TY - THES A1 - Wiencierz, Anne Maria T1 - Entwicklung eines Dual-Luciferase-Reportergen-Assays zum Nachweis der Induktion antioxidativer Enzyme durch Nahrungsbestandteile T1 - Establishment of a reporter gene assay for the determination of induction of antioxidative enzymes by food components N2 - Die Induktion antioxidativer Enzyme gilt als eine Möglichkeit, die antioxidative Kapazität von Zellen zu steigern und dadurch mit oxidativem Stress assoziierten Erkrankungen (z. B. Herz-Kreislauf-Erkrankungen, Neurodegeneration, Atherosklerose) vorzubeugen. Ausgehend davon wurde in der vorliegenden Arbeit der Dual-Luciferase-Reportergen-(DLR)-Assay zum Nachweis der Induktion der antioxidativen Enzyme Katalase (CAT), zytosolische Glutathion-Peroxidase (GPX1) und Kupfer-Zink-Superoxid-Dismutase (SOD1) entwickelt. Im Zuge dessen wurden drei Säugetierzelllinien (CaCo2, IEC-18, V79) auf ihre Eignung zur Modellzelllinie untersucht. Aufgrund der Transfektionseffizienz wurde die Fibroblastenzelllinie V79 ausgewählt. Zur Gewährleistung eines hohen Substanzdurchsatzes des DLR-Assays wurden bei der Etablierung Parameter wie Kulturplattenformat, DNA-Menge, Luciferasen-Kinetik berücksichtigt. Nach erfolgreicher Etablierung des Versuchs im 96-Well-Format wurden L-Carnitin, Catechin, Epigallocatechingallat, Genistein, Wasserstoffperoxid (H2O2), Natrium-Ascorbat, Paraquat, Quercetin, 12-O-Tetradecanoylphorbol-13-Acetat (TPA) und Trolox in nicht-zytotoxischen Konzentrationen hinsichtlich der Aktivierung des Ratten-CAT-, des humanen GPX1- und des humanen SOD1-Promotors untersucht. Die Bestimmung der maximal tolerierbaren Behandlungskonzentration erfolgte im Vorfeld mittels Resazurintest. Von den zehn Verbindungen zeichneten sich drei Substanzen als potente Induktoren für die SOD1 und die GPX1 aus. Die 24-stündige Behandlung von mit Reportergenkonstrukten transient transfizierten V79-Zellen mit 100 µM Paraquat resultierte in einer Verdopplung der relativen SOD1-Promotor-Aktivität und einer Erhöhung der relativen GPX1-Promotor-Aktivität auf 1,6 bzw. 1,7. Die Stimulation mit 20 µM Genistein oder 10 µM Quercetin führte wiederum zu einer Verdopplung bis Verdreifachung der relativen SOD1- und GPX1-Promotor-Aktivität. Der Promotor der Rattenkatalase konnte demgegenüber nur durch 50 µM H2O2 aktiviert werden (1,5fach). Für diesen DLR-Assays bieten sich folglich Genistein, Quercetin wie auch H2O2 als Referenzsubstanzen an. Um aber eine qualitative Charakterisierung der einzelnen Verbindungen hinsichtlich ihres Induktionspotentials zu gewährleisten, sollten von allen getesteten Substanzen Dosis-Wirkungskurven aufgenommen werden. Zudem wird für den routinemäßigen Einsatz die Verwendung stabil transfizierter Zellen zur Vermeidung von mit der Transfektion verbundenen experimentellen Schwankungen empfohlen. N2 - The induction of antioxidative enzymes might be an opportunity to elevate the cellular antioxidative capacity and, thus, to prevent oxidative stress associated diseases (e. g. cardio-vascular disease, neurodegenerative disease, atherosclerosis). Based on this idea the dual luciferase reporter gene (DLR) assay was developed to demonstrate the induction of three antioxidative enzymes: catalase (CAT), cytosolic glutathione peroxidase (GPX1), and copper-zinc superoxide dismutase (SOD1). In the course of the development three mammalian cell lines (CaCo2, IEC-18, V79) were tested for their ability to serve as a model cell line. The line V79 was chosen due to the transfection efficiency. To give consideration to a high-throughput several parameters were studied (e. g. format of the cultural plates, amount of DNA, kinetics of the luciferases) and the DLR assay was successfully established in 96 well plates. Subsequently, L-carnitine, catechin, epigallocatechin gallate, genistein, hydrogen peroxide (H2O2), sodium ascorbate, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and trolox were tested in non-cytotoxic concentrations for the activation of the rat CAT, human GPX1 and human SOD1 promoter. The maximally tolerable concentrations were determined by resazurin test in advance. Three out of these ten compounds were identified as potent inducers of GPX1 and SOD1. Stimulation of reporter gene construct transient transfected V79 cells for 24 hours with 100 µM paraquat caused a duplication of the relative GPX1 promoter activity and a 1.6-/1.7-fold increase of the relative SOD1 promoter activity. The incubation with 20 µM gen-istein or 10 µM quercetin resulted in duplication to triplication of both, the relative GPX1 and SOD1 promoter activity. In contrast, the rat CAT promoter was activated by 50 µM H2O2 (1.5-fold). Consequently, genistein, quercetin, and H2O2 are considered to be suitable reference substances for this DLR assay. To further characterize the inducing potential of the tested compounds all of them should be tested in different concentrations. Furthermore, for the routinely performed DLR assay it is recommended to use stably transfected cells to eliminate transfection caused variations. KW - Katalase KW - Glutathion-Peroxidase KW - Superoxiddismutase KW - Reportergen-Assay KW - Nahrungsbestandteile KW - catalase KW - glutathione peroxidase KW - superoxide dismutase KW - reporter gene assay KW - natural compounds Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-27901 ER - TY - GEN A1 - Gupta, Saurabh A1 - Dong, Yanni A1 - Dijkwel, Paul P. A1 - Müller-Röber, Bernd A1 - Gechev, Tsanko S. T1 - Genome-Wide Analysis of ROS Antioxidant Genes in Resurrection Species Suggest an Involvement of Distinct ROS Detoxification Systems during Desiccation T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - Abiotic stress is one of the major threats to plant crop yield and productivity. When plants are exposed to stress, production of reactive oxygen species (ROS) increases, which could lead to extensive cellular damage and hence crop loss. During evolution, plants have acquired antioxidant defense systems which can not only detoxify ROS but also adjust ROS levels required for proper cell signaling. Ascorbate peroxidase (APX), glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) are crucial enzymes involved in ROS detoxification. In this study, 40 putative APX, 28 GPX, 16 CAT, and 41 SOD genes were identified from genomes of the resurrection species Boea hygrometrica, Selaginella lepidophylla, Xerophyta viscosa, and Oropetium thomaeum, and the mesophile Selaginella moellendorffi. Phylogenetic analyses classified the APX, GPX, and SOD proteins into five clades each, and CAT proteins into three clades. Using co-expression network analysis, various regulatory modules were discovered, mainly involving glutathione, that likely work together to maintain ROS homeostasis upon desiccation stress in resurrection species. These regulatory modules also support the existence of species-specific ROS detoxification systems. The results suggest molecular pathways that regulate ROS in resurrection species and the role of APX, GPX, CAT and SOD genes in resurrection species during stress. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 763 KW - abiotic stress KW - desiccation KW - resurrection plants KW - ROS KW - ascorbate peroxidase KW - glutathione peroxidase KW - catalase KW - superoxide dismutase Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-437299 SN - 1866-8372 IS - 763 ER - TY - JOUR A1 - Gupta, Saurabh A1 - Dong, Yanni A1 - Dijkwel, Paul P. A1 - Müller-Röber, Bernd A1 - Gechev, Tsanko S. T1 - Genome-Wide Analysis of ROS Antioxidant Genes in Resurrection Species Suggest an Involvement of Distinct ROS Detoxification Systems during Desiccation JF - International Journal of Molecular Sciences N2 - Abiotic stress is one of the major threats to plant crop yield and productivity. When plants are exposed to stress, production of reactive oxygen species (ROS) increases, which could lead to extensive cellular damage and hence crop loss. During evolution, plants have acquired antioxidant defense systems which can not only detoxify ROS but also adjust ROS levels required for proper cell signaling. Ascorbate peroxidase (APX), glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) are crucial enzymes involved in ROS detoxification. In this study, 40 putative APX, 28 GPX, 16 CAT, and 41 SOD genes were identified from genomes of the resurrection species Boea hygrometrica, Selaginella lepidophylla, Xerophyta viscosa, and Oropetium thomaeum, and the mesophile Selaginella moellendorffi. Phylogenetic analyses classified the APX, GPX, and SOD proteins into five clades each, and CAT proteins into three clades. Using co-expression network analysis, various regulatory modules were discovered, mainly involving glutathione, that likely work together to maintain ROS homeostasis upon desiccation stress in resurrection species. These regulatory modules also support the existence of species-specific ROS detoxification systems. The results suggest molecular pathways that regulate ROS in resurrection species and the role of APX, GPX, CAT and SOD genes in resurrection species during stress. KW - abiotic stress KW - desiccation KW - resurrection plants KW - ROS KW - ascorbate peroxidase KW - glutathione peroxidase KW - catalase KW - superoxide dismutase Y1 - 2019 U6 - https://doi.org/10.3390/ijms20123101 SN - 1422-0067 SN - 1661-6596 VL - 20 IS - 12 PB - Molecular Diversity Preservation International CY - Basel ER -