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1.
Neuroimage ; 301: 120882, 2024 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-39362505

RESUMO

BioLuminescent OptoGenetics ("BL-OG") is a chemogenetic method that can evoke optogenetic reactions in the brain non-invasively. In BL-OG, an enzyme that catalyzes a light producing reaction (i.e., a luciferase) is tethered to an optogenetic element that is activated in response to bioluminescent light. Bioluminescence is generated by injecting a chemical substrate (luciferin, e.g., h-Coelenterazine; h-CTZ) that is catalyzed by the luciferase. By directly injecting the luciferin into the brain, we show that bioluminescent light is proportional to spiking activity, and this relationship scales as a function of luciferin dosage. Here, we build on these previous observations by characterizing the temporal dynamics and dose response curves of bioluminescence generated by luminopsins (LMOs), a proxy of BL-OG effects, to intravenous (IV) injections of the luciferin. We imaged bioluminescence through a thinned skull of mice running on a wheel, while delivering h-CTZ via the tail vein with different dosage concentrations and injection rates. The data reveal a systematic relationship between strength of bioluminescence and h-CTZ dosage, with higher concentration generating stronger bioluminescence. We also found that bioluminescent activity occurs rapidly (< 60 s after IV injection) regardless of concentration dosage. However, as expected, the onset time of bioluminescence is delayed as the injection rate decreases. Notably, the strength and time decay of bioluminescence is invariant to the injection rate of h-CTZ. Taken together, these data show that BL-OG effects are highly consistent across injection parameters of h-CTZ, highlighting the reliability of BL-OG as a minimally invasive neuromodulation method.


Assuntos
Medições Luminescentes , Optogenética , Animais , Optogenética/métodos , Camundongos , Medições Luminescentes/métodos , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagem , Pirazinas/administração & dosagem , Pirazinas/farmacocinética , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Imidazóis/farmacocinética , Substâncias Luminescentes , Masculino , Luciferina de Vaga-Lumes/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos Endogâmicos C57BL
2.
Int J Mol Sci ; 25(16)2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39201296

RESUMO

Luminescent technology based on the luciferin-luciferase reaction has been extensively employed across various disciplines as a quantitative imaging modality. Owing to its non-invasive imaging capacity, it has evolved as a valuable in vivo bioimaging tool, particularly in small animal models in fields such as gene and cell therapies. We have previously successfully generated rats with a systemic expression of the luciferase gene at the Rosa26 locus. In this study, we transplanted bone marrow from these rats into micro-mini pigs and used in vivo imaging to non-invasively analyze the dynamics of the transplanted cells. In addition, we established that the rat-to-pig transplantation system is a discordant system, similar to the pig-to-human transplantation system. Thus, rat-to-pig transplantation may provide a clinically appropriate large animal model for pig-to-human xenotransplantation.


Assuntos
Transplante de Medula Óssea , Luciferases , Porco Miniatura , Transplante Heterólogo , Animais , Suínos , Ratos , Transplante de Medula Óssea/métodos , Transplante Heterólogo/métodos , Luciferases/metabolismo , Luciferases/genética , Humanos , Medições Luminescentes/métodos , Xenoenxertos , Luciferina de Vaga-Lumes/metabolismo , Luciferina de Vaga-Lumes/química
3.
J Am Chem Soc ; 146(19): 13406-13416, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38698549

RESUMO

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K+. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems.


Assuntos
Luciferases , Medições Luminescentes , Potássio , Potássio/metabolismo , Potássio/química , Animais , Medições Luminescentes/métodos , Camundongos , Luciferases/química , Luciferases/metabolismo , Humanos , Engenharia de Proteínas , Substâncias Luminescentes/química , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismo
4.
Int J Mol Sci ; 24(6)2023 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-36982148

RESUMO

Bioluminescence-based probes have long been used to quantify and visualize biological processes in vitro and in vivo. Over the past years, we have witnessed the trend of bioluminescence-driven optogenetic systems. Typically, bioluminescence emitted from coelenterazine-type luciferin-luciferase reactions activate light-sensitive proteins, which induce downstream events. The development of coelenterazine-type bioluminescence-induced photosensory domain-based probes has been applied in the imaging, sensing, and control of cellular activities, signaling pathways, and synthetic genetic circuits in vitro and in vivo. This strategy can not only shed light on the mechanisms of diseases, but also promote interrelated therapy development. Here, this review provides an overview of these optical probes for sensing and controlling biological processes, highlights their applications and optimizations, and discusses the possible future directions.


Assuntos
Fenômenos Biológicos , Medições Luminescentes , Medições Luminescentes/métodos , Luciferases/genética , Luciferases/metabolismo , Luciferina de Vaga-Lumes/metabolismo
5.
Int J Mol Sci ; 23(24)2022 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-36555716

RESUMO

The application of in vivo bioluminescent imaging in infectious disease research has significantly increased over the past years. The detection of transgenic parasites expressing wildtype firefly luciferase is however hampered by a relatively low and heterogeneous tissue penetrating capacity of emitted light. Solutions are sought by using codon-optimized red-shifted luciferases that yield higher expression levels and produce relatively more red or near-infrared light, or by using modified bioluminescent substrates with enhanced cell permeability and improved luminogenic or pharmacokinetic properties. In this study, the in vitro and in vivo efficacy of two modified bioluminescent substrates, CycLuc1 and AkaLumine-HCl, were compared with that of D-luciferin as a gold standard. Comparisons were made in experimental and insect-transmitted animal models of leishmaniasis (caused by intracellular Leishmania species) and African trypanosomiasis (caused by extracellular Trypanosoma species), using parasite strains expressing the red-shifted firefly luciferase PpyRE9. Although the luminogenic properties of AkaLumine-HCl and D-luciferin for in vitro parasite detection were comparable at equal substrate concentrations, AkaLumine-HCl proved to be unsuitable for in vivo infection follow-up due to high background signals in the liver. CycLuc1 presented a higher in vitro luminescence compared to the other substrates and proved to be highly efficacious in vivo, even at a 20-fold lower dose than D-luciferin. This efficacy was consistent across infections with the herein included intracellular and extracellular parasitic organisms. It can be concluded that CycLuc1 is an excellent and broadly applicable alternative for D-luciferin, requiring significantly lower doses for in vivo bioluminescent imaging in rodent models of leishmaniasis and African trypanosomiasis.


Assuntos
Parasitos , Tripanossomíase Africana , Animais , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Parasitos/metabolismo , Medições Luminescentes/métodos , Luciferases/genética , Luciferases/metabolismo , Luciferinas , Luciferina de Vaga-Lumes/metabolismo
6.
Angew Chem Int Ed Engl ; 61(46): e202209670, 2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36169114

RESUMO

Luciferin is one of Nature's most widespread luminophores, and enzymes that catalyze luciferin luminescence are the basis of successful commercial "glow" assays for gene expression and metabolic ATP formation. Herein we report an electrochemical method to promote firefly's luciferin luminescence in the absence of its natural biocatalyst-luciferase. We have gained experimental and computational insights on the mechanism of the enzyme-free luciferin electrochemiluminescence, demonstrated its spectral tuning from green to red by means of electrolyte engineering, proven that the colour change does not require, as still debated, a keto/enol isomerization of the light emitter, and gained evidence of the electrostatic-assisted stabilization of the charge-transfer excited state by double layer electric fields. Luciferin's electrochemiluminescence, as well as the in situ generation of fluorescent oxyluciferin, are applied towards an optical measurement of diffusion coefficients.


Assuntos
Luciferina de Vaga-Lumes , Luciferinas , Luciferases/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Luminescência , Catálise , Medições Luminescentes
7.
Int J Mol Sci ; 23(15)2022 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-35955470

RESUMO

This study demonstrates that the luciferin of the firefly squid Watasenia scintillans, which generally reacts with Watasenia luciferase, reacted with human albumin to emit light in proportion to the albumin concentration. The luminescence showed a peak wavelength at 540 nm and was eliminated by heat or protease treatment. We used urine samples collected from patients with diabetes to quantify urinary albumin concentration, which is essential for the early diagnosis of diabetic nephropathy. Consequently, we were able to measure urinary albumin concentrations by precipitating urinary proteins with acetone before the reaction with luciferin. A correlation was found with the result of the immunoturbidimetric method; however, the Watasenia luciferin method tended to produce lower albumin concentrations. This may be because the Watasenia luciferin reacts with only intact albumin. Therefore, the quantification method using Watasenia luciferin is a new principle of urinary albumin measurement that differs from already established methods such as immunoturbidimetry and high-performance liquid chromatography.


Assuntos
Decapodiformes , Vaga-Lumes , Albuminas/metabolismo , Albuminúria/diagnóstico , Animais , Decapodiformes/química , Vaga-Lumes/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Humanos , Luciferinas
8.
Sci Rep ; 12(1): 14815, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-36045277

RESUMO

Luciferin biosynthetic origin and alternative biological functions during the evolution of beetles remain unknown. We have set up a bioluminescent sensing method for luciferin synthesis from cysteine and benzoquinone using E. coli and Pichia pastoris expressing the bright Amydetes vivianii firefly and P. termitilluminans click beetle luciferases. In the presence of D-cysteine and benzoquinone, intense bioluminescence is quickly produced, indicating the expected formation of D-luciferin. Starting with L-cysteine and benzoquinone, the bioluminescence is weaker and delayed, indicating that bacteria produce L-luciferin, and then racemize it to D-luciferin in the presence of endogenous esterases, CoA and luciferase. In bacteria the p-benzoquinone toxicity (IC50 ~ 25 µM) is considerably reduced in the presence of cysteine, maintaining cell viability at 3.6 mM p-benzoquinone concomitantly with the formation of luciferin. Transcriptional analysis showed the presence of gene products involved with the sclerotization/tanning in the photogenic tissues, suggesting a possible link between these pathways and bioluminescence. The lack of two enzymes involved with the last steps of these pathways, indicate the possible accumulation of toxic quinone intermediates in the lanterns. These results and the abundance of cysteine producing enzymes suggest that luciferin first appeared as a detoxification byproduct of cysteine reaction with accumulated toxic quinone intermediates during the evolution of sclerotization/tanning in Coleoptera.


Assuntos
Besouros , Luciferina de Vaga-Lumes , Luciferases de Vaga-Lume , Quinonas , Animais , Besouros/metabolismo , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vaga-Lumes/genética , Luciferina de Vaga-Lumes/metabolismo , Luciferases/genética , Luciferases/metabolismo , Luciferases de Vaga-Lume/metabolismo , Luciferinas , Medições Luminescentes , Quinonas/metabolismo , Saccharomycetales/metabolismo
9.
Nat Commun ; 12(1): 2680, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976191

RESUMO

Bioluminescent imaging (BLI) is one of the most powerful and widely used preclinical imaging modalities. However, the current technology relies on the use of transgenic luciferase-expressing cells and animals and therefore can only be applied to a limited number of existing animal models of human disease. Here, we report the development of a "portable bioluminescent" (PBL) technology that overcomes most of the major limitations of traditional BLI. We demonstrate that the PBL method is capable of noninvasive measuring the activity of both extracellular (e.g., dipeptidyl peptidase 4) and intracellular (e.g., cytochrome P450) enzymes in vivo in non-luciferase-expressing mice. Moreover, we successfully utilize PBL technology in dogs and human cadaver, paving the way for the translation of functional BLI to the noninvasive quantification of biological processes in large animals. The PBL methodology can be easily adapted for the noninvasive monitoring of a plethora of diseases across multiple species.


Assuntos
Fenômenos Biológicos , Diagnóstico por Imagem/métodos , Medições Luminescentes/métodos , Modelos Animais , Animais , Animais Geneticamente Modificados , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Cães , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismo , Humanos , Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes/instrumentação , Estrutura Molecular , Reprodutibilidade dos Testes
10.
Cells ; 10(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668735

RESUMO

A major obstacle in studying the interplay between cancer cells and the immune system has been the examination of proposed biological pathways and cell interactions in a dynamic, physiologically relevant system in vivo. Intravital imaging strategies are one of the few molecular imaging techniques that can follow biological processes at cellular resolution over long periods of time in the same individual. Bioluminescence imaging has become a standard preclinical in vivo optical imaging technique with ever-expanding versatility as a result of the development of new emission bioluminescent reporters, advances in genomic techniques, and technical improvements in bioluminescence imaging and processing methods. Herein, we describe an advance of technology with a molecular imaging window chamber platform that combines bioluminescent and fluorescent reporters with intravital macro-imaging techniques and bioluminescence spectral unmixing in real time applied to heterogeneous living systems in vivo for evaluating tumor signaling dynamics and immune cell enzyme activities concurrently.


Assuntos
Comunicação Celular , Microscopia Intravital , Imagem Molecular , Neoplasias/imunologia , Transdução de Sinais , Animais , Luciferina de Vaga-Lumes/metabolismo , Genes Reporter , Luciferases/metabolismo , Medições Luminescentes , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pâncreas/patologia , Reprodutibilidade dos Testes
11.
Chem Soc Rev ; 50(9): 5668-5705, 2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-33735357

RESUMO

Bioluminescence is the fascinating natural phenomenon by which living creatures produce light. Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent systems but the luciferin-luciferase pairs of only 11 systems have been characterised to-date, whilst other novel systems are currently under investigation. The different luciferin-luciferase pairs have different light emission wavelengths and hence are suitable for various applications. The last decade or so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is now routinely used for gene assays, the detection of protein-protein interactions, high-throughput screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to control neurons. There has also been an increase in blue-sky research such as the engineering of various light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This review focuses on the past, present, and future applications of bioluminescence. We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist.


Assuntos
Biotecnologia , Luciferina de Vaga-Lumes/química , Luminescência , Medições Luminescentes , Animais , Vaga-Lumes , Luciferina de Vaga-Lumes/metabolismo
12.
Biochemistry ; 60(8): 563-572, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33599497

RESUMO

Bioluminescence imaging with luciferase-luciferin pairs is routinely used to monitor cellular functions. Multiple targets can be visualized in tandem using luciferases that process unique substrates, but only a handful of such orthogonal probes are known. Multiplexed studies require additional robust, light-emitting molecules. In this work, we report new luciferins for orthogonal imaging that comprise disubstituted cores. These probes were found to be bright emitters with various engineered luciferases. The unique patterns of light output also provided insight into enzyme-substrate interactions necessary for productive emission. Screening studies identified mutant luciferases that could preferentially process the disubstituted analogues, enabling orthogonal imaging with existing bioluminescent reporters. Further mutational analyses revealed the origins of substrate selectivity. Collectively, this work provides insights into luciferase-luciferin features relevant to bioluminescence and expands the number of probes for multicomponent tracking.


Assuntos
Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismo , Luciferases/metabolismo , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Células HEK293 , Humanos , Medições Luminescentes , Estrutura Molecular
13.
Int J Mol Sci ; 21(20)2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-33050422

RESUMO

: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate-coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization-Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme-substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.


Assuntos
Imidazóis/farmacologia , Luciferases/metabolismo , Medições Luminescentes/métodos , Pirazinas/farmacologia , Pesquisa , Cálcio/metabolismo , Ativação Enzimática , Luciferina de Vaga-Lumes/metabolismo , Expressão Gênica , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Estresse Oxidativo
14.
Sci Rep ; 10(1): 15882, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985577

RESUMO

Fireflies are among the most charismatic insects for their spectacular bioluminescence, but the origin and evolution of bioluminescence remain elusive. Especially, the genic basis of luciferin (D-luciferin) biosynthesis and light patterns is largely unknown. Here, we present the high-quality reference genomes of two fireflies Lamprigera yunnana (1053 Mb) and Abscondita terminalis (501 Mb) with great differences in both morphology and luminous behavior. We sequenced the transcriptomes and proteomes of luminous organs of two species. We created the CRISPR/Cas9-induced mutants of Abdominal B gene without luminous organs in the larvae of A. terminalis and sequenced the transcriptomes of mutants and wild-types. Combining gene expression analyses with comparative genomics, we propose a more complete luciferin synthesis pathway, and confirm the convergent evolution of bioluminescence in insects. Using experiments, the function of the firefly acyl-CoA thioesterase (ACOT1) to convert L-luciferin to D-luciferin was validated for the first time. Comparisons of three-dimension reconstruction of luminous organs and their differentially expressed genes among two species suggest that two positive genes in the calcium signaling pathway and structural difference of luminous organs may play an important role in the evolution of flash pattern. Altogether, our results provide important resources for further exploring bioluminescence in insects.


Assuntos
Evolução Biológica , Vaga-Lumes/genética , Luciferina de Vaga-Lumes/metabolismo , Animais , Vaga-Lumes/metabolismo , Proteoma , Especificidade da Espécie , Transcriptoma
15.
Cell Chem Biol ; 27(8): 904-920, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32795417

RESUMO

Bioluminescence has long been used to image biological processes in vivo. This technology features luciferase enzymes and luciferin small molecules that produce visible light. Bioluminescent photons can be detected in tissues and live organisms, enabling sensitive and noninvasive readouts on physiological function. Traditional applications have focused on tracking cells and gene expression patterns, but new probes are pushing the frontiers of what can be visualized. The past few years have also seen the merger of bioluminescence with optogenetic platforms. Luciferase-luciferin reactions can drive light-activatable proteins, ultimately triggering signal transduction and other downstream events. This review highlights these and other recent advances in bioluminescence technology, with an emphasis on tool development. We showcase how new luciferins and engineered luciferases are expanding the scope of optical imaging. We also highlight how bioluminescent systems are being leveraged not just for sensing-but also controlling-biological processes.


Assuntos
Medições Luminescentes/métodos , Animais , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Imidazóis/química , Luz , Luciferases/genética , Luciferases/metabolismo , Microscopia de Fluorescência , Neoplasias/diagnóstico por imagem , Imagem Óptica , Pirazinas/química
16.
Nat Chem Biol ; 16(12): 1385-1393, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32778841

RESUMO

Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.


Assuntos
Envelhecimento/genética , Diagnóstico por Imagem/métodos , Luciferina de Vaga-Lumes/química , Corantes Fluorescentes/química , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Potencial da Membrana Mitocondrial/genética , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Dioxóis/farmacologia , Feminino , Luciferina de Vaga-Lumes/metabolismo , Corantes Fluorescentes/metabolismo , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Neoplasias Mamárias Experimentais/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Nigericina/farmacologia , Compostos de Piridínio
17.
Angew Chem Int Ed Engl ; 59(38): 16485-16489, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32543104

RESUMO

Mechanochemical analogues have recently been established for several enzymatic reactions, but they require periodic interruption of the reaction for sampling, dissolution, and (bio)chemical analysis to monitor their progress. By applying a mechanochemical procedure to induce bioluminescence analogous to that used by the marine ostracod Cypridina (Vargula) hilgendorfii, here we demonstrate that the light emitted by a bioluminescent reaction can be used to directly monitor the progress of a mechanoenzymatic reaction without sampling. Mechanical treatment of Cypridina luciferase with luciferin generates bright blue light which can be readily detected and analyzed spectroscopically. This mechanically assisted bioluminescence proceeds through a mechanism identical to that of bioluminescence in solution, but has higher activation energy due to being diffusion-controlled in the viscous matrix. The results suggest that luciferases could be used as light-emissive reporters of mechanoenzymatic reactions.


Assuntos
Luciferases/metabolismo , Medições Luminescentes , Animais , Crustáceos , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismo , Luciferases/química , Estrutura Molecular
18.
Sci Rep ; 10(1): 9608, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541805

RESUMO

Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are  located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other  luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.


Assuntos
Dípteros/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Luciferases/metabolismo , Animais , Cromatografia por Troca Iônica , Dípteros/enzimologia , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/isolamento & purificação , Perfilação da Expressão Gênica , Luciferases/química , Luciferases/isolamento & purificação , Medições Luminescentes , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Espectrometria de Fluorescência
19.
Nat Biotechnol ; 38(8): 944-946, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32341562

RESUMO

Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants.


Assuntos
Luciferina de Vaga-Lumes/metabolismo , Nicotiana/genética , Plantas Geneticamente Modificadas/metabolismo , Ácidos Cafeicos/metabolismo , Fungos/genética , Fungos/metabolismo
20.
Biochem Biophys Res Commun ; 526(2): 404-409, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32223929

RESUMO

Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λmax = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-H2O2 system, but not in sodium hypochlorite.


Assuntos
Apoproteínas/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Imidazóis/metabolismo , Proteínas Luminescentes/metabolismo , Pirazinas/metabolismo , Apoproteínas/biossíntese , Apoproteínas/química , Escherichia coli/metabolismo , Luciferina de Vaga-Lumes/química , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Imidazóis/química , Medições Luminescentes , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/química , Pirazinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
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