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Academic literature on the topic 'RoGFP2'

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Published: 8 June 2024

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  1. Journal articles
  2. Dissertations / Theses
  3. Book chapters
  4. Conference papers
  5. Reports

Journal articles on the topic "RoGFP2":

1

Albrecht, Simone C., Mirko C. Sobotta, Daniela Bausewein, Isabel Aller, Rüdiger Hell, Tobias P. Dick, and Andreas J. Meyer. "Redesign of Genetically Encoded Biosensors for Monitoring Mitochondrial Redox Status in a Broad Range of Model Eukaryotes." Journal of Biomolecular Screening 19, no. 3 (August 16, 2013): 379–86. http://dx.doi.org/10.1177/1087057113499634.

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The development of genetically encoded redox biosensors has paved the way toward chemically specific, quantitative, dynamic, and compartment-specific redox measurements in cells and organisms. In particular, redox-sensitive green fluorescent proteins (roGFPs) have attracted major interest as tools to monitor biological redox changes in real time and in vivo. Most recently, the engineering of a redox relay that combines glutaredoxin (Grx) with roGFP2 as a translational fusion (Grx1-roGFP2) led to a biosensor for the glutathione redox potential ( EGSH). The expression of this probe in mitochondria is of particular interest as mitochondria are the major source of oxidants, and their redox status is closely connected to cell fate decisions. While Grx1-roGFP2 can be expressed in mammalian mitochondria, it fails to enter mitochondria in various nonmammalian model organisms. Here we report that inversion of domain order from Grx1-roGFP2 to roGFP2-Grx1 yields a biosensor with perfect mitochondrial targeting while fully maintaining its biosensor capabilities. The redesigned probe thus allows extending in vivo observations of mitochondrial redox homeostasis to important nonmammalian model organisms, particularly plants and insects.
2

Liu, Ting-Hang, Mohammad A. Yaghmour, Miin-Huey Lee, Thomas M. Gradziel, Johan H. J. Leveau, and Richard M. Bostock. "An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces." Phytopathology® 110, no. 2 (February 2020): 297–308. http://dx.doi.org/10.1094/phyto-07-19-0237-r.

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The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant–microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 μM for oxidation by H2O2 and menadione, respectively, and 10 μM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.
3

Xu, Xiuling, Katharina von Löhneysen, Katrin Soldau, Deborah Noack, Andrew Vu, and Jeffrey S. Friedman. "A novel approach for in vivo measurement of mouse red cell redox status." Blood 118, no. 13 (September 29, 2011): 3694–97. http://dx.doi.org/10.1182/blood-2011-03-342113.

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Abstract Maintenance of a reducing redox balance is a critical physiologic function of red cells (RBC) that can be perturbed in variety of RBC pathologies. Here we describe a new approach to evaluate in vivo RBC redox status using a redox sensitive GFP (roGFP2) sensor under control of a β-globin mini-promoter, directing expression specifically to erythroid cells. RoGFP2 expressing RBCs demonstrate ratiometric and reversible shifts in fluorescence on exposure to oxidants and reductants. We demonstrate that roGFP2 expressing RBC can be used to monitor thiol redox status during in vitro phenylhydrazine treatment and over the course of in vivo RBC aging, where a shift to a more oxidized state is observed in older cells. Thus, roGFP2 transgenic mice are a new and versatile tool that can be used to probe how RBC redox status responds in the context of drug therapy, physiologic stressors and pathologic states.
4

Xu, Xiuling, Katharina von Loehneysen, Deborah Noack, Andrew Vu, and Jeff S. Friedman. "A Novel Approach for In Vivo Measurement of Red Cell Redox Status." Blood 116, no. 21 (November 19, 2010): 2036. http://dx.doi.org/10.1182/blood.v116.21.2036.2036.

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Abstract Abstract 2036 Maintenance of a reducing redox balance is a critical physiologic function of red cell metabolic machinery. Perturbation of this balance, whether inherited or acquired, is found in a variety of red cell pathologies. Methods for evaluation of red cell redox status include direct approaches such as determining glutathione (GSH, GSSG) levels, and indirect approaches such as measuring fluorescence of oxidation sensitive dyes. Here we describe an alternative method for evaluation of red cell redox status that can be used in vivo and in real-time assays. Engineered variants of GFP possessing two solvent accessible cysteine residues function as molecular redox sensors with distinct fluorescence characteristics. Excitation spectrum shifts upon the oxidation of cysteine residues forming a disulfide. A higher ratio of fluorescence when comparing excitation at 405nm versus 488nm indicates rising levels of oxidized GFP and a shift in cellular redox status. To validate redox GFPs in erythroid cells, we first performed in vitro assays with MEL cells over-expressing several related GFP sensors (ro-GFPs), selecting the brightest molecule (roGFP2) for further study. The sensor function of roGFP2 in MEL cells was verified by stimulation with exogenous oxidant (1mM H202) or reductant (10 mM DTT) as shown in the figure below. In order to create a physiologic in vivo model for study of red cell redox status, transgenic mice expressing roGFP2 specifically in the erythroid lineage were generated. roGFP2 expressing red cells demonstrate the expected shift in fluorescence upon exposure to H202 or t-butyl peroxide in a short-term assay. In vivo, we have measured red cell lifespan (using biotin-labeling) in roGFP2 transgenic animals to follow redox status of red cells as a function of cell age. Expression of roGFP2 has no effect on red cell survival. Interestingly, when comparing old red cells (age > 50days) with younger cells (age < 50days), a shift in GFP fluorescence ratio indicating that a higher fraction of the sensor is oxidized in the aged cells was observed. This observation is consistent with the hypothesis that metabolic changes, in particular a decline in ability to reduce oxidative damage, contribute to red cell senescence. We are generating several murine strains with defined red cell defects also expressing roGFP2 in order to assess the role of changes in intra-erythrocyte redox status in a range of pathologic conditions. In vitro and in vivo assays using roGFP2 transgenic cells/mice are in process to determine the potential utility of this system as a screen for hematoxicity of drugs and other compounds. Figure 1 Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Figure 1. Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Disclosures: No relevant conflicts of interest to declare.
5

de Cubas, Laura, Valeriy V. Pak, Vsevolod V. Belousov, José Ayté, and Elena Hidalgo. "The Mitochondria-to-Cytosol H2O2 Gradient Is Caused by Peroxiredoxin-Dependent Cytosolic Scavenging." Antioxidants 10, no. 5 (May 6, 2021): 731. http://dx.doi.org/10.3390/antiox10050731.

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Fluorescent protein-based reporters used to measure intracellular H2O2 were developed to overcome the limitations of small permeable dyes. The two major families of genetically encoded redox reporters are the reduction-oxidation sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins and HyPer and derivatives. We have used the most sensitive probes of each family, roGFP2-Tpx1.C169S and HyPer7, to monitor steady-state and fluctuating levels of peroxides in fission yeast. While both are able to monitor the nanomolar fluctuations of intracellular H2O2, the former is two-five times more sensitive than HyPer7, and roGFP2-Tpx1.C169S is partially oxidized in the cytosol of wild-type cells while HyPer7 is fully reduced. We have successfully expressed HyPer7 in the mitochondrial matrix, and it is ~40% oxidized, suggesting higher steady-state levels of peroxides, in the low micromolar range, than in the cytosol. Cytosolic HyPer7 can detect negligible H2O2 in the cytosol from mitochondrial origin unless the main H2O2 scavenger, the cytosolic peroxiredoxin Tpx1, is absent, while mitochondrial HyPer7 is oxidized to the same extent in wild-type and ∆tpx1 cells. We conclude that there is a bidirectional flux of H2O2 across the matrix and the cytosol, but Tpx1 rapidly and efficiently scavenges mitochondrial-generated peroxides and stops their steady-state cytosolic levels rising.
6

Xu, Xiuling, and Jeff S. Friedman. "In Vivo Monitoring of Red Cell Redox Status to Screen for Potential Hematotoxicity of Anti-Malarial Drugs." Blood 118, no. 21 (November 18, 2011): 2099. http://dx.doi.org/10.1182/blood.v118.21.2099.2099.

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Abstract Abstract 2099 Redox equilibrium is an important determinant of malaria pathology and host defensive response to malaria parasites. Many anti-malarial drugs are reported to increase oxidative stress in red cells (RBC). Drug-induced hemolysis, particularly in G6PD deficient individuals, limits the utility of approved 8-aminoquinolones such as primaquine. The search for derivatives of primaquine that maintain efficacy without RBC toxicity is hampered by lack of a predictive assay for hemolytic potential. In order to monitor in vivo RBC redox changes in response to anti-malaria drugs, we have established a transgenic mouse line specifically expressing a redox sensitive GFP (roGFP2) in RBC1. RoGFP2 is an engineered EGFP with 2 cysteines introduced at amino acid positions 147 and 2042. When oxidized, the 2 cysteines form a disulfide bond, resulting in a protein conformational change that alters the spectral properties of the GFP. By following the ratio of fluorescence emission at 520nm after excitation at 405 vs 488nm, the intracellular redox potential in live cells can be determined. Here we apply this novel mouse model to follow in vivo RBC redox status upon exposure of transgenic mice to a series of 8-AQÕs and control compounds including known hemolytic agents such as dapsone. Unlike in vitro studies, these whole animal experiments incorporate metabolic transformation of inert parent compounds, pharmacokinetics and a time-course that closely models clinical hemolytic reactions in susceptible individuals exposed to the same drugs. roGFP2 transgenic mice (N=4 per group) were given test compounds (primaquine, chloroquine, dapsone and 1161B*) by gavage 2X/day at a total dose of 50mg/Kg over a period of 5 days. Peripheral RBC were analyzed using a LSRII FACS (BD) to determine the 405/488 ratio, and thus follow the redox status as shown in the figure below. Chloroquine treatment did not cause detectable change in roGFP2 signal, in agreement with previous data that chloroquine does not induce ROS production or hematotoxicity. Treatment with 1161B induced a steady increase in roGFP2 ratio that was first evident at 3 days of treatment, while primaquine treatment has little effect. Dapsone serves as a positive control in this assay, as a known hemolytic agent requiring metabolic activation to produce a redox cycling metabolite.3 Treatment with dapsone produced a time dependent shift to a more oxidized state of roGFP2 that was first evident after 2 days of drug administration. Consistent with these results, CBCÕs obtained one week after start of treatment showed moderate and mild hemolysis in those mice receiving dapsone and 1161B, respectively. In summary, roGFP2 transgenic mice provide a promising platform for assessment of hemolytic potential of drugs—here demonstrated using dapsone and 8-aminoquinolones. While the mice used in this study were GFP transgenic on a wild-type background, studies are in progress to assess whether strains with specific RBC defects (such as G6PD hypomorphs, PRDX2 KO andSOD1 KO) are more sensitive to hemolytic insults. Preliminary studies indicate that PRDX2 and SOD1 KO mice are more sensitive to primaquine, but not chloroquine, according to the roGFP2 ratio changes. G6PD deficient animals may prove particularly useful for discriminating compounds such as primaquine that are safe for normal individuals, but cause hemolysis in the context of enzyme deficiency. *1161B is the B enantiomer of an 8-aminoquinolone derivative with better efficacy and reduced hematoxicity4,5 in mice, when compared to the racemic mixture of the same compound. Disclosures: No relevant conflicts of interest to declare.
7

Fernández-Puente, Escarlata, and Jesús Palomero. "Genetically Encoded Biosensors to Monitor Intracellular Reactive Oxygen and Nitrogen Species and Glutathione Redox Potential in Skeletal Muscle Cells." International Journal of Molecular Sciences 22, no. 19 (October 8, 2021): 10876. http://dx.doi.org/10.3390/ijms221910876.

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Reactive oxygen and nitrogen species (RONS) play an important role in the pathophysiology of skeletal muscle and are involved in the regulation of intracellular signaling pathways, which drive metabolism, regeneration, and adaptation in skeletal muscle. However, the molecular mechanisms underlying these processes are unknown or partially uncovered. We implemented a combination of methodological approaches that are funded for the use of genetically encoded biosensors associated with quantitative fluorescence microscopy imaging to study redox biology in skeletal muscle. Therefore, it was possible to detect and monitor RONS and glutathione redox potential with high specificity and spatio-temporal resolution in two models, isolated skeletal muscle fibers and C2C12 myoblasts/myotubes. Biosensors HyPer3 and roGFP2-Orp1 were examined for the detection of cytosolic hydrogen peroxide; HyPer-mito and HyPer-nuc for the detection of mitochondrial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for registration of the glutathione redox potential in mitochondria and cytosol. G-geNOp was proven to detect cytosolic nitric oxide. The fluorescence emitted by the biosensors is affected by pH, and this might have masked the results; therefore, environmental CO2 must be controlled to avoid pH fluctuations. In conclusion, genetically encoded biosensors and quantitative fluorescence microscopy provide a robust methodology to investigate the pathophysiological processes associated with the redox biology of skeletal muscle.
8

García-Quirós, Estefanía, Juan de Dios Alché, Barbara Karpinska, and Christine H. Foyer. "Glutathione redox state plays a key role in flower development and pollen vigour." Journal of Experimental Botany 71, no. 2 (September 26, 2019): 730–41. http://dx.doi.org/10.1093/jxb/erz376.

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roGFP2 measurements demonstrate that ungerminated Arabidopsis pollen grains are highly oxidized. Glutathione-deficient flowers show increased cellular oxidation with poor pollen germination/tube growth, suggesting that the reduced state underpins growth functions.
9

Morgan, Bruce, Mirko C. Sobotta, and Tobias P. Dick. "Measuring EGSH and H2O2 with roGFP2-based redox probes." Free Radical Biology and Medicine 51, no. 11 (December 2011): 1943–51. http://dx.doi.org/10.1016/j.freeradbiomed.2011.08.035.

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Costa, Cláudio F., Celien Lismont, Serhii Chornyi, Hongli Li, Mohamed A. F. Hussein, Hans R. Waterham, and Marc Fransen. "Functional Analysis of GSTK1 in Peroxisomal Redox Homeostasis in HEK-293 Cells." Antioxidants 12, no. 6 (June 7, 2023): 1236. http://dx.doi.org/10.3390/antiox12061236.

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Peroxisomes serve as important centers for cellular redox metabolism and communication. However, fundamental gaps remain in our understanding of how the peroxisomal redox equilibrium is maintained. In particular, very little is known about the function of the nonenzymatic antioxidant glutathione in the peroxisome interior and how the glutathione antioxidant system balances with peroxisomal protein thiols. So far, only one human peroxisomal glutathione-consuming enzyme has been identified: glutathione S-transferase 1 kappa (GSTK1). To study the role of this enzyme in peroxisomal glutathione regulation and function, a GSTK1-deficient HEK-293 cell line was generated and fluorescent redox sensors were used to monitor the intraperoxisomal GSSG/GSH and NAD+/NADH redox couples and NADPH levels. We provide evidence that ablation of GSTK1 does not change the basal intraperoxisomal redox state but significantly extends the recovery period of the peroxisomal glutathione redox sensor po-roGFP2 upon treatment of the cells with thiol-specific oxidants. Given that this delay (i) can be rescued by reintroduction of GSTK1, but not its S16A active site mutant, and (ii) is not observed with a glutaredoxin-tagged version of po-roGFP2, our findings demonstrate that GSTK1 contains GSH-dependent disulfide bond oxidoreductase activity.
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Journal articles Dissertations / Theses

Dissertations / Theses on the topic "RoGFP2":

1

Dehaene, Noémie. "Functional analysis of a cytoplasmic male sterility in Arabidopsis thaliana." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS418/document.

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Les stérilités mâles cytoplasmiques (SMC) résultent d'une incompatibilité nucléo-cytoplasmique. Le cytoplasme (presque toujours la mitochondrie) peut porter un gène de stérilité mâle, et le noyau peut restaurer la fertilité pollinique ou non. Les mécanismes physiologiques conduisant à la mort pollinique restent largement incompris. Plusieurs hypothèses ont été proposées, parmi lesquelles une déficience en ATP. Une SMC gamétophytique a été découverte chez A. thaliana. Une phase ouverte de lecture codant possiblement un peptide de 117 acides aminés, appelée orf117Sha, a été identifiée comme facteur de stérilité candidat.Au cours de ma thèse, j'ai cherché à valider le rôle de l'orf117Sha, et à comprendre comment une anomalie mitochondriale pouvait induire cette SMC. Aucune différence n'a pu être détectée au niveau de l'ARNm de l'orf117Sha entre les lignées stérile et restaurée, mais sa protéine semble accumulée uniquement dans la lignée stérile. La phénocopie par transgénèse de la SMC a suggéré un effet délétère de l'ORF117SHA dans les gamétophytes mâle et femelle.La description cytologique de la SMC montre une mort pollinique progressive à partir du stade binucléé. Auparavant, les mitochondries du pollen gonflent puis éclatent, et le développement s'arrête. L’utilisation de senseurs génétiquement encodés mesurant la concentration en ATP (ATeam) et l'état redox du glutathion (roGFP2-Grx1) a permis la mesure de ces facteurs en microscopie confocale, dans des tissus végétatifs et dans le pollen. La production d'ATP ne semble pas affectée dans la lignée stérile, contredisant l'hypothèse de l'ATP. Le glutathion mitochondrial est suroxydé dans la lignée stérile, à la fois dans les tissus végétatifs étudiés et le pollen, qui serait liée à la SMC car annulée par la restauration génétique de fertilité.Avec cette étude, j'apporte des arguments en faveur de l'orf117Sha dans l'induction de la SMC Sha, et je décris les évènements préalables à l'avortement du grain de pollen. Mes résultats permettent de mieux comprendre les évènements physiologiques conduisant à la mort du pollen
This work aims at better understand the events leading to pollen abortion in a recently discovered gametophytic cytoplasmic male sterility (CMS) in Arabidopsis thaliana. Although CMS have been widely used in hybrid seed production in many crops, the physiological mechanisms leading to pollen death by the mitochondrial sterilizing genes in the permissive (maintainer) nuclear backgrounds are poorly understood. Association genetics previously identified orf117Sha as a candidate mitochondrial CMS-associated gene.In a first part, I analyzed the expression of the orf117Sha gene in sterile plants and in fertile plants carrying nuclear genes restoring male fertility. I observed unusual features of its mRNA, but detected no difference at this level between sterile and restored plants. Oppositely, the ORF117SHA protein seems to be accumulated specifically in the sterile line, supporting its role in CMS. A phenocopy attempt by transgenesis suggested a possible link between a female and male gametophytic lethality and the ORF117SHA, even though few individuals could be analyzed.In a second part, I observed pollen development in sterile plants and fertile controls using different cytological approaches. My results show a progressive pollen death starting from the binucleate stage in the sterile. Prior to abortion, pollen mitochondria swell before rupture, and the development stops. I used confocal microscopy combined with genetically encoded sensors to explore specific physiological features in pollen and vegetative tissues of sterile plants. With ATeam, which allows the assessment of ATP content in the cytosol, I could challenge the generally accepted hypothesis of an ATP deficiency leading to pollen abortion in CMS. Indeed, the ATP production does not seem to be affected in the sterile line. With a mitochondria-addressed roGFP2-Grx1, I was able to assess the redox state of the glutathione pool in vegetative tissues and in the male gametophyte. I observed an overoxydation of the glutathione pool in mitochondria of the sterile line, in vegetative tissue investigated and in the pollen grain. This overoxydation seems to be linked to the CMS as it is annihilated by the presence of restorer genes.My results pave the way for further exploration of the links between the sterility protein, mitochondrial morphology changes, mitochondrial overoxydation, and pollen development arrest and death in the A. thaliana CMS
2

Caubrière, Damien. "Développement de nouveaux biosenseurs redox pour composés soufrés." Electronic Thesis or Diss., Université de Lorraine, 2022. http://www.theses.fr/2022LORR0359.

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Au cours des dix dernières années, le développement de biosenseurs redox fluorescents a permis de générer des outils permettant d'étudier les dynamiques in vivo de molécules comme les formes réduite et oxydée du glutathion ou le peroxyde d'hydrogène. La cystéine étant un métabolite clé du métabolisme du soufre, l'objectif de ce projet de thèse était de développer un biosenseur redox fluorescent spécifique de la cystéine en couplant une oxydoréductase à la roGFP2 (reduction-oxidation green fluorescent protein). Tout d'abord les activités de plusieurs isoformes de cystéine désulfurases (CD) et des protéines à domaine rhodanese (Rhd), catalysant respectivement la désulfuration de la cystéine et des réactions de trans-persulfuration ont été analysées in vitro afin de déterminer si elles pouvaient constituer de bons candidats pour cette activité oxydoréductase. Ces analyses ont mis en évidence qu'une protéine chimérique naturelle bactérienne possédant des domaines CD et Rhd oxyde efficacement la roGFP2, au travers de réactions de trans-persulfuration depuis la cystéine vers la roGFP2. Cette protéine candidate a ensuite été fusionnée à la roGFP2 pour générer le biosenseur CD-Rhd-roGFP2. In vitro, cette protéine est sensible à l'oxydation en présence de concentrations physiologiques en cystéine alors que l'oxydation par le thiosulfate, autre substrat potentiel du domaine Rhd, est négligeable. D'une part, les réactions de trans-persulfuration entre les domaines protéiques menant à l'oxydation de la roGFP2 ne sont pas ou très peu inhibées par les systèmes réducteurs physiologiques. Néanmoins, le système glutathion-glutarédoxine réduit spécifiquement la roGFP2. L'expression de ce biosenseur chez la bactérie Escherichia coli, a révélé une réponse dynamique en réponse à des ajouts exogènes de cystéine ou de cystine ouvrant la voie à des études similaires dans les organites d'autres organismes modèles eucaryotes
Over the last decade, the development of fluorescent redox biosensors has provided tools to study the in vivo dynamics of molecules such as the reduced and oxidized forms of glutathione or hydrogen peroxide. Cysteine being a key metabolite of sulfur metabolism, this PhD project aimed at developing a fluorescent redox biosensor specific for cysteine by coupling an oxidoreductase to roGFP2 (reduction-oxidation green fluorescent protein). First, the activities of several isoforms of cysteine desulfurases (CD) and rhodanese-domain containing proteins (Rhd), catalyzing cysteine desulfuration and trans-persufidation reactions, respectively, were analyzed in vitro in order to determine whether they could constitute good candidates for this oxidoreductase activity. These analyses revealed that a natural chimeric protein possessing both CD and Rhd domains efficiently oxidizes roGFP2, by catalyzing trans-persulfidation reactions from cysteine to roGFP2. This candidate protein was then fused to roGFP2 to generate the CD-Rhd-roGFP2 biosensor. In vitro, this protein is sensitive to oxidation in the presence of physiological concentrations of cysteine whereas oxidation by thiosulfate, another potential substrate of the Rhd domain, is negligible. In addition, the trans-persulfidation reactions between the protein domains leading to the oxidation of roGFP2 are not inhibited by physiological reducing systems. Nevertheless, the glutathione/glutaredoxin system specifically reduces roGFP2. The expression of this biosensor in the bacterium Escherichia coli revealed a dynamic response of the biosensor to exogenous addition of cysteine or cystine, paving the way for similar studies in organelles from other eukaryotic model organisms
3

Schneider, Jannis Frederic [Verfasser], Lars I. [Gutachter] Leichert, and Joachim [Gutachter] Rassow. "Untersuchung des Redoxzustandes des Periplasmas und Aufklärung früher redox-regulatorischer Ereignisse in Escherichia coli während der eukaryotischen Immunantwort unter Verwendung von roGFP2 basierten Sonden / Jannis Frederic Schneider ; Gutachter: Lars I. Leichert, Joachim Rassow ; Medizinische Fakultät." Bochum : Ruhr-Universität Bochum, 2021. http://d-nb.info/1235224279/34.

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4

Maciejuk, Anna-Maria. "Measurements of redox potential during apoptosis." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28862.

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Consensus opinion suggests that apoptosis occurs when the intracellular redox potential reaches its oxidative range, i.e. when the balance between oxidants and reductants is disturbed. An understanding of the links between redox potential and the induction of apoptosis in cells could improve our understanding of the process and help to predict therapeutic responses. This study investigates the changes in redox potential at distinct stages of apoptosis induced in the human cervical cancer cell line, HeLa. Stages of the apoptotic process were defined by loss of mitochondrial membrane polarisation (ΔΨm), membrane phosphatidyl serine exposure, caspase-3 activation, and nuclear fragmentation. To measure real-time redox potential change in apoptotic cells two independent methods were used: (1) expression of redox-responsive green fluorescent protein (roGFP2) measured by flow cytometry and (2) redox-responsive nanosensors detected by surface enhanced Raman spectroscopy (SERS). roGFP2 measurements showed that HeLa cells demonstrate a shift towards an oxidative redox state during the later stages of apoptosis and this was preceded by loss of ΔΨm. The relationship between these two events was investigated by transient inhibition of mitochondrial permeability transition pore opening using the inhibitor bongkrekic acid (BKA) pre-treatment. At the cell population level, transient exclusion of the mitochondrial contribution delayed two key events of apoptosis in the first two hours measured by nuclear fragmentation and loss of ΔΨm. However, BKA treatment did not affect redox potential, reported by roGFP2, when compared with controls. Therefore, this suggests that mitochondria do not contribute towards the overall redox potential change in apoptosis. To gain insight into the significance of redox change at the earliest stages of apoptosis, single cell studies were performed. SERS, employing simultaneous redox potential and intracellular pH measurements using two synthetic nanosensors AQ-NS and MBA-NS, showed that BKA pre-treatment resulted in increased alkalinity and the cells were consequently protected from induction of apoptosis in the first thirty minutes of the kinase inhibitor staurosporine treatment. Measurements with SERS nanosensors allowed for adjustment for pH, which provides a clearer insight into redox potential dynamics, with consideration of the environment, and accurate quantitative assessment of redox at early stages of apoptosis. Together these data suggest that while roGFP2 is a valid method to use at a population level, SERS is a more sensitive method for measuring the redox potential of the cell at the early stages of apoptosis.
5

Edwards, Sarah. "Disulfide-Mediated Modifications of roGFP and their Impact on Its Use as a Redox Sensor." Thesis, The University of Arizona, 2011. http://hdl.handle.net/10150/144316.

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Wagener, Kerstin Charlotte [Verfasser], Michael [Akademischer Betreuer] Müller, Michael [Gutachter] Müller, and Stefan [Gutachter] Jakobs. "Transgene Redoxindikator-Mäuse mit mitochondrialer roGFP1-Expression: Phänotypisierung, neuronales Verteilungsmuster und Sensorfunktionalität / Kerstin Charlotte Wagener ; Gutachter: Michael Müller, Stefan Jakobs ; Betreuer: Michael Müller." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://d-nb.info/1149956364/34.

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Souza, Arnaldo Henrique de. "Modulação redox, função e sobrevivência de células β-pancreáticas: evidência sobre o papel da enzima NADPH oxidase-2 (NOX2) em um modelo in vitro de glicotoxicidade." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/42/42137/tde-06092016-094234/.

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O estresse oxidativo e a enzima NADPH oxidase-2 (NOX2) estão associados com a diminuição da massa funcional de células-β em pacientes com diabetes do tipo 2 (DT2). Neste estudo, testamos o papel da NOX2 sobre a glicotoxicidade em células-β. Ilhotas de camundongo C57BL/6J nocautes ou não para NOX2 (NOX2-KO e WT, respectivamente) foram isoladas e cultivadas por até 3 semanas em 10 ou 30 mmol/l de glucose (G10 e G30, respectivamente). A secreção de insulina foi maior nas ilhotas NOX2-KO vs. WT sem apresentar diferenças metabólicas ou do potencial redox da glutationa citosólica (EGSH). O cultivo de ilhotas em G30 aumenta a concentração de H2O2 e a oxidação de tióis no compartimento citosólico, seguido por aumento de apoptose de células-β, mas, preservando a reposta máxima secretória. Estas respostas foram quase idênticas em ambos os tipos de ilhotas. Em conclusão, a NOX2 regula negativamente a secreção de insulina em ilhotas de camundongos C57BL/6J, mas não é um componente crítico para a sobrevivência de células β em um modelo in vitro de glicotoxicidade.
Oxidative stress and NADPH oxidase-2 (NOX2) enzyme are associated to the decline of the functional β-cell mass in type 2 diabetes (T2D). Here, we tested the role of NOX2 on β-cell glucotoxicity. NOX2 knockout (NOX2 KO) and wild type (WT) C57BL/6J mice islets were isolated and cultured up to 3 weeks at 10 or 30 mmol/l glucose concentrations (G10 and G30, respectively). The insulin secretion was higher in NOX2-KO vs. WT islets despite similar metabolic and cytosolic glutathione-redox potential (EGSH) changes. The prolonged culture at G30 increases the H2O2 concentration and cytosolic thiol oxidation, followed by increased βcell apoptosis but preserving maximal secretory response. These responses were almost identical in both types of islets. In conclusion, NOX2 is a negative regulator of insulin secretion in C57BL/6J mouse islets, but is not a critical component for β-cell survival in a model of glucotoxicity in vitro.
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Kolbrink, Benedikt [Verfasser], Michael [Akademischer Betreuer] Müller, and Jochen [Akademischer Betreuer] Staiger. "Charakterisierung eines transgenen Mausmodells mit spezifischer zytosolischer Expression des optischen Redox-Indikators roGFP1 in Neuronen / Benedikt Kolbrink. Gutachter: Michael Müller ; Jochen Staiger. Betreuer: Michael Müller." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1077096410/34.

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Kizina, Kathrin Michaela. "Funktionelles ROS/Redox Imaging, basierend auf genetisch-kodierten optischen Sensoren, exzitationsratiometrischer Zwei-Photonen-Mikroskopie und Fluoreszenzlebenszeiten." Doctoral thesis, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0003-C139-3.

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Wagener, Kerstin Charlotte. "Transgene Redoxindikator-Mäuse mit mitochondrialer roGFP1-Expression: Phänotypisierung, neuronales Verteilungsmuster und Sensorfunktionalität." Doctoral thesis, 2017. http://hdl.handle.net/11858/00-1735-0000-0023-3F99-F.

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Book chapters on the topic "RoGFP2":

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Ugalde, José Manuel, Lara Fecker, Markus Schwarzländer, Stefanie J. Müller-Schüssele, and Andreas J. Meyer. "Live Monitoring of ROS-Induced Cytosolic Redox Changes with roGFP2-Based Sensors in Plants." In Methods in Molecular Biology, 65–85. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2469-2_5.

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Lismont, Celien, Paul A. Walton, and Marc Fransen. "Quantitative Monitoring of Subcellular Redox Dynamics in Living Mammalian Cells Using RoGFP2-Based Probes." In Methods in Molecular Biology, 151–64. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6937-1_14.

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Buratti, Stefano, Matteo Grenzi, Giorgia Tortora, Sara Paola Nastasi, Elisa Dell’Aglio, Andrea Bassi, and Alex Costa. "Noninvasive In Planta Live Measurements of H2O2 and Glutathione Redox Potential with Fluorescent roGFPs-Based Sensors." In ROS Signaling in Plants, 45–64. New York, NY: Springer US, 2024. http://dx.doi.org/10.1007/978-1-0716-3826-2_4.

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Conference papers on the topic "RoGFP2":

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Niculescu, Mihai Alexandru, Stefan Ruseti, and Mihai Dascalu. "RoGPT2: Romanian GPT2 for Text Generation." In 2021 IEEE 33rd International Conference on Tools with Artificial Intelligence (ICTAI). IEEE, 2021. http://dx.doi.org/10.1109/ictai52525.2021.00183.

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Wierer, S., K. Elgass, S. Bieker, U. Zentgraf, A. J. Meixner, and F. Schleifenbaum. "Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation." In SPIE BiOS, edited by Daniel L. Farkas, Dan V. Nicolau, and Robert C. Leif. SPIE, 2011. http://dx.doi.org/10.1117/12.873753.

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Reports on the topic "RoGFP2":

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Friedman, Haya, Chris Watkins, Susan Lurie, and Susheng Gan. Dark-induced Reactive Oxygen Species Accumulation and Inhibition by Gibberellins: Towards Inhibition of Postharvest Senescence. United States Department of Agriculture, December 2009. http://dx.doi.org/10.32747/2009.7613883.bard.

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Abstract:
Dark-induced senescence could pose a major problem in export of various crops including cuttings. The assumption of this work was that ROS which is increased at a specific organelle can serve as a signal for activation of cell senescence program. Hormones which reduce senescence in several crops like gibberellic acid (GA) and possibly cytokinin (CK) may reduce senescence by inhibiting this signal. In this study we worked on Pelargonium cuttings as well as Arabidopsis rosette. In Pelargonium the increase in ROS occurred concomitantly with increase in two SAGs, and the increase persisted in isolated chloroplasts. In Arabidopsis we used two recentlydeveloped technologies to examine these hypotheses; one is a transcriptome approach which, on one hand, enabled to monitor expression of genes within the antioxidants network, and on the other hand, determine organelle-specific ROS-related transcriptome footprint. This last approach was further developed to an assay (so called ROSmeter) for determination of the ROS-footprint resulting from defined ROS stresses. The second approach involved the monitoring of changes in the redox poise in different organelles by measuring fluorescence ratio of redox-sensitive GFP (roGFP) directed to plastids, mitochondria, peroxisome and cytoplasm. By using the roGFP we determined that the mitochondria environment is oxidized as early as the first day under darkness, and this is followed by oxidation of the peroxisome on the second day and the cytoplast on the third day. The plastids became less oxidized at the first day of darkness and this was followed by a gradual increase in oxidation. The results with the ROS-related transcriptome footprint showed early changes in ROS-related transcriptome footprint emanating from mitochondria and peroxisomes. Taken together these results suggest that the first ROS-related change occurred in mitochondria and peroxisomes. The analysis of antioxidative gene’s network did not yield any clear results about the changes occurring in antioxidative status during extended darkness. Nevertheless, there is a reduction in expression of many of the plastids antioxidative related genes. This may explain a later increase in the oxidation poise of the plastids, occurring concomitantly with increase in cell death. Gibberellic acid (GA) prevented senescence in Pelargonium leaves; however, in Arabidopsis it did not prevent chlorophyll degradation, but prevented upregulation of SAGs (Apendix Fig. 1). Gibberellic acid prevented in Pelargonium the increase in ROS in chloroplast, and we suggested that this prevents the destruction of the chloroplasts and hence, the tissue remains green. In Arabidopsis, reduction in endogenous GA and BA are probably not causing dark-induced senescence, nevertheless, these materials have some effect at preventing senescence. Neither GA nor CK had any effect on transcriptome footprint related to ROS in the various organelles, however while GA reduced expression of few general ROS-related genes, BA mainly prevented the decrease in chloroplasts genes. Taken together, GA and BA act by different pathways to inhibit senescence and GA might act via ROS reduction. Therefore, application of both hormones may act synergistically to prevent darkinduced senescence of various crops.

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