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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Bakayan, Adil, Picaud, Sandrine, Malikova, Natalia P., Tricoire, Ludovic, Lambolez, Bertrand, Vysotski, Eugene S., Peyrieras, Nadine
Заглавие : RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities
Колич.характеристики :20 с
Коллективы : Centre National de la Recherche ScientifiqueCentre National de la Recherche Scientifique (CNRS); Agence Nationale de la RechercheFrench National Research Agency (ANR) [ANR-10-INBS-04, ANR-11-EQPX-029]; Fondation pour la Recherche sur le Cerveau/Rotary Club de France; RFBRRussian Foundation for Basic Research (RFBR) [20-04-00085]
Место публикации : Int. J. Mol. Sci.: MDPI, 2020. - Vol. 21, Is. 21. - Ст.7846. - ISSN 1422-0067(eISSN), DOI 10.3390/ijms21217846
Примечания : Cited References:53. - This work was supported by grants from Centre National de la Recherche Scientifique (AAP Prematuration CNRS 2016, to A.B. and N.P.; equipment transfer to S.P. and B.L.), from Agence Nationale de la Recherche (AAP Prematuration FCS/IDEX Paris Saclay, to A.B. and N.P., France BioImaging infrastructure ANR-10-INBS-04, ANR-11-EQPX-029 to N.P.), from Fondation pour la Recherche sur le Cerveau/Rotary Club de France (B.L.), and from RFBR (project number 20-04-00085 to N.P.M. and E.S.V.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Предметные рубрики: IN-VIVO
PHOTOPROTEIN AEQUORIN
CA2+-REGULATED PHOTOPROTEINS
SPREADING
Аннотация: Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Bakayan A., Picaud S., Malikova N. P., Tricoire L., Lambolez B., Vysotski E. S., Peyrieras N.
Заглавие : Redquorinxs mutants with enhanced calcium sensitivity and bioluminescence output efficiently report cellular and neuronal network activities
Место публикации : Int. J. Mol. Sci.: MDPI AG, 2020. - Vol. 21, Is. 21. - Ст.7846. - С. 1-22. - ISSN 16616596 (ISSN), DOI 10.3390/ijms21217846
Аннотация: Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+? dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP?aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty?four amino acid positions in and around EF?hand Ca2+?binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C?terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild?type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild?type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt?Redquorin, and one, RedquorinXS?Q159T, outperformed GA. Finally, wide?field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS?Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS?Q159T is a red light?emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Scopus
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Pande, Shubhra, Kratasyuk, Valentina A., Medvedeva, Nadezhda N., Kolenchukova, Oxana A., Salmina, Alla B.
Заглавие : Nutritional biomarkers: Current view and future perspectives
Колич.характеристики :15 с
Коллективы : Russian Foundation for Basic Research [16-06-00-439]; Russian Ministry of Education [M 2.2.3]; Russian Foundation for Basic Research; Krasnoyarsk Regional Fund for Scientific and Technical Activity Support [16-44-242126]
Место публикации : Crit. Rev. Food Sci. Nutr.: TAYLOR & FRANCIS INC, 2018. - Vol. 58, Is. 18. - С. 3055-3069. - ISSN 1040-8398, DOI 10.1080/10408398.2017.1350136. - ISSN 1549-7852(eISSN)
Примечания : Cited References:163. - The research was partially supported by the Russian Foundation for Basic Research [Grant No 16-06-00-439], the Russian Ministry of Education, Post-Doctoral Program of Project "5-100" [Grant No. M 2.2.3] and the Russian Foundation for Basic Research in collaboration with Krasnoyarsk Regional Fund for Scientific and Technical Activity Support [project No 16-44-242126].
Предметные рубрики: PROMOTES CELL-SURVIVAL
FATTY-ACID OXIDATION
CALORIE RESTRICTION
Ключевые слова (''Своб.индексиров.''): sirtuins--nad--biomarker--proteomics--calorie restriction--longitivity
Аннотация: There is a poor relationship between nutrient intake and existing nutritional biomarkers due to variety of factors affecting their sensitivity and specificity. To explore the impact of nutrients at molecular level and devising a sensitive biomarker, proteomics is a central technology with sirtuins as one of the most promising nutritional biomarker. Sirtuins (seven mammalian sirtuins reported so far) have been reported to perform protein deacetylases and ADP-ribosyltransferases activity. It is distributed in different cellular compartments thereby controlling several metabolic processes. Sirtuins are oxidized nicotinamide adenine dinucleotide dependent, which implicates a direct effect of the metabolic state of the cell on its activity. Calorie restriction upregulates the mammalian sirtuin protein levels in variety of tissues and organs where it acts upon both histone and nonhistone substrates. Sirtuin senses nutrient availability and impacts gluconeogenesis, glycolysis, and insulin sensitivity. It deacetylates and inhibits the nuclear receptor that activates fat synthesis and adipogenesis in the body, leading to fat loss and bringing favorable cellular and health changes. Sirtuins mediates intracellular response that promotes cell survival, DNA damage repair thereby increasing the cell longitivity. The activation of sirtuins brings a wide spectrum of other health benefits and its activity levels are indicative of nutritional status as well as disease progression in cancer, inflammation, obesity, cardiovascular diseases, and viral infections. There are several foods that activate sirtuin activity and offer significant health benefits by their consumption.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova, Svetlana V., Larionova, Marina D., Burakova, Ludmila P., Vysotski, Eugene S.
Заглавие : The smallest natural high-active luciferase: Cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa
Колич.характеристики :6 с
Коллективы : Bayer AG (Germany); Russian Science Foundation [14-14-01119]
Место публикации : Biochem. Biophys. Res. Commun.: ACADEMIC PRESS INC ELSEVIER SCIENCE, 2015. - Vol. 457, Is. 1. - С. 77-82. - ISSN 0006-291X, DOI 10.1016/j.bbrc.2014.12.082. - ISSN 1090-2104(eISSN)
Примечания : Cited References:20. - The cloning of cDNA encoding MLuc7 luciferase of M. longa was supported by Bayer AG (Germany); all other studies - by the grant 14-14-01119 of the Russian Science Foundation. We declare that authors have no conflict of interest.
Предметные рубрики: CDNA CLONING
SECRETED LUCIFERASE
ESCHERICHIA-COLI
EXPRESSION
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--copepod luciferase--mammalian--expression--real-time imaging
Аннотация: Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 S-S bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of similar to 3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. (C) 2014 Elsevier Inc. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova S.V., Burakova L.P., Golz S..., Malikova N.P., Frank L.A., Vysotski E.S.
Заглавие : The light-sensitive photoprotein berovin from the bioluminescent ctenophore Beroe abyssicola: a novel type of Ca2+-regulated photoprotein
Колич.характеристики :15 с
Место публикации : FEBS J.: WILEY-BLACKWELL, 2012. - Vol. 279, Is. 5. - С. 856-870. - ISSN 1742-464X, DOI 10.1111/j.1742-4658.2012.08476.x
Примечания : Cited References: 63. - The authors thank Natalia Chervyakova from Department of Zoology of Invertebrates of Moscow State University for the photo of the White Sea ctenophore Beroe abyssicola. This work was supported by RFBR grant 09-04-00172, by grant 64987.2010.4, Molecular and Cellular Biology program of RAS, and Bayer Pharma AG (Germany).
Предметные рубрики: CALCIUM-ACTIVATED PHOTOPROTEINS
C-TERMINAL PROLINE
SEQUENCE-ANALYSIS
MNEMIOPSIS-SP
COELENTERAZINE-BINDING
ANGSTROM RESOLUTION
RECOMBINANT OBELIN
CRYSTAL-STRUCTURES
EXCITED-STATE
CDNA CLONING
Ключевые слова (''Своб.индексиров.''): bioluminescence--calcium--coelenterazine--luciferase--mammalian expression
Аннотация: Light-sensitive Ca2+-regulated photoproteins are responsible for the bright bioluminescence of ctenophores. Using functional screening, four full-size cDNA genes encoding the same 208-amino-acid polypeptide were isolated from two independent cDNA libraries prepared from two Beroe abyssicola specimens. Sequence analysis revealed three canonical EF-hand calcium-binding sites characteristic of Ca2+-regulated photoproteins, but a very low degree of sequence identity (2729%) with aequorin-type photoproteins, despite functional similarities. Recombinant berovin was expressed in Escherichia coli cells, purified, converted to active photoprotein and characterized. Active berovin has absorption maxima at 280 and 437 nm. The Ca2+-discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (?max = 491 nm) and a change in pH over the range 6.09.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca2+-discharged protein (?ex = 350 nm) is pH sensitive: at neutral pH the maximum is at 420 nm and at alkaline pH there are two maxima at 410 and 485 nm. Like native ctenophore photoproteins, recombinant berovin is also inactivated by light. The Ca2+ concentrationeffect curve is a sigmoid with a slope on a loglog plot of similar to 2.5. Although this curve for berovin is very similar to those obtained for obelin and aequorin, there are evident distinctions: berovin responds to calcium changes at lower concentrations than jellyfish photoproteins and its Ca2+-independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova S.V., Burakova L.P., Vysotski E.S.
Заглавие : High-active truncated luciferase of copepod Metridia longa
Колич.характеристики :6 с
Место публикации : Biochem. Biophys. Res. Commun.: ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012. - Vol. 417, Is. 1. - С. 98-103. - ISSN 0006-291X, DOI 10.1016/j.bbrc.2011.11.063
Примечания : Cited References: 31. - This study was supported by the Grants 16.512.11.2141 and 64987.2010.4 of the Ministry of Education and Science of Russian Federation.
Предметные рубрики: COELENTERAZINE-BINDING PROTEIN
REPORTER-GENE-EXPRESSION
RENILLA LUCIFERASE
GAUSSIA LUCIFERASE
LIGHT-EMITTER
IN-VIVO
BIOLUMINESCENCE
PHOTOPROTEINS
CDNA
SUBSTRATE
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--mammalian expression--secretion
Аннотация: The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coil cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme. (C) 2011 Elsevier Inc. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova S.V., Burakova L.P., Frank L.A., Golz S..., Korostileva K.A., Vysotski E.S.
Заглавие : Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria: cDNA cloning, expression, and characterization of novel recombinant protein
Колич.характеристики :9 с
Коллективы :
Место публикации : Photochem. Photobiol. Sci.: ROYAL SOC CHEMISTRY, 2010. - Vol. 9, Is. 6. - С. 757-765. - ISSN 1474-905X, DOI 10.1039/c0pp00023j
Примечания : Cited References: 42. - We thank Dr John Lee (University of Georgia) for constructive suggestions. This work was supported by the Russian Foundation for Basic Research (Grants: 08-04-92209 and 09-04-12022), "Molecular and Cell Biology" program of RAS, and Bayer AG (Germany).
Предметные рубрики: ENERGY-TRANSFER
CA2+-REGULATED PHOTOPROTEINS
RENILLA BIOLUMINESCENCE
ANGSTROM RESOLUTION
SEQUENCE-ANALYSIS
CRYSTAL-STRUCTURE
EXCITED-STATE
AEQUORIN
PURIFICATION
OBELIN
Аннотация: The bioluminescent systems of many marine organisms are comprised of two proteins - the Ca2+-regulated photoprotein and green-fluorescent protein (GFP). This work reports the cloning of the full-size cDNA encoding GFP (cgreGFP) from jellyfish Clytia gregaria, its expression and properties of the recombinant protein. The overall degree of identity between the amino acid sequence of the novel cgreGFP and the sequence of GFP (avGFP) from Aequorea victoria is 42% (similarity - 64%) despite these GFPs originating from jellyfish that both belong to the same class, Hydrozoa. However although the degree of identity is low, three residues, Ser-Tyr-Gly, which form the chromophore are identical in both GFPs. The cgreGFP displayed two absorption peaks at 278 and 485 nm, and the fluorescence maximum at 500 nm. The fluorescence quantum yield was determined to be 0.86, the brightness to be 54 mM(-1) cm(-1). For the first time we have also demonstrated an efficient radiationless energy transfer in vitro between clytin and cgreGFP in solution at micromolar concentrations. The cgreGFP may be a useful intracellular fluorescent marker, as it was able to be expressed in mammalian cells.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Stepanyuk G.A., Xu H..., Wu C.K., Markova S.V., Lee J..., Vysotski E.S., Wang B.C.
Заглавие : Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells
Колич.характеристики :7 с
Коллективы :
Место публикации : Protein Expr. Purif.: ACADEMIC PRESS INC ELSEVIER SCIENCE, 2008. - Vol. 61, Is. 2. - С. 142-148. - ISSN 1046-5928, DOI 10.1016/j.pep.2008.05.013
Примечания : Cited References: 34. - This work was supported by the National Institutes of Health (Grant 1P50 GM62407), University of Georgia Research Foundation and Georgia Research Alliance, the Russian Foundation for Basic Research and Taiwan National Science Council (Grant 06-0489502) and the program for "Molecular and Cellular Biology" of Russian Academy of Sciences.
Предметные рубрики: VARGULA-HILGENDORFII LUCIFERASE
CRYSTAL-STRUCTURE
RENILLA-RENIFORMIS
GAUSSIA LUCIFERASE
BIOLUMINESCENT REPORTER
OBELIN BIOLUMINESCENCE
ANGSTROM RESOLUTION
MAMMALIAN-CELLS
GENE-EXPRESSION
IN-VIVO
Аннотация: Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2-3mg/L This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence. indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis. (c) 2008 Elsevier Inc. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Lamarcq L.H., Scherer B.J., Phelan M.L., Kalnine N.N., Nguyen Y.H., Kabakova T..., Chen X.Y., Tan M..., Chang C..., Berlon C..., Campos-Gonzalez R..., Gao G.J., Golz S..., Vysotski E.S., Farmer A.A.
Заглавие : Large-scale, high-throughput validation of short hairpin RNA sequences for RNA interference
Колич.характеристики :11 с
Место публикации : J. Biomol. Screen: SAGE PUBLICATIONS INC, 2006. - Vol. 11, Is. 3. - С. 236-246. - ISSN 1087-0571, DOI 10.1177/1087057105284342
Примечания : Cited References: 50
Предметные рубрики: ENZYME FRAGMENT COMPLEMENTATION
ORFEOME VERSION 1.1
SMALL NUCLEAR-RNA
MAMMALIAN-CELLS
GENE-EXPRESSION
SIRNA SEQUENCES
POLYMERASE-III
SELECTION
TRANSCRIPTION
MICROARRAYS
Ключевые слова (''Своб.индексиров.''): rnai--high-throughput screening--target validation--shrna--reporter assay
Аннотация: (shRNAs) is described. Using this approach, 464 shRNAs auainst 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAS against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for position-specific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNA interference (RNAi).
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10.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Stepanyuk G.A., Golz S..., Markova S.V., Frank L.A., Lee J..., Vysotski E.S.
Заглавие : Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein
Колич.характеристики :7 с
Место публикации : FEBS Lett.: ELSEVIER SCIENCE BV, 2005. - Vol. 579, Is. 5. - С. 1008-1014. - ISSN 0014-5793, DOI 10.1016/j.febslet.2005.01.004
Примечания : Cited References: 49
Предметные рубрики: FIREFLY LUCIFERASE
SEQUENCE-ANALYSIS
CA2+-REGULATED PHOTOPROTEINS
CA2+-DISCHARGED PHOTOPROTEIN
VIOLET BIOLUMINESCENCE
INTRACELLULAR CALCIUM
ENDOPLASMIC-RETICULUM
ANGSTROM RESOLUTION
CRYSTAL-STRUCTURE
APOAEQUORIN CDNA
Ключевые слова (''Своб.индексиров.''): coelenterazine--calcium--reporter protein--mammalian expression--fluorescence spectrum
Аннотация: The bioluminescence spectra from the Ca2+-regulated photoproteins aequorin (lambda(max) = 469 nm) and obelin (lambda(max) = 482 nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter wavelength with F88Y having lambda(max) = 453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to lambda(max) = 501 nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection. (C) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova S.V., Golz S..., Frank L.A., Kalthof B..., Vysotski E.S.
Заглавие : Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa - A novel secreted bioluminescent reporter enzyme
Колич.характеристики :6 с
Место публикации : J. Biol. Chem.: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2004. - Vol. 279, Is. 5. - С. 3212-3217. - ISSN 0021-9258, DOI 10.1074/jbc.M309639200
Примечания : Cited References: 37
Предметные рубрики: VARGULA-HILGENDORFII LUCIFERASE
GREEN FLUORESCENT PROTEIN
GENE-EXPRESSION
FIREFLY LUCIFERASE
PROMOTER ACTIVITY
MAMMALIAN-CELLS
RECEPTOR
CANCER
PHOTOPROTEINS
LUMINESCENCE
Аннотация: Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max)=480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultra-high throughput screening technologies.
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13.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Setkov N.A., Epifanova O.I.
Заглавие : Brief exposures of resting fibroblasts to okadaic acid stimulate DNA synthesis
Колич.характеристики :13 с
Место публикации : Cell Prolif.: BLACKWELL SCIENCE LTD, 1997. - Vol. 30, Is. 1. - P7-19. - ISSN 0960-7722, DOI 10.1111/j.1365-2184.1997.tb00912.x
Примечания : Cited References: 32
Предметные рубрики: PDGF-INDUCED PROLIFERATION
HAMSTER-EMBRYO CELLS
PROTEIN-PHOSPHORYLATION
PHOSPHATASE INHIBITORS
MOUSE FIBROBLASTS
3T3 CELLS
FUSION
TRANSFORMATION
INDUCTION
ARREST
Аннотация: To study further the factors providing for cellular quiescence, we used okadaic acid (OA) at concentrations (0.1, 1, 10 or 100 nM) inhibiting type 1 and/or type 2A protein phosphatases in mammalian cell cultures. Brief (2 h) exposure of resting (0.2% serum for 72 h) NIH 3T3 mouse fibroblasts to OA with subsequent incubation of cells in a medium with 0.2% serum, stimulated DNA synthesis at all concentrations studied. Maximal stimulation was observed following pre-incubation of resting cells with 10 nM OA. Treatment of cycling cells (10% serum) with OA (2 h pulses at 12 h intervals for 72 h) prevented their exit to the resting state on transfer to a medium with 0.2% serum. Brief exposures of resting cells to OA did not affect the rate of protein synthesis. OA pulses in the late pre-replicative period had no effect on the entry of serum-stimulated cells into the S phase. Cell fusion experiments with resting (serum-deprived) and proliferating (serum-stimulated) NIH 3T3 cells, using radioautography with a double-labelling technique, revealed that pre-incubation of resting cells with OA for 2 h before and after fusion abrogates their ability to suppress the onset of DNA synthesis in the nuclei of proliferating cells in heterodikaryons. The results indicate that protein phosphatases of type 1 and/or 2A may be involved in the growth-arrest machinery that provides for cellular quiescence.
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