAs. The addition of a single methyl group to A9 of these tRNAs was sufficient to restore nearly native levels of amino-acylation and EF-Tu-binding activity as well as tertiary structure, suggesting that m(1)A(9w) is a key residue for the activity of T-armless tRNAs. Thus, m(1)A(9) is indispensable for the structure and function of T-armless tRNAs of nematode mitochondrial origin.

DOI： 10.1093/nar/gki309

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4) TC), was prepared, and its degradation in 50 mM KOH at 60 degreesC was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was regarded as an intermediate before the strand break. The formation of this intermediate was compared with intermediates from the degradation of other alkali-labile lesions such as the abasic site, thymine glycol, and 5,6-dihydrothymine. The results strongly suggested that the first step of the alkali degradation of the (6- 4) photoproduct was the hydrolysis between the N3 and C4 positions of the 5'-pyrimidine component. Analyses by NMR spectroscopy and mass spectrometry supported the chemical structure of this product. Assays of the complex formation with XPC . HR23B and the translesion synthesis by DNA polymerase eta revealed that the biochemical properties are indistinguishable between the intact and hydrolyzed photoproducts.

DOI： 10.1074/jbc.M307186200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

The introduction of modified or labeled nucleotides into RNA is a powerful RNA engineering tool as it enables us to investigate how native RNA modifications affect RNA function and structure. It also helps in the structural analysis of RNA. A modified nucleotide can be introduced into a specific position of RNA by the method of two-step enzymatic ligation of RNA fragments. However, this method requires a complicated purification step between the two ligation steps that results in low yields of the ligation product. Here we have developed a new ligation technique employing periodate oxide that eliminates this purification step. This increases the total yield of the ligation product and makes it a faster procedure.

DOI： 10.1093/nar/gng145

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記述言語：英語   出版者・発行元：NATURE PUBLISHING GROUP  

DOI： 10.1038/nsb0803-669a

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記述言語：英語  

PubMed

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掲載種別：研究論文（学術雑誌）  

Scopus

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J-GLOBAL

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掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu.EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode-bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

The translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNAs to ribosomes by recognizing the tRNA acceptor and T stems. However, the unusual truncation observed in some animal mitochondrial tRNAs seems to prevent recognition by a canonical EF-Tu. For instance, nematode mitochondria contain tRNAs lacking a T or D arm. We recently found an atypical EF-Tu (EF-Tu1) specific for nematode mitochondrial tRNAs that lack the T arm. We have now discovered a second factor, EF-Tu2, which binds only to tRNAs that lack a D arm. EF-Tu2 seems unique in its amino acid specificity because it recognizes the aminoacyl moiety of seryl-tRNAs and the tRNA structure itself. Such EF-Tu evolution might explain tRNA structural divergence in animal mitochondria.

DOI： 10.1038/nsb826

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CAMBRIDGE UNIV PRESS  

In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 31 terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G2649.C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 8degrees) between the G264.C311 and C262.G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262.G312 and G413.C313 base pairs (45 +/- 10degrees), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 Angstrom resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.

DOI： 10.1017/S1355838202026043

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The tertiary structure of Ascaris suum mitochondrial tRNA(VCU)(Ser) was examined by nuclear magnetic resonance analysis using its transcript, since tRNA(UCU)(Ser), lacking the D arm and possessing a truncated T arm, is the shortest of all the known tRNAs. Most basepairs in the proposed secondary structure of tRNA(UCU)(Ser) were shown to exist, but the connector region comprising the truncated D loop and the extra loop was flexible. This flexibility, would enable adjustment of the mutual distance between the 3'-terminus and the anticodon consistent with that of usual tRNAs. Thus, tRNA(UCU)(Ser) appears to function in a similar way to that of usual tRNAs in the ribosome. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE AMERICA INC  

An unnatural base pair of 2-amino-6-(2-thienyl) purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/nass/1.1.237

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans, Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu, Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs, This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase, In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chain-terminator method supplemented with the deoxynucleoside triphosphate of y, The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

DOI： 10.1093/nass/42.1.191

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

Most nematode mitochondrial (mt) tRNAs have a TV-replacement loop (TV loop) which replaces the normal T arm and the variable loop in standard tRNAs with a less-structured loop. The tertiary structure of such tRNAs has been discussed theoretically with reference to the crystal structure of yeast tRNA(Phe) [Wolstenholme et al, (1994) Nucleic Acids Res. 22, 4300-4306] and examined experimentally by chemical and enzymatic probing [Watanabe et al. (1994) J, Biol. Chem. 269, 22902-22906]. The results suggest that most regions of the tRNA other than the TV loop are folded in a similar manner to yeast tRNA(Phe). To confirm this notion more clearly, the tertiary structure of Ascaris suum mt tRNA(Met) was analyzed by NMR using various synthetic tRNAs site-specifically labeled with stable isotopes, which were prepared by a combination of chemical synthesis and enzymatic ligation, Tertiary interactions involving G(L2), G(L3), U(L4), and U8 were observed in the NMR spectra of the labeled tRNAs, but those relating to G(L5) were not. On the basis of these results, a possible tertiary structural model of nematode mitochondrial tRNA(Met) was constructed.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPAN BIOCHEMICAL SOC  

RNA samples corresponding to Ascaris suum mitochondrial tRNA(Met) were chemically and automatically synthesized in amounts sufficient for NMR measurement. Conventional and rapid deprotection methods gave tRNA samples with the same amino acid-accepting activity as those prepared by other method; enzymatic synthesis, and enzymatic ligation of chemically synthesized fragments. The synthetic tRNA showed the same H-1-NMR spectrum in the iminoproton region as the ligated tRNA, This rapid and reliable preparation method thus provides biologically active tRNA for NMR measurement, and further, it is applicable for synthesis of other large synthetic RNAs, by combining the site-specific isotopic labeling method.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS UNITED KINGDOM  

Ascaris suum mitochondrial tRNA(Met) lacking the entire T stem was prepared by enzymatic ligation of two chemically synthesized RNA fragments. The synthetic tRNA could be charged with methionine by A.suum mitochondrial extract, although the charging activity was considerably low compared with that of the native tRNA, probably due to lack of modification, Enzymatic probing of the synthetic tRNA showed a very similar digestion pattern to that of the native tRNA(Met), which has already been concluded to take an L-shape-like structure [Watanabe et al, (1994) J. Biol. Chem., 269, 22902-22906], These results suggest that the synthetic tRNA possesses almost the same conformation as the native one, irrespective of the presence or absence of modified residues. The method of preparing the bizarre tRNA used here will provide a useful tool for elucidating the tertiary structure of such tRNAs, because they can be obtained without too much difficulty in the amounts necessary for physicochemical studies such as NMR spectroscopy.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MARCEL DEKKER INC  

Sulfated polysaccharide which had a relatively high degree of sulfation showed cytotoxicity to 3T3-L1 fibroblasts. Acidic and basic fibroblast growth factors inhibited the cell damage caused by the sulfated polysaccharides, while epidermal growth factor and platelet growth factor had no effects.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CHEMICAL SOC JAPAN  

Effects of synthetic polyanions on the mitogenic activity of fibroblast growth factors were investigated. Poly(vinylsulfonate) contributed to potentiate the activity of the acidic fibroblast growth factor, while the carboxyl group was quite effective for basic fibroblast growth factor.

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DOI： 10.1007/978-94-017-8896-0_19

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記述言語：日本語   出版者・発行元：日本DDS学会  

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その他リンク： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23651221/

記述言語：日本語  

CiNii Article

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記述言語：日本語   出版者・発行元：日本生化学会  

CiNii Article

CiNii Books

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その他リンク： https://search.jamas.or.jp/link/ui/2009151587

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その他リンク： https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-19021034/

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記述言語：日本語   出版者・発行元：(株)日本臨床社  

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出願番号：特願2008-1174884  出願日：2008年4月28日

公開番号：特開2008-301812 

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出願番号：特願2007-37041  出願日：2007年2月16日

公開番号：特開2008-301812 

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出願番号：特願2006-240828  出願日：2006年9月5日

公開番号：特開2008-061538 

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出願番号：特願2005-102868  出願日：2005年3月31日

特許番号/登録番号：特許4406731 

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出願番号：特願2005-103703  出願日：2005年3月31日

特許番号/登録番号：特許4709971 

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出願番号：特願2004-359328  出願日：2004年12月13日

公開番号：特開2006-166709 

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