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- ホタル関連文献目録：欧文編 ネット上要旨集（年版 ver.2）
- A Spineless Biologist
- Lampyrid: The Journal of Bioluminescent Beetle Research 2011, v. 1 (Hardcover)
- eLife digest
However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation SFLC as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively.
Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.
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Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH.
Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H and E constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. Firefly luciferase -based dynamic bioluminescence imaging: a noninvasive technique to assess tumor angiogenesis.
Bioluminescence imaging BLI is emerging as a cost-effective, high-throughput, noninvasive, and sensitive imaging modality to monitor cell growth and trafficking. We describe the use of dynamic BLI as a noninvasive method of assessing vessel permeability during brain tumor growth. The maximum intensity was used as an indirect measurement of tumor growth.
Using a modified Evans blue perfusion protocol, we calculated the relative permeability of the vascular tree at various time points. Daily maximum intensity correlated strongly with tumor volume. At postinjection day 23, histology and BLI demonstrated an exponential growth of the tumor mass. Background Human interactome is predicted to contain , to , protein-protein interactions, PPIs. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics. Results Split Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E.
Finally, the stability of the probe was investigated. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu.
Mass spectrometry analysis and transcriptome sequencing reveal glowing squid crystal proteins are in the same superfamily as firefly luciferase. The Japanese firefly squid Hotaru-ika Watasenia scintillans produces intense blue light from photophores at the tips of two arms. These photophores are densely packed with protein microcrystals that catalyse the bioluminescent reaction using ATP and the substrate coelenterazine disulfate. The squid is the only organism known to produce light using protein crystals. We extracted microcrystals from arm tip photophores and identified the constituent proteins using mass spectrometry and transcriptome libraries prepared from arm tip tissue.
The crystals contain three proteins, wsluc1—3, all members of the ANL superfamily of adenylating enzymes. We propose that wsluc1—3 form a complex that crystallises inside the squid photophores, and that in the crystal one or more of the proteins catalyses the production of light using coelenterazine disulfate and ATP.
These results suggest that ANL superfamily enzymes have independently evolved in distant species to produce light using unrelated substrates. Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate ATP. A manual on the procedures and instruments developed for the adenosine triphosphate ATP luciferase assay is presented.
Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics. Moyle, Richard L.
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Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA- target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach.
The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA- target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined.
The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miRb, with as few as six technical replicates. The assay can be extended to assess other small RNA- target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence.
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Protein sterilization method of firefly luciferase using reduced pressure and molecular sieves. The sterilization of the protein fruitfly luciferase under conditions that prevent denaturation is examined. Denaturation is prevented by heating the protein in contact with molecular seives and under a reduced pressure of the order of 0.
ホタル関連文献目録：欧文編 ネット上要旨集（年版 ver.2）
Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases. Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference RNAi offers a promising approach to treating tumor patients.
In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure.
These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells. Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase. Luciferase is the key component of light production in bioluminescence process.
A Spineless Biologist
Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above Tm on the structure and dynamical properties of luciferase enzyme compared to temperature at K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at K. Increased inter domain hydrogen bonds by three at K suggests that inter domain contact is strengthened.
Lampyrid: The Journal of Bioluminescent Beetle Research 2011, v. 1 (Hardcover)
The appearance of simultaneous strong salt bridge and hydrogen bond between K and D and increased existence probability between R and E could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K and D experimentally.
balmoralcampestre.com/cmo-adiestrar-a-un-perro.php Also the significant reduction in SASA for experimentally important residues K, D and T which are involved in active site region was observed. Principle component analysis PCA in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation.
According to these results we proposed two strategies to improve thermal stability of functional luciferase. Identification and characterization of the Luc2-type luciferase in the Japanese firefly , Luciola parvula, involved in a dim luminescence in immobile stages.
Nocturnal Japanese fireflies , Luciola parvula, emit from their lanterns a yellow light, one of the most red-shifted colors found among fireflies. Previously, we isolated and characterized two different types of luciferase gene, Luc1 and Luc2, from the fireflies Luciola cruciata and Luciola lateralis; Luc1 is responsible for the green-yellow luminescence of larval and adult lanterns, whereas Luc2 is responsible for the dim greenish glow of eggs and pupal bodies.
The biological role of firefly lanterns in adults is related to sexual communication, but why the eggs and pupae glow remains uncertain.
In this study, we isolated the gene Luc2 from L. A semi-quantitative reverse transcription polymerase chain reaction showed that Luc1 was predominantly expressed in larvae, prepupae, pupae and adults, whereas Luc2 was expressed in eggs, prepupae, pupae and adult females. Enzymatic analyses showed that the luminescent color of Luc1 matches the visual sensitivity of L. These results suggest that the biological role of Luc2 expressed in immobile stages is not intraspecific communication.
The sensitive BRET system for the homogeneous immunoassay of a low-molecular weight antigen was developed using progesterone as an example. The methods for the synthesis of the luciferase -progesterone Luc-Pg conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody AF-Ab were developed. Both conjugates retained their functional properties, had high antigen-antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen.
Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.
Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for in vivo imaging of stem cells. One critical issue for noninvasive imaging of transplanted bioluminescent cells is the large amount of light absorption in tissue when emission wavelengths below nm are used. Luciferase with a red-shifted spectrum can potentially bypass this limitation. We assessed and compared a mutant of firefly luciferase Ppy RE9, PRE9 against the yellow luciferase luc2 gene for use in cell transplantation studies.