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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Fritsler Y., Bartsev S., Belozor O., Ant. S., And. S.
Заглавие : Modifying the Models of Calcium Dynamics in Astrocytes by Ryanodine Release
Место публикации : Math. Biol. Bioinform.: Russian Academy of Sciences,Department of the Earth Sciences, 2021. - Vol. 16, Is. 1. - С. 86-100. - ISSN 19946538 (ISSN), DOI 10.17537/2021.16.86
Аннотация: The influence of ryanodine channels on the cytosole Ca2+dynamics was studied. We added the equations for ryanodine receptors and voltage-gated calcium channels into the original De Pitta et al. model of Ca2+. The derived model was shown to have significantly wider range of predictions: we derived the frequency of cytosole calcium spontaneous oscillations (which are absent in the original De Pitta et al. model) for various existing models of Ca2+signalling in astrocytes. Particularly, the initial De Pitta et al. results can be converted to either Lavrentovich and Hemkin model or in the Dupont et al model predictions. The absence of the Ca2+oscillations in astrocytes with the active ryanodine channels only was recently reported. This behaviour can be achieved in our model predictions for the certain values of parameters, which are supposedly responsible for the bifurcation landscape between the oscillatory and non-oscillatory dynamics of cytosol Ca2+in astrocytes. We also investigated the interplay between the spontaneous and glutamate-triggered oscillations. © 2021. All Rights Reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Malikova N. P., Eremeeva E. V., Gulnov D. V., Natashin P. V., Nemtseva E. V., Vysotski E. S.
Заглавие : Specific Activities of Hydromedusan Ca2+-Regulated Photoproteins
Место публикации : Photochem. Photobiol.: John Wiley and Sons Inc, 2021. - Article in press. - ISSN 00318655 (ISSN), DOI 10.1111/php.13556
Аннотация: Nowadays the recombinant Ca2+-regulated photoproteins originating from marine luminous organisms are widely applied to monitor calcium transients in living cells due to their ability to emit light on Ca2+ binding. Here we report the specific activities of the recombinant Ca2+-regulated photoproteins—aequorin from Aequorea victoria, obelins from Obelia longissima and Obelia geniculata, clytin from Clytia gregaria and mitrocomin from Mitrocoma cellularia. We demonstrate that along with bioluminescence spectra, kinetics of light signals and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of bioluminescent reactions. The highest specific activities were found for obelins and mitrocomin, whereas those of aequorin and clytin were shown to be lower. To determine the factors influencing the variations in specific activities the fluorescence quantum yields for Ca2+-discharged photoproteins were measured and found to be quite different varying in the range of 0.16–0.36. We propose that distinctions in specific activities may result from different efficiencies of singlet excited state generation and different fluorescence quantum yields of coelenteramide bound within substrate-binding cavity. This in turn may be conditioned by variations in the amino acid environment of the substrate-binding cavities and hydrogen bond distances between key residues and atoms of 2-hydroperoxycoelenterazine. © 2021 American Society for Photobiology
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Larionova, Marina D., Wu, Lijie, Eremeeva, Elena, V, Natashin, Pavel, V, Gulnov, Dmitry, V, Nemtseva, Elena, V, Liu, Dongsheng, Liu, Zhi-Jie, Vysotski, Eugene S.
Заглавие : Crystal structure of semisynthetic obelin-v
Колич.характеристики :16 с
Коллективы : National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [32011530076]; Russian Foundation for Basic ResearchRussian Foundation for Basic Research (RFBR) [20-04-00085, 20-44-240006, 20-54-53011]
Место публикации : Protein Sci.: WILEY, 2021. - Article in press. - ISSN 0961-8368, DOI 10.1002/pro.4244. - ISSN 1469-896X(eISSN)
Примечания : Cited References:69. - National Natural Science Foundation of China, Grant/Award Number: 32011530076; Russian Foundation for Basic Research, Grant/Award Numbers: 20-04-00085, 20-44-240006, 20-54-53011
Предметные рубрики: CA2+-REGULATED PHOTOPROTEIN OBELIN
PHOTOLUMINESCENCE QUANTUM YIELD
Аннотация: Coelenterazine-v (CTZ-v), a synthetic derivative with an additional benzyl ring, yields a bright bioluminescence of Renilla luciferase and its "yellow" mutant with a significant shift in the emission spectrum toward longer wavelengths, which makes it the substrate of choice for deep tissue imaging. Although Ca2+-regulated photoproteins activated with CTZ-v also display red-shifted light emission, in contrast to Renilla luciferase their bioluminescence activities are very low, which makes photoproteins activated by CTZ-v unusable for calcium imaging. Here, we report the crystal structure of Ca2+-regulated photoprotein obelin with 2-hydroperoxycoelenterazine-v (obelin-v) at 1.80 angstrom resolution. The structures of obelin-v and obelin bound with native CTZ revealed almost no difference; only the minor rearrangement in hydrogen-bond pattern and slightly increased distances between key active site residues and some atoms of 2-hydroperoxycoelenterazine-v were found. The fluorescence quantum yield (phi(FL)) of obelin bound with coelenteramide-v (0.24) turned out to be even higher than that of obelin with native coelenteramide (0.19). Since both obelins are in effect the enzyme-substrate complexes containing the 2-hydroperoxy adduct of CTZ-v or CTZ, we reasonably assume the chemical reaction mechanisms and the yields of the reaction products (phi(R)) to be similar for both obelins. Based on these findings we suggest that low bioluminescence activity of obelin-v is caused by the low efficiency of generating an electronic excited state (phi(S)). In turn, the low phi(S) value as compared to that of native CTZ might be the result of small changes in the substrate microenvironment in the obelin-v active site.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva, Elena, V, Jiang, Tianyu, Malikova, Natalia P., Li, Minyong, Vysotski, Eugene S.
Заглавие : Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents
Колич.характеристики :21 с
Коллективы : RFBRRussian Foundation for Basic Research (RFBR); NSFCNational Natural Science Foundation of China (NSFC) [20-54-53011]; Russian Foundation for Basic ResearchRussian Foundation for Basic Research (RFBR) [18-44-242001]; Krasnoyarsk Regional Fund of Science; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [81874308]; Shandong Natural Science FoundationNatural Science Foundation of Shandong Province [ZR2018ZC0233]; Government of Krasnoyarsk Territory
Место публикации : Int. J. Mol. Sci.: MDPI, 2020. - Vol. 21, Is. 15. - Ст.5446. - ISSN 1422-0067(eISSN), DOI 10.3390/ijms21155446
Примечания : Cited References:50. - The reported study was funded by RFBR and NSFC according to the research project No. 20-54-53011 (E.V.E. and N.P.M.), Russian Foundation for Basic Research (No. 18-44-242001), Government of Krasnoyarsk Territory, Krasnoyarsk Regional Fund of Science (E.S.V.), the National Natural Science Foundation of China (No. 81874308), and the Shandong Natural Science Foundation (No. ZR2018ZC0233) (M.L.).
Предметные рубрики: CA2+-REGULATED PHOTOPROTEINS
SPECTROSCOPIC PROPERTIES
Аннотация: Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues.
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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.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva E. V., Jiang T., Malikova N. P., Li M., Vysotski E. S.
Заглавие : Bioluminescent properties of semi-synthetic obelin and aequorin activated by coelenterazine analogues with modifications of C-2, C-6, and C-8 substituents
Место публикации : Int. J. Mol. Sci.: MDPI AG, 2020. - Vol. 21, Is. 15. - Ст.5446. - С. 1-21. - ISSN 16616596 (ISSN), DOI 10.3390/ijms21155446
Аннотация: Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Ding, Bo-Wen, Eremeeva, Elena V., Vysotski, Eugene S., Liu, Ya-Jun
Заглавие : Luminescence Activity Decreases Whenv-coelenterazine Replaces Coelenterazine in Calcium-Regulated Photoprotein-A Theoretical and Experimental Study
Колич.характеристики :14 с
Коллективы : National Natural Science Foundation of ChinaNational Natural Science Foundation of China [21911530094, 21673020, 21973005]; RFBRRussian Foundation for Basic Research (RFBR) [19-54-53004, 20-54-53011]; China Postdoctoral Science FoundationChina Postdoctoral Science Foundation [2018M630100]
Место публикации : Photochem. Photobiol.: WILEY, 2020. - Article in press. - ISSN 0031-8655, DOI 10.1111/php.13280. - ISSN 1751-1097(eISSN)
Примечания : Cited References:68. - This study was sponsored by the National Natural Science Foundation of China (Grant No. 21911530094, 21673020 and 21973005) and RFBR (Grant No. 19-54-53004 and 20-54-53011). Ding also thank the support from the China Postdoctoral Science Foundation (Grant No. 2018M630100).
Предметные рубрики: RECOMBINANT SEMISYNTHETIC AEQUORINS
OBELIN BIOLUMINESCENCE
MECHANISTIC
Аннотация: Calcium-regulated photoproteins are found in at least five phyla of organisms. The light emitted by those photoproteins can be tuned by mutating the photoprotein and/or by modifying the substrate coelenterazine (CTZ). Thirty years ago, Shimomura observed that the luminescence activity of aequorin was dramatically reduced when the substrate CTZ was replaced by its analogv-CTZ. The latter is formed by adding a phenyl ring to the pi-conjugated moiety of CTZ. The decrease in luminescence activity has not been understood until now. In this paper, through combined quantum mechanics and molecular mechanics calculations as well as molecular dynamics simulations, we discovered the reason for this observation. Modification of the substrate changes the conformation of nearby aromatic residues and enhances the pi-pi stacking interactions between the conjugated moiety ofv-CTZ and the residues, which weakens the charge transfer to form light emitter and leads to a lower luminescence activity. The microenvironments of CTZ in obelin and in aequorin are very similar, so we predicted that the luminescence activity of obelin will also dramatically decrease when CTZ is replaced byv-CTZ. This prediction has received strong evidence from currently theoretical calculations and has been verified by experiments.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Ding B. -W., Eremeeva E. V., Vysotski E. S., Liu Y. -J.
Заглавие : Luminescence Activity Decreases When v-coelenterazine Replaces Coelenterazine in Calcium-Regulated Photoprotein—A Theoretical and Experimental Study
Место публикации : Photochem. Photobiol.: Blackwell Publishing Inc., 2020. - Article in press. - ISSN 00318655 (ISSN), DOI 10.1111/php.13280
Аннотация: Calcium-regulated photoproteins are found in at least five phyla of organisms. The light emitted by those photoproteins can be tuned by mutating the photoprotein and/or by modifying the substrate coelenterazine (CTZ). Thirty years ago, Shimomura observed that the luminescence activity of aequorin was dramatically reduced when the substrate CTZ was replaced by its analog v-CTZ. The latter is formed by adding a phenyl ring to the ?-conjugated moiety of CTZ. The decrease in luminescence activity has not been understood until now. In this paper, through combined quantum mechanics and molecular mechanics calculations as well as molecular dynamics simulations, we discovered the reason for this observation. Modification of the substrate changes the conformation of nearby aromatic residues and enhances the ?-? stacking interactions between the conjugated moiety of v-CTZ and the residues, which weakens the charge transfer to form light emitter and leads to a lower luminescence activity. The microenvironments of CTZ in obelin and in aequorin are very similar, so we predicted that the luminescence activity of obelin will also dramatically decrease when CTZ is replaced by v-CTZ. This prediction has received strong evidence from currently theoretical calculations and has been verified by experiments. © 2020 American Society for Photobiology
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Lopatina T. S., Aleksandrova Y. V., Anishchenko O. V., Gribovskaya I. V., Oskina N. A., Zotina T. A., Zadereev E. S.
Заглавие : The effect of deicing salt solutes on Moina macrocopa and Allium cepa in a toxicity test experiment
Место публикации : Vestn. Tomsk. Gos. Univ. Biol.: Tomsk State University, 2020. - Is. 51. - С. 162-178. - ISSN 19988591 (ISSN), DOI 10.17223/19988591/51/9
Аннотация: Chloride salts are the most commonly used deicing materials for winter maintenance of roads. Numerous studies indicate a significant increase in the salinity of aquatic ecosystems associated with the long-term use of deicing materials in countries located in cold climates. The functioning of ecosystems largely depends on salinity, since salinity is one of the key factors determining the species composition, the structure of food webs and the productivity of aquatic communities. Given the growing threat of salinization of groundwater and surface waters, it is extremely important to study the effect of deicing materials on the biota and functioning of aquatic ecosystems. The aim of this research is to determine the threshold concentrations of solutions of the deicing salt mixture “Bionord” containing sodium and calcium chlorides, at which negative effects on the development of animal and plant test objects are observed. In this study, we used the salt-containing mixture “Bionord” as a model deicer. Similarly, with the most commonly used ice melting chemicals, the «Bionord» salt mixture contains a large amount of sodium and calcium chlorides (about 85% of the total weight). To evaluate the toxicity of the deicer solutions, we used acute and chronic toxicity tests with cladoceran Moina macrocopa (Straus, 1820) (Cladocera: Moinidae) and standard onion-based test with Allium cepa L. (Liliopsida: Amaryllidaceae) (Allium-test). In acute and chronic toxicity tests with Cladocera, the females on the first day of their life (body size 0.5-0.6 mm) were placed individually in jars with aged (not less than for 72 h) tap water with a volume of 20 ml with the addition of a deicer at a certain concentration. A group of animals that was placed in the medium without the deicer was used as a control. In the acute toxicity tests, we used the following concentration of the deicer: 1.3; 2.5; 4.0, 5.0; 6.0; 8.0; 10.0 g/l. The mortality of animals was recorded 24 and 48 hours after the start of the experiment. The concentration of the deicer (LC50) at which 50% of animals was observed to die, compared to the control, was determined in the acute toxicity test. In a chronic toxicity test, animals were tested in the following range of concentrations of the deicer: 0.3; 0.6; 1.3; 2.5; 5.0; 6.0 and 8.0 g/l. The chronic toxicity test was conducted until the death of all test animals. Based on the data obtained in the chronic toxicity test, the specific growth rate of juvenile females, average fecundity, and average life span of M. macrocopa were calculated for each concentration of the deicer. Bulbs of onions of the Stuttgartenrisen variety with a diameter of 1.8 ± 0.1 cm and a weight of 2.27 ± 0.17 g were used in the onion test. Bulbs with their bottoms were placed in test tubes containing 20 ml of a solute of the deicer or tap water for 48 hours. Three bulbs were tested for each concentration and for the control. The following concentrations of the deicer were used in the onion test: 1.0; 2.5; 5.0; 7.0; 10.0; 15.0; 20.0; 50.0 g/l. The general toxic and cytotoxic effects were evaluated in the onion test. The average root length and the total root length on each bulb were used as indicators of the total toxicity of the solutions of deicer. To evaluate proliferative activity, we calculated the mitotic index as the fraction of dividing cells in the apical root meristem to the total number of cells. Based on the results of the experiments, we determined median effective mixture concentrations (EC50) at which there is a 50% decrease, compared to the control, in the values of root growth indicators: average root length, sum of root lengths on each bulb and mitotic index. Median lethal concentration (LC50) of the deicing salt determined in the 48-hour acute toxicity test with females of M. macrocopa was equal to 5.1 g/l. In the chronic test, we showed that the exposure to the solutions of the deicing salt in the range of concentrations from 0.3 to 5.0 g/l does not affect the life span, specific growth rate of juveniles and fecundity of females of M. macrocopa. The median effective concentration (EC50) of the deicing salt determined in the Allium-tests were 6.3, 5.2 and 10.4 g/l for the sum of root lengths, average root length on each bulb and proliferative activity at the tips of roots (mitotic index), respectively (See Table 1 and 2). Complete inhibition of onion root growth was observed at the concentration of the decider equal to 20 g/l, while the death of all test animals in the acute toxicity test occurred at the concentration of the deicer equal to 8,0 g/l (See Fig. 1). Thus, we demonstrated that similar concentrations of the deicer induced 50% inhibition of the growth of onion roots and 50% mortality of cladocerans. These values, in general, corresponded to a critical salinity of 5-8 % above which qualitative changes occur both in the external and internal condition of aquatic animals. The electrical conductivity of the deicer solutions, which had a negative effect on the selected test species, coincides with the previously obtained values of the electrical conductivity of sodium chloride solutions harmful to cladocerans. We can assume that the main mechanism of the effect of the deicing material that we study is associated with the biological effect of its chlorine and sodium salts. Taking this into account, the value of electrical conductivity measured for solutions of deicing salt can be used to assess its negative potential effects. We estimated that in the absence of timely cleaning, regulated by the rules for using the material, the runoff from each square meter of the treated surface can lead to the pollution of 8-13 liters of fresh water. Thus, the basic requirement for the use of deicing salts on roads is the need to comply with the cleaning regime of the treated surfaces. Otherwise, the gradual accumulation of sodium and calcium chlorides in water bodies can cause an increase in salinity which will affect the survival of freshwater aquatic organisms and lead to serious disturbances in the functioning of aquatic ecosystems. © 2020 Tomsk State University. All rights reserved.
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10.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Lopatina, Tatiana S., Aleksandrova, Yuliyana, V, Anishchenko, Olesya, V, Gribovskaya, Iliada, V, Oskina, Nataliya A., Zotina, Tatiana A., Zadereev, Egor S.
Заглавие : The effect of deicing salt solutes on Moina macrocopa and Allium cepa in a toxicity test experiment
Колич.характеристики :17 с
Коллективы : Russian Foundation for Basic ResearchRussian Foundation for Basic Research (RFBR); Government of Krasnoyarsk Krai; Krasnoyarsk Krai Fund for Supporting Scientific and Scientific-Technical Activities [19-44-240014]
Место публикации : Vestn. Tomsk. Gos. Univ. Biol.: TOMSKIJ GOSUDARSTVENNYI UNIV, 2020. - Is. 51. - С. 162-178. - ISSN 1998-8591, DOI 10.17223/19988591/51/9. - ISSN 2311-2077(eISSN)
Примечания : Cited References:29. - This work was supported by the joint grant from the Russian Foundation for Basic Research, the Government of Krasnoyarsk Krai, and the Krasnoyarsk Krai Fund for Supporting Scientific and Scientific-Technical Activities (Grant No 19-44-240014).
Предметные рубрики: DAPHNIA-MAGNA
NACL SALINITY
ROAD SALTS
WATER
FRESH
CHLORIDE
Аннотация: Chloride salts are the most commonly used deicing materials for winter maintenance of roads. Numerous studies indicate a significant increase in the salinity of aquatic ecosystems associated with the long-term use of deicing materials in countries located in cold climates. The functioning of ecosystems largely depends on salinity, since salinity is one of the key factors determining the species composition, the structure of food webs and the productivity of aquatic communities. Given the growing threat of salinization of groundwater and surface waters, it is extremely important to study the effect of deicing materials on the biota and functioning of aquatic ecosystems. The aim of this research is to determine the threshold concentrations of solutions of the deicing salt mixture "Bionord" containing sodium and calcium chlorides, at which negative effects on the development of animal and plant test objects are observed. In this study, we used the salt-containing mixture "Bionord" as a model deicer. Similarly, with the most commonly used ice melting chemicals, the "Bionord" salt mixture contains a large amount of sodium and calcium chlorides (about 85% of the total weight). To evaluate the toxicity of the deicer solutions, we used acute and chronic toxicity tests with cladoceran Moina macrocopa (Straus, 1820) (Cladocera: Moinidae) and standard onion-based test with Allium cepa L. (Liliopsida: Amaryllidaceae) (Allium-test). In acute and chronic toxicity tests with Cladocera, the females on the first day of their life (body size 0.5-0.6 mm) were placed individually in jars with aged (not less than for 72 h) tap water with a volume of 20 ml with the addition of a deicer at a certain concentration. A group of animals that was placed in the medium without the deicer was used as a control. In the acute toxicity tests, we used the following concentration of the deicer: 1.3; 2.5; 4.0, 5.0; 6.0; 8.0; 10.0 g/l. The mortality of animals was recorded 24 and 48 hours after the start of the experiment. The concentration of the deicer (LC50) at which 50% of animals was observed to die, compared to the control, was determined in the acute toxicity test. In a chronic toxicity test, animals were tested in the following range of concentrations of the deicer: 0.3; 0.6; 1.3; 2.5; 5.0; 6.0 and 8.0 g/l. The chronic toxicity test was conducted until the death of all test animals. Based on the data obtained in the chronic toxicity test, the specific growth rate of juvenile females, average fecundity, and average life span of M. macrocopa were calculated for each concentration of the deicer. Bulbs of onions of the Stuttgartenrisen variety with a diameter of 1.8 +/- 0.1 cm and a weight of 2.27 +/- 0.17 g were used in the onion test. Bulbs with their bottoms were placed in test tubes containing 20 ml of a solute of the deicer or tap water for 48 hours. Three bulbs were tested for each concentration and for the control. The following concentrations of the deicer were used in the onion test: 1.0; 2.5; 5.0; 7.0; 10.0; 15.0; 20.0; 50.0 g/l. The general toxic and cytotoxic effects were evaluated in the onion test. The average root length and the total root length on each bulb were used as indicators of the total toxicity of the solutions of deicer. To evaluate proliferative activity, we calculated the mitotic index as the fraction of dividing cells in the apical root meristem to the total number of cells. Based on the results of the experiments, we determined median effective mixture concentrations (EC50) at which there is a 50% decrease, compared to the control, in the values of root growth indicators: average root length, sum of root lengths on each bulb and mitotic index. Median lethal concentration (LC50) of the deicing salt determined in the 48-hour acute toxicity test with females of Al. macrocopa was equal to 5.1 g/l. In the chronic test, we showed that the exposure to the solutions of the deicing salt in the range of concentrations from 0.3 to 5.0 g/l does not affect the life span, specific growth rate of juveniles and fecundity of females of M. macrocopa. The median effective concentration (EC50) of the deicing salt determined in the Allium-tests were 6.3, 5.2 and 10.4 g/l for the sum of root lengths, average root length on each bulb and proliferative activity at the tips of roots (mitotic index), respectively (See Table 1 and 2). Complete inhibition of onion root growth was observed at the concentration of the decider equal to 20 g/l, while the death of all test animals in the acute toxicity test occurred at the concentration of the deicer equal to 8,0 g/l (See Fig. 1). Thus, we demonstrated that similar concentrations of the deicer induced 50% inhibition of the growth of onion roots and 50% mortality of cladocerans. These values, in general, corresponded to a critical salinity of 5-8 %o above which qualitative changes occur both in the external and internal condition of aquatic animals. The electrical conductivity of the deicer solutions, which had a negative effect on the selected test species, coincides with the previously obtained values of the electrical conductivity of sodium chloride solutions harmful to cladocerans. We can assume that the main mechanism of the effect of the deicing material that we study is associated with the biological effect of its chlorine and sodium salts Taking this into account, the value of electrical conductivity measured for solutions of deicing salt can be used to assess its negative potential effects. We estimated that in the absence of timely cleaning, regulated by the rules for using the material, the runoff from each square meter of the treated surface can lead to the pollution of 8-13 liters of fresh water. Thus, the basic requirement for the use of deicing salts on roads is the need to comply with the cleaning regime of the treated surfaces. Otherwise, the gradual accumulation of sodium and calcium chlorides in water bodies can cause an increase in salinity which will affect the survival of freshwater aquatic organisms and lead to serious disturbances in the functioning of aquatic ecosystems.
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11.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Huser B. J., Futter M. N., Bogan D., Brittain J. E., Culp J. M., Goedkoop W., Gribovskaya I., Karlsson J., Lau D. C.P., Ruhland K. M., Schartau A. K., Shaftel R., Smol J. P., Vrede T., Lento J.
Заглавие : Spatial and temporal variation in Arctic freshwater chemistry—Reflecting climate-induced landscape alterations and a changing template for biodiversity
Место публикации : Freshw. Biol.: Blackwell Publishing Ltd, 2020. - Article in press. - ISSN 00465070 (ISSN), DOI 10.1111/fwb.13645
Аннотация: Freshwater chemistry across the circumpolar region was characterised using a pan-Arctic data set from 1,032 lake and 482 river stations. Temporal trends were estimated for Early (1970–1985), Middle (1986–2000), and Late (2001–2015) periods. Spatial patterns were assessed using data collected since 2001. Alkalinity, pH, conductivity, sulfate, chloride, sodium, calcium, and magnesium (major ions) were generally higher in the northern-most Arctic regions than in the Near Arctic (southern-most) region. In particular, spatial patterns in pH, alkalinity, calcium, and magnesium appeared to reflect underlying geology, with more alkaline waters in the High Arctic and Sub Arctic, where sedimentary bedrock dominated. Carbon and nutrients displayed latitudinal trends, with lower levels of dissolved organic carbon (DOC), total nitrogen, and (to a lesser extent) total phosphorus (TP) in the High and Low Arctic than at lower latitudes. Significantly higher nutrient levels were observed in systems impacted by permafrost thaw slumps. Bulk temporal trends indicated that TP was higher during the Late period in the High Arctic, whereas it was lower in the Near Arctic. In contrast, DOC and total nitrogen were both lower during the Late period in the High Arctic sites. Major ion concentrations were higher in the Near, Sub, and Low Arctic during the Late period, but the opposite bulk trend was found in the High Arctic. Significant pan-Arctic temporal trends were detected for all variables, with the most prevalent being negative TP trends in the Near and Sub Arctic, and positive trends in the High and Low Arctic (mean trends ranged from +0.57%/year in the High/Low Arctic to ?2.2%/year in the Near Arctic), indicating widespread nutrient enrichment at higher latitudes and oligotrophication at lower latitudes. The divergent P trends across regions may be explained by changes in deposition and climate, causing decreased catchment transport of P in the south (e.g. increased soil binding and trapping in terrestrial vegetation) and increased P availability in the north (deepening of the active layer of the permafrost and soil/sediment sloughing). Other changes in concentrations of major ions and DOC were consistent with projected effects of ongoing climate change. Given the ongoing warming across the Arctic, these region-specific changes are likely to have even greater effects on Arctic water quality, biota, ecosystem function and services, and human well-being in the future. © 2020 The Authors. Freshwater Biology published by John Wiley & Sons Ltd.
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12.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Huser, Brian J., Futter, Martyn N., Bogan, Daniel, Brittain, John E., Culp, Joseph M., Goedkoop, Willem, Gribovskaya, Iliada, Karlsson, Jan, Lau, Danny C. P., Ruhland, Kathleen M., Schartau, Ann Kristin, Shaftel, Rebecca, Smol, John P., Vrede, Tobias, Lento, Jennifer
Заглавие : Spatial and temporal variation in Arctic freshwater chemistry-Reflecting climate-induced landscape alterations and a changing template for biodiversity
Колич.характеристики :16 с
Коллективы : Environment and Climate Change Canada; Cumulative Impact Monitoring Program, Government of Northwest Territories
Место публикации : Freshw. Biol.: WILEY, 2020. - Article in press. - ISSN 0046-5070, DOI 10.1111/fwb.13645. - ISSN 1365-2427(eISSN)
Примечания : Cited References:98. - Environment and Climate Change Canada; Cumulative Impact Monitoring Program, Government of Northwest Territories
Предметные рубрики: DISSOLVED ORGANIC-CARBON
PERMAFROST THAW
CHEMICAL LIMNOLOGY
Аннотация: Freshwater chemistry across the circumpolar region was characterised using a pan-Arctic data set from 1,032 lake and 482 river stations. Temporal trends were estimated for Early (1970-1985), Middle (1986-2000), and Late (2001-2015) periods. Spatial patterns were assessed using data collected since 2001. Alkalinity, pH, conductivity, sulfate, chloride, sodium, calcium, and magnesium (major ions) were generally higher in the northern-most Arctic regions than in the Near Arctic (southern-most) region. In particular, spatial patterns in pH, alkalinity, calcium, and magnesium appeared to reflect underlying geology, with more alkaline waters in the High Arctic and Sub Arctic, where sedimentary bedrock dominated. Carbon and nutrients displayed latitudinal trends, with lower levels of dissolved organic carbon (DOC), total nitrogen, and (to a lesser extent) total phosphorus (TP) in the High and Low Arctic than at lower latitudes. Significantly higher nutrient levels were observed in systems impacted by permafrost thaw slumps. Bulk temporal trends indicated that TP was higher during the Late period in the High Arctic, whereas it was lower in the Near Arctic. In contrast, DOC and total nitrogen were both lower during the Late period in the High Arctic sites. Major ion concentrations were higher in the Near, Sub, and Low Arctic during the Late period, but the opposite bulk trend was found in the High Arctic. Significant pan-Arctic temporal trends were detected for all variables, with the most prevalent being negative TP trends in the Near and Sub Arctic, and positive trends in the High and Low Arctic (mean trends ranged from +0.57%/year in the High/Low Arctic to -2.2%/year in the Near Arctic), indicating widespread nutrient enrichment at higher latitudes and oligotrophication at lower latitudes. The divergent P trends across regions may be explained by changes in deposition and climate, causing decreased catchment transport of P in the south (e.g. increased soil binding and trapping in terrestrial vegetation) and increased P availability in the north (deepening of the active layer of the permafrost and soil/sediment sloughing). Other changes in concentrations of major ions and DOC were consistent with projected effects of ongoing climate change. Given the ongoing warming across the Arctic, these region-specific changes are likely to have even greater effects on Arctic water quality, biota, ecosystem function and services, and human well-being in the future.
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13.

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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14.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva, Elena V., Vysotski, Eugene S.
Заглавие : Exploring Bioluminescence Function of the Ca2+-regulated Photoproteins with Site-directed Mutagenesis
Колич.характеристики :16 с
Коллективы : Russian Foundation for Basic Research [17-04-00764]; Russian Academy of Sciences [0356-2017-0017]
Место публикации : Photochem. Photobiol.: WILEY, 2019. - Vol. 95, Is. 1. - С. 8-23. - ISSN 0031-8655, DOI 10.1111/php.12945. - ISSN 1751-1097(eISSN)
Примечания : Cited References:88. - This work was supported by grant 17-04-00764 of Russian Foundation for Basic Research and the state budgetallocated to the fundamental research at the Russian Academy of Sciences (project 0356-2017-0017).
Предметные рубрики: CALCIUM-BINDING PHOTOPROTEIN
GREEN-FLUORESCENT PROTEIN
JELLYFISH
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--aequorin--obelin--clytin--mitrocomin--ef-hand protein
Аннотация: Site-directed mutagenesis is a powerful tool to investigate the structure-function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site-directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+-regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.
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15.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Burakova L. P., Vysotski E. S.
Заглавие : Recombinant Ca2+-regulated photoproteins of ctenophores: current knowledge and application prospects
Место публикации : Appl. Microbiol. Biotechnol.: Springer Verlag, 2019. - Vol. 103, Is. 15. - С. 5929-5946. - ISSN 01757598 (ISSN) , DOI 10.1007/s00253-019-09939-0
Аннотация: Bright bioluminescence of ctenophores is conditioned by Ca2+-regulated photoproteins. Although they share many properties characteristic of hydromedusan Ca2+-regulated photoproteins responsible for light emission of marine animals belonging to phylum Cnidaria, a substantial distinction still exists. The ctenophore photoproteins appeared to be extremely sensitive to light—they lose the ability for bioluminescence on exposure to light over the entire absorption spectrum. Inactivation is irreversible because keeping the inactivated photoprotein in the dark does not recover its activity. The capability to emit light can be restored only by incubation of inactivated photoprotein with coelenterazine in the dark at alkaline pH in the presence of oxygen. Although these photoproteins were discovered many years ago, only the cloning of cDNAs encoding these unique bioluminescent proteins in the early 2000s has provided a new impetus for their studies. To date, cDNAs encoding Ca2+-regulated photoproteins from four different species of luminous ctenophores have been cloned. The amino acid sequences of ctenophore photoproteins turned out to completely differ from those of hydromedusan photoproteins (identity less than 29%) though also similar to them having three EF-hand Ca2+-binding sites. At the same time, these photoproteins reveal the same two-domain scaffold characteristic of hydromedusan photoproteins. This review is an attempt to systemize and critically evaluate the data scattered through various articles regarding the structural features of recombinant light-sensitive Ca2+-regulated photoproteins of ctenophores and their bioluminescent and physicochemical properties as well as to compare them with those of hydromedusan photoproteins. In addition, we also discuss the prospects of their biotechnology applications. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature.
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16.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Vysotski E., Markova S., Natashin P., Stepanyuk G., Lee J., Malikova N., Liu Z.
Заглавие : Hydrogen bond network near OH group of 6-(p-hydroxyphenyl) substituent of coelenterazine determines the bioluminescence spectra differences among hydromedusan calcium-regulated photoproteins
Колич.характеристики :2 с
Коллективы : RFBR [17-04-00764]; China-Russia international collaboration grant from Chinese Academy of Sciences; Natural Science Foundation of China
Место публикации : FEBS Open Bio: WILEY, 2018. - Vol. 8. - С. 435-436. - ISSN 2211-5463
Примечания : Cited References:0. - This work was supported by RFBR grant 17-04-00764 and a China-Russia international collaboration grant from the Chinese Academy of Sciences and the Natural Science Foundation of China.
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17.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Rodionova N. S., Rota E., Tsarkova A. S., Petushkov V. N.
Заглавие : Progress in the Study of Bioluminescent Earthworms
Место публикации : Photochem. Photobiol.: Blackwell Publishing Inc., 2017. - Vol. 93, Is. 2. - С. 416-428. - ISSN 00318655 (ISSN) , DOI 10.1111/php.12709
Аннотация: Even though bioluminescent oligochaetes rarely catch people's eyes due to their secretive lifestyle, glowing earthworms sighting reports have come from different areas on all continents except Antarctica. A major breakthrough in the research of earthworm bioluminescence occurred in the 1960s with the studies of the North American Diplocardia longa. Comparative studies conducted on 13 earthworm species belonging to six genera showed that N-isovaleryl-3-aminopropanal (Diplocardia luciferin) is the common substrate for bioluminescence in all examined species, while luciferases appeared to be responsible for the color of bioluminescence. The second momentous change in the situation has occurred with the discovery in Siberia (Russia) of two unknown luminous enchytraeids. The two bioluminescent systems belong to different types, have different spectral characteristics and localization, and different temperature and pH optima. They are unique, and this fact is confirmed by the negative results of all possible cross-reactions. The bioluminescent system of Henlea sp. comprises four essential components: luciferase, luciferin, oxygen and calcium ion. For Friderica heliota, the luminescent reaction requires five components: luciferase, luciferin, ATP, magnesium ion and oxygen. Along with luciferin, more than a dozen analogues were isolated from worm biomass. These novel peptide-like natural compounds represent an unprecedented chemistry found in terrestrial organisms. © 2017 The American Society of Photobiology
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18.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva E. V., Vysotski E. S.
Заглавие : Bioluminescent and biochemical properties of Cys-free Ca2+-regulated photoproteins obelin and aequorin
Место публикации : J. Photochem. Photobiol. B Biol.: Elsevier B.V., 2017. - Vol. 174. - С. 97-105. - ISSN 10111344 (ISSN) , DOI 10.1016/j.jphotobiol.2017.07.021
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenteramide--coelenterazine--cysteine--photoprotein--serine
Аннотация: Bioluminescence of a variety of marine coelenterates is determined by Ca2+-regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca2+-regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or ?-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coli, purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a two-fold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild-type aequorin. In contrast, Cys-free obelin retains only ~ 10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a “fast” component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (Tm) were determined to be 53.3 ± 0.2 and 44.6 ± 0.4 °C for aequorin and Cys-free aequorin, and 49.1 ± 0.1 and 28.8 ± 0.3 °C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield. © 2017 Elsevier B.V.
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19.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva, Elena V., Vysotski, Eugene S.
Заглавие : Bioluminescent and biochemical properties of Cys-free Ca2+-regulated photoproteins obelin and aequorin
Колич.характеристики :9 с
Коллективы : Russian Academy of Sciences [03562016-0712, 0356-2015-0103]; RFBR [17-04-00764]
Место публикации : J. Photochem. Photobiol. B-Biol.: ELSEVIER SCIENCE SA, 2017. - Vol. 174. - С. 97-105. - ISSN 1011-1344, DOI 10.1016/j.jphotobio1.2017.07.021
Примечания : Cited References:54. - This work was supported by the state budget allocated to the fundamental research at the Russian Academy of Sciences (projects 03562016-0712 and 0356-2015-0103) and the RFBR grant 17-04-00764.
Предметные рубрики: SEQUENCE-ANALYSIS
APO-OBELIN
INTRINSIC FLUORESCENCE
COELENTERAZINE
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--photoprotein--coelenteramide--cysteine--serine
Аннотация: Bioluminescence of a variety of marine coelenterates is determined by Ca2+-regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca2+-regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or p-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coil, purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a two-fold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild -type aequorin. In contrast, Cys-free obelin retains only 10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a "fast" component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (Tm) were determined to be 53.3 0.2 and 44.6 0.4 C for aequorin and Cys-free aequorin, and 49.1 0.1 and 28.8 0.3 C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield.
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20.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Burakova L. P., Stepanyuk G. A., Eremeeva E. V., Vysotski E. S.
Заглавие : Role of certain amino acid residues of the coelenterazine-binding cavity in bioluminescence of light-sensitive Ca2+-regulated photoprotein berovin
Место публикации : Photochem. Photobiol. Sci. - 2016. - Vol. 15, Is. 5. - С. 691-704. - ISSN 1474905X (ISSN) , DOI 10.1039/c6pp00050a
Аннотация: Bright bioluminescence of ctenophores is caused by Ca2+-regulated photoproteins. Although these photoproteins are functionally identical to and share many properties of cnidarian photoproteins, like aequorin and obelin, and retain the same spatial architecture, they are extremely sensitive to light, i.e. lose the ability to bioluminesce on exposure to light over the entire absorption spectrum. In addition, the degree of identity of their amino acid sequences with those of cnidarian photoproteins is only 29.4%. This suggests that the residues involved in bioluminescence of ctenophore and cnidarian photoproteins significantly differ. Here we describe the bioluminescent properties of berovin mutants with substitution of the residues located in the photoprotein internal cavity. Since the spatial structure of berovin bound with a substrate is not determined yet, to identify these residues we have modeled it with an accommodated substrate using the structures of some cnidarian Ca2+-regulated photoproteins with bound coelenterazine or coelenteramide as templates in order to obtain an adequate sampling and to take into account all possible conformers and variants for ligand-protein docking. Based on the impact of substitutions on the bioluminescent properties and model structures we speculate that within the internal cavity of ctenophore photoproteins, coelenterazine is bound as a 2-peroxy anion adduct which is stabilized owing to Coulomb interaction with a positively charged guanidinium group of Arg41 paired with Tyr204. In this case, the bioluminescence reaction is triggered by only calcium-induced conformational changes leading to the disturbance of charge-charge interaction. © 2016 The Royal Society of Chemistry and Owner Societies.
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