Advance on detection method and application based on bioluminescence of luciferase / 中国药科大学学报

@#Bioluminescence is a widespread phenomenon in nature, and luminescent organisms can be found both on land and in the ocean. Among them, luciferase based bioluminescence systems have been widely studied, inspiring the exploration of genetic and epigenetic aspects and the development of a series of related assays for in vivo and in vitro studies. This paper summarizes the recent developments of luciferase based bioluminescence assays in terms of bioluminescence systems, types of luciferases, and the development and application of luciferase bioluminescence assays.

Synthesis, in vitro evaluation and in vivo imaging of a highly selective hydrazine bioluminescent probe based on luciferin-luciferase system / 药学学报

A highly sensitive and selective bioluminescent probe for hydrazine (BPH) was designed, synthesized and evaluated for detection of hydrazine in vitro and in vivo. BPH was designed to include a specific recognition group (acetyl) of hydrazine at an appropriate modification site of the optical reporter hydroxyluciferin (D-luciferin), which showed excellent performance both in selectivity and sensitivity to hydrazine. The results showed that the bioluminescent probe BPH developed in this study is an innovative and widely applicable tool for detecting hydrazine in complex natural environment or in animals.

Impact of Coix seed oil on fluorescence excretion pharmacokinetics and protein expression in doxorubicin-resistant cells MCF-7/DOX / 药学学报

This study was designed to explore the mechanism of Coix seed oil (Coix) impact on the drug resistance, bioluminescence imaging (BLI) and the efflux of D-luciferin potassium salt, the substrate of ABC transporters, in doxorubicin-resistant breast cancer cells. Multidrug resistance (MDR) gene and protein expression were analyzed in the cells by q-PCR and Western blot. First, in order to investigate the effect of the efflux function by ABC protein, a cell line with overexpressed luciferase was established in MCF-7 cell line. BLI was used to monitor the efflux kinetics of D-luciferin potassium salt before and after Coix treament. The results showed that the efflux of D-fluorescein potassium from MCF-7/DOXFluc was lessened when pretreated with Coix, which means that Coix may decrease the efflux of other chemotherapies using ABC transporters. Both of the results of q-PCR and Western blot showed that gene and protein expression of ABC transporters such as ABCG2, ABCC1 and ABCB1 were down-regulated by Coix treatment. The efficacy of Coix reversing MDR was verified with the chemotherapy medication doxorubicin (DOX). MTT assay showed that Coix increased the inhibitory effect of DOX on proliferation of MCF-7/DOX, and the optimal combination of ratio was 25 times that of DOX. The results suggest that Coix may reverse MDR of the substrate of ABC transporters from two aspects, one is to cut down the ABC protein efflux function, and the other is to decrease the quantity of ABC gene and protein expression.

Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado

ABSTRACT Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera) belonging to the Lampyridae (fireflies), Elateridae (click-beetles), and Phengodidae (railroad-worms) families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the "luminous termite mounds" in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.

Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency (k cat/K m) for lauric acid hydroxylation mainly due to an increase in K m. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in k cat and an increase in K m. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.