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Биолюминесцентная детекция стрессовых воздействий на живые системытезисы доклада
- Авторы: Угарова Н.Н., Ломакина Г.Ю.
- Сборник: Биоантиоксидант: материалы Х Международной конференции, посвященной 105-летию со дня рождения академика Н.М. Эмануэля
- Тезисы
- Год издания: 2020
- Издательство: РУДН
- Местоположение издательства: М.
- Первая страница: 95
- Последняя страница: 98
- Аннотация: Various methods of detection are used for investigation of stress effects on living systems. The bioluminescent methods are very attractive due to high sensitivity, specificity and possibility of its application in noninvasive analysis of the cells and intact animals in real-time, in vivo and in situ. This report contains the examples from literature and the author’s results, dealing with application of bioluminescent systems for detection of the effeсt of different chemical and biological agents on biosystems.Bioluminescence is the light emission in the visible region of the spectrum. It is produced during biochemical processes (as a rule – oxidation of low molecular weight substrates – luciferins), catalyzed by enzymes – luciferases. The cells of native or recombinant microorganisms with constitutive expression lux-genes, the light intensity of which reflects structure-functional activity of this luminescent system existing in these cells, were used as biosensors in 60-nd of the last century for integral estimation of toxic or bactericide effects on luminescent bacterial cell. The observed inhibition of luminescence was caused by depression of the cell viability due to disturbance of its energy providing or the cell destruction. In Russia, the state standard exists for estimation of integral toxicity of different water samples and water extracts from environmental objects with the use of test-system «Ecolum» - lyophilized microorganisms Escherichia coli, transformed by hybrid plasmid pUC19 with cloned luxCDABE genes P.leiognathi 54D10. Firefly and click beetle luciferases use D-luciferin, ATP and oxygen as their substrates. Due to high activity and absolute specificity to ATP, firefly luciferase is widely used in test-systems for detection of ATP – marker of the cell viability. The proportionality between the ATP content and cell viability in the cell samples was established. So, bioluminescent method permits to rapidly detect the changes in intracellular processes under action of various stress agents (radiation, heating, toxins, antibiotics) by measuring the ATP content in the samples.Firefly luciferase and ATP were applied as reporters for investigation of stress effect of membrane active agent – digitonin, complexing with cholesterol of cell membranes and causing the disintegration of the membrane structure, the formation of pores, and the release of intracellular components in the reaction medium. The most important initial stage of the interaction of cell membranes with digitonin was studied with HEK293 cells transiently transfected with the pcDNALuc plasmid expressing firefly luciferase. In real time, the kinetic curves of accumulation of luciferase and ATP in the extracellular space in the presence of different digitonin concentrations were recorded in second and minute interval of time. Thus, the bioluminescence method offered to study in situ changes in the permeability of cell membranes under the action of membrane-active effectors in the early stages of cells’ lysis process.Optical reporters are widely used in cell biology to tag and monitor molecular processes non-invasively in living cells or in whole intact animals. While less bright than fluorescent probes, bioluminescent probes have several distinct advantages since they do not require external illumination and have extremely low background signals. The particular benefit of these luciferin-luciferase systems is the ability to perform continuous long-term real-time monitoring in live cell cultures and in intact animals up to several weeks and with up to a single cell resolution.The luciferase reporters may be divided into two groups. First group of luciferases includes the luciferases with high brightness of bioluminescence and long half-life in the cell. Specific types of cells (cancer cells or pathogenic bacteria, viruses or genetic material) are marked constitutively with luciferase, introduced into living organism and then imaged in intact animals by bioluminescence to monitor cell location and their number during long time. As a result, the information is obtained with quantitative characteristics of tumor growth and spread of pathogens in living animal.Second group of luciferase reporter is applied to monitor changes in gene expression and protein-protein interactions in real time under action of different stress agents. In this case high luciferase activity is also needed, but with relatively short half-life luciferase to respond quickly to changes in gene expression of these transcriptional reporters. To monitor luciferase activity in live cells in real time, special reporter vector is used that includеs promoter sequences from the genes of interest and luciferase gene. This vector is transfected into cell, and real-time monitoring of luciferase activity in live cells proceeds.Several factors determine the temporal properties of these inducible reporters: 1) transcriptional activity (mRNA synthesis), 2) time curve of mRNA, 3) time curve of luciferase expression, 4) time curve of luciferase activity (half-life of luciferase). Depending on the structure of reporter on can have different time curves for bioluminescence intensity: from half-time 7-8 hours till 2.5 hours. To correctly monitor temporal changes in gene expression it is necessary to use luciferases with short half-life, fast response to transcriptional changes and high bioluminescent activity. The short half-life is usually achieved by incorporation of protein degradation tags (so-called degron tags) into C-terminal of luciferase. These tags increase the rate of proteolytic degradation of luciferase in mammalian cells and decrease the luciferase half-time. At the same time these tags lower the luciferase activity. To overcome these difficulties, several mutants of luciferase Photinus pyralis were obtained that surpass the commercial codon-optimized variants of luciferases: Eluc – luciferase from click beetle with green emission in vivo and luc2 – luciferase from P.pyralis firefly with orange emission - in brightness, short half-life and fast response to transcriptional changes. The mutant “GR” provides a faster response (≈ like the luc2 with PEST tag) and low half-life of luciferase activity in mammalian cells. Mutant RV/luc also increases the brightness and shows a fast response like luc2 with two degron tags.
- Добавил в систему: Ломакина Галина Юрьевна