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Yoshihiro Nakajima – One of the best experts on this subject based on the ideXlab platform.

  • correlation between luminescence intensity and cytotoxicity in cell based cytotoxicity assay using Luciferase
    Analytical Biochemistry, 2017
    Co-Authors: Shinobu Wakuri, Yoshihiro Nakajima, Kohji Yamakage, Yasuhiro Kazuki, Kanako Kazuki, Mitsuo Oshimura, Sachiyo Aburatani, Moritoshi Yasunaga
    Abstract:

    Abstract The Luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle Luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using Luciferase does not depend on the stability of Luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle Luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that Luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of Luciferases are simultaneously used in the cytotoxicity assay.

  • highly sensitive Luciferase reporter assay using a potent destabilization sequence of calpain 3
    Journal of Biotechnology, 2015
    Co-Authors: Moritoshi Yasunaga, Takako Noguchi, Yoshihiro Ohmiya, Mitsuo Oshimura, Kazutoshi Murotomi, Tomomi Yamazaki, Shigeaki Nishii, Tetsuya Ohbayashi, Kazuki Niwa, Yoshihiro Nakajima
    Abstract:

    Abstract Reporter assays that use Luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the Luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of Luciferase protein in the cells, which results in an increase of sensitivity of the Luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST ( t 1/2  = 9.8 to 2.8 h), the half-life of C9-fused ELuc was significantly shorter ( t 1/2  = 1.0 h) than that of PEST-fused ELuc when measurements were conducted at 37 °C. In addition, firefly Luciferase ( luc2 ) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrnecrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a Luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.

  • bioluminescence assays multicolor Luciferase assay secreted Luciferase assay and imaging Luciferase assay
    Expert Opinion on Drug Discovery, 2010
    Co-Authors: Yoshihiro Nakajima, Yoshihiro Ohmiya
    Abstract:

    Importance of the field: By selecting the most appropriate bioluminescence assay, the researcher can study the underlying molecular mechanisms of a physiological system and the effects of a drug throughout the body.Areas covered in this review: This review covers three Luciferase assay systems: the multicolor Luciferase assay, secreted Luciferase assay and imaging Luciferase assay. These assays are applied to drug screening in vitro, in cellulo and in vivo.What the reader will gain: Different solutions for reporter assay in vitro, in cellulo and in vivo are presented. A suitable bioluminescence system depending on the assay purpose is also discussed.Take home message: Bioluminescence is a manifold system based on the different types of luciferin and its Luciferase. Namely, luciferin catalyzed by corresponding Luciferases resulted in the production of different color lights. We must understand the manifold mechanisms of bioluminescence reaction.

Yoshihiro Ohmiya – One of the best experts on this subject based on the ideXlab platform.

  • Tibetan Firefly Luciferase with Low Temperature Adaptation.
    Photochemistry and Photobiology, 2016
    Co-Authors: Yasuo Mitani, Ryo Futahashi, Xingcai Liang, Yoshihiro Ohmiya
    Abstract:

    Fireflies are widespread all over the world and a numerous numbers of Luciferases have been isolated and characterized. In this study, we identified and characterized the Luciferase and Luciferase-like genes from a Tibetan firefly collected in Shangri-La, China. The altitude of this area is more than 3,300 meters. We saw this Tibetan firefly flying with strong luminescence after sunset at ~10°C. We analyzed the transcriptome of Tibetan firefly using head, thorax, abdomen (without light organ), and light organ tissue by RNA sequencing. We identified one Luciferase gene, which was almost identical to Luciferase from fireflies Pyrocoelia species, and expressed specifically in the light organ. Interestingly, the optimal temperature of the Tibetan firefly recombinant Luciferase was 10°C. The Km for D-luciferin and ATP of the recombinant Luciferase was 23 and 154 μM, respectively. The optimal pH was around 7.0 to 7.5. The emission peak was 556 nm at pH 8.0, while it shifted to 606 nm at pH 6.0. We also found a Luciferase-like gene with 43% identical amino acids to the Tibetan firefly Luciferase, which was scarcely expressed in any portion of the adult body. No Luciferase activity was detected for this Luciferase-like protein. This article is protected by copyright. All rights reserved.

  • highly sensitive Luciferase reporter assay using a potent destabilization sequence of calpain 3
    Journal of Biotechnology, 2015
    Co-Authors: Moritoshi Yasunaga, Takako Noguchi, Yoshihiro Ohmiya, Mitsuo Oshimura, Kazutoshi Murotomi, Tomomi Yamazaki, Shigeaki Nishii, Tetsuya Ohbayashi, Kazuki Niwa, Yoshihiro Nakajima
    Abstract:

    Abstract Reporter assays that use Luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the Luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of Luciferase protein in the cells, which results in an increase of sensitivity of the Luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST ( t 1/2  = 9.8 to 2.8 h), the half-life of C9-fused ELuc was significantly shorter ( t 1/2  = 1.0 h) than that of PEST-fused ELuc when measurements were conducted at 37 °C. In addition, firefly Luciferase ( luc2 ) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a Luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.

  • bioluminescence assays multicolor Luciferase assay secreted Luciferase assay and imaging Luciferase assay
    Expert Opinion on Drug Discovery, 2010
    Co-Authors: Yoshihiro Nakajima, Yoshihiro Ohmiya
    Abstract:

    Importance of the field: By selecting the most appropriate bioluminescence assay, the researcher can study the underlying molecular mechanisms of a physiological system and the effects of a drug throughout the body.Areas covered in this review: This review covers three Luciferase assay systems: the multicolor Luciferase assay, secreted Luciferase assay and imaging Luciferase assay. These assays are applied to drug screening in vitro, in cellulo and in vivo.What the reader will gain: Different solutions for reporter assay in vitro, in cellulo and in vivo are presented. A suitable bioluminescence system depending on the assay purpose is also discussed.Take home message: Bioluminescence is a manifold system based on the different types of luciferin and its Luciferase. Namely, luciferin catalyzed by corresponding Luciferases resulted in the production of different color lights. We must understand the manifold mechanisms of bioluminescence reaction.

Masaaki Ikeda – One of the best experts on this subject based on the ideXlab platform.

  • Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting Luciferases
    BMC Biotechnology, 2008
    Co-Authors: Takako Noguchi, Masaaki Ikeda, Yoshihiro Ohmiya, Yoshihiro Nakajima
    Abstract:

    Background Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color Luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle Luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time.

  • Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting Luciferases
    BMC Biotechnology, 2008
    Co-Authors: Takako Noguchi, Masaaki Ikeda, Yoshihiro Ohmiya, Yoshihiro Nakajima
    Abstract:

    BACKGROUND: Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color Luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle Luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time.\n\nRESULTS: Two Rat-1 cell lines were established that stably express either green- or red-emitting Luciferases under the control of the mBmal1 promoter, a canonical clock gene. We cocultured these cell lines, and gene expression profiles in both were monitored simultaneously. The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured. Furthermore, the independent rhythms were synchronized by medium change as an external stimulus.\n\nCONCLUSION: Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts.

  • multicolor Luciferase assay system one step monitoring of multiple gene expressions with a single substrate
    BioTechniques, 2005
    Co-Authors: Yoshihiro Nakajima, Masaaki Ikeda, Takuma Kimura, Kazunori Sugata, Toshiteru Enomoto, Atsushi Asakawa, H Kubota, Yoshihiro Ohmiya
    Abstract:

    Reporter assays that use Luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly Luciferase and Renilla Luciferase are used for monitoring single genegene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because of one of the two reporting Luciferases must be used as an internal control. We have developed a novel reporter assay system in which three Luciferases that emit green, orange, and red light with a single substrate are used as reporter genes. The activities of the Luciferases can be measured simultaneously and quantitatively with optical filters. This system enables us to simply and rapidly monitor multiple gene expressions in a one-step reaction.

Takako Noguchi – One of the best experts on this subject based on the ideXlab platform.

  • highly sensitive Luciferase reporter assay using a potent destabilization sequence of calpain 3
    Journal of Biotechnology, 2015
    Co-Authors: Moritoshi Yasunaga, Takako Noguchi, Yoshihiro Ohmiya, Mitsuo Oshimura, Kazutoshi Murotomi, Tomomi Yamazaki, Shigeaki Nishii, Tetsuya Ohbayashi, Kazuki Niwa, Yoshihiro Nakajima
    Abstract:

    Abstract Reporter assays that use Luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the Luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of Luciferase protein in the cells, which results in an increase of sensitivity of the Luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST ( t 1/2  = 9.8 to 2.8 h), the half-life of C9-fused ELuc was significantly shorter ( t 1/2  = 1.0 h) than that of PEST-fused ELuc when measurements were conducted at 37 °C. In addition, firefly Luciferase ( luc2 ) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a Luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.

  • Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting Luciferases
    BMC Biotechnology, 2008
    Co-Authors: Takako Noguchi, Masaaki Ikeda, Yoshihiro Ohmiya, Yoshihiro Nakajima
    Abstract:

    Background Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color Luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle Luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time.

  • Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting Luciferases
    BMC Biotechnology, 2008
    Co-Authors: Takako Noguchi, Masaaki Ikeda, Yoshihiro Ohmiya, Yoshihiro Nakajima
    Abstract:

    BACKGROUND: Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color Luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle Luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time.\n\nRESULTS: Two Rat-1 cell lines were established that stably express either green- or red-emitting Luciferases under the control of the mBmal1 promoter, a canonical clock gene. We cocultured these cell lines, and gene expression profiles in both were monitored simultaneously. The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured. Furthermore, the independent rhythms were synchronized by medium change as an external stimulus.\n\nCONCLUSION: Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts.

Anthony K. Campbell – One of the best experts on this subject based on the ideXlab platform.