Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors. Conventional redox-sensitive GFPs (roGFPs) are limited by undefined ...specificity and slow response to changes in redox potential. In this study we demonstrate that the fusion of human glutaredoxin-1 (Grx1) to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple. The Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential (E(GSH)) in different cellular compartments with high sensitivity and temporal resolution. The biosensor detected nanomolar changes in oxidized glutathione (GSSG) against a backdrop of millimolar reduced glutathione (GSH) on a scale of seconds to minutes. It facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.
The cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation ...of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H₂O₂ or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 m m cytosolic glutathione buffer would contain only 25 n m oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stress-induced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.
Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress ...conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that almost equal to65% of the total GR activity is attributed to GR1, whereas almost equal to35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (EGSH) in the cytosol. Using this tool, it is shown that EGSH in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.
The Arabidopsis (Arabidopsis thaliana) phytoalexin-deficient mutant pad2-1 displays enhanced susceptibility to a broad range pathogens and herbivorous insects that correlates with deficiencies in the ...production of camalexin, indole glucosinolates, and salicylic acid (SA). The pad2-1 mutation is localized in the GLUTAMATE-CYSTEINE LIGASE (GCL) gene encoding the enzyme of glutathione biosynthesis. While pad2-1 glutathione deficiency is not caused by a decrease in GCL transcripts, analysis of GCL protein level revealed that pad2-1 plants contained only 48% of the wild-type protein amount. In contrast to the wild type, the oxidized form of GCL was dominant in pad2-1, suggesting a distinct redox environment. This finding was corroborated by the expression of GRXl-roGFP2, showing that the cytosolic glutathione redox potential was significantly less negative in pad2-1. Analysis of oxidative stress-related gene expression showed a higher transcript accumulation in pad2-1 of GLUTATHIONE REDUCTASE, GLUTATHIONE-S-TRANSFERASE, and RESPIRATORY BURST OXIDASE HOMOLOG D in response to the oomycete Phytophthora brassicae. Interestingly, oligogalacturonide elicitation in pad2-1 revealed a lower membrane depolarization that was found to act upstream of an impaired hydrogen peroxide production. This impaired hydrogen peroxide production was also observed during pathogen infection and correlated with a reduced hypersensitive response in pad2-1. In addition, a lack of pathogen-triggered expression of the ISOCHORISMATE SYNTHASE1 gene, coding the SA-biosynthetic enzyme isochorismate synthase, was identified as the cause of the SA deficiency in pad2-1. Together, our results indicate that the padl-l mutation is related to a decrease in GCL protein and that the resulting glutathione deficiency negatively affects important processes of disease resistance.
Families of internationally adopted children may face specific problems with which general practitioners (GPs) may not be familiar. The aim of the study was to explore problems faced by families ...before, during and after the arrival of their internationally adopted child and to assess the usefulness of a specific medical structure for internationally adopted children, which could be a resource for the GP.
We conducted a qualitative study using individual semistructured guided conversations and interviewed 21 families that had adopted a total of 26 children internationally in the Puy de Dome department, France, in 2003. Quantitative data were used to describe the pathologies diagnosed and the investigations performed.Our study showed that the history of these families, from the start of the adoption project to its achievement, is complex and warrants careful analysis. Health-care providers should not only consider the medical aspects of adoption, but should also be interested in the histories of these families, which may play a role in the forming of attachments between the adoptee and their adoptive parents and prevent further trouble during the development of the child. We also showed that adoptive parents have similar fears or transient difficulties that may be resolved quickly by listening and reassurance. Most such families would support the existence of a specific medical structure for internationally adopted children, which could be a resource for the general practitioner. However, the health-care providers interviewed were divided on the subject and expressed their fear that a special consultation could be stigmatizing to children and families.
A specific consultation with well-trained and experienced practitioners acting in close collaboration with GPs and paediatricians may be of help in better understanding and supporting adopted children and their families.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
La coopération entre disciplines est en train de se développer à tous les niveaux de la recherche et de l’enseignement en sciences humaines. Même s’il y a encore quelque distance entre les intentions ...affichées et les pratiques effectives, elle est installée comme une tendance irréversible. L’hypothèse de cet article est qu’il ne s’agit pas de la fin des disciplines, mais d’une reconfiguration profonde : nous passons de disciplines fragmentées et fondées sur le modèle pyramidal, à des disciplines interactives (= « faire avec ») dans laquelle la personne, l’acteur réflexif, et le « travail sur soi » sont pleinement sollicités. C’est pour cette raison que le choix méthodologique de ce petit essai est celui de l’auto-ethnographie.
La coopération entre disciplines est en train de se développer à tous les niveaux de la recherche et de l’enseignement en sciences humaines. Même s’il y a encore quelque distance entre les intentions ...affichées et les pratiques effectives, elle est installée comme une tendance irréversible. L’hypothèse de cet article est qu’il ne s’agit pas de la fin des disciplines, mais d’une reconfiguration profonde : nous passons de disciplines fragmentées et fondées sur le modèle pyramidal, à des disciplines interactives (= « faire avec ») dans laquelle la personne, l’acteur réflexif, et le « travail sur soi » sont pleinement sollicités. C’est pour cette raison que le choix méthodologique de ce petit essai est celui de l’auto-ethnographie.
• A highly negative glutathione redox potential (EGSH
) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox ...regulation and ironasulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear.
• We dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing.
• Our data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ATP-binding cassette (ABC) transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability.
• We identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.
Electric properties of dust devils Franzese, Gabriele; Esposito, Francesca; Lorenz, Ralph ...
Earth and planetary science letters,
07/2018, Letnik:
493
Journal Article
Recenzirano
•First concurrent measurements of E-field, dust abundance and meteorological data.•First statistical study of induced E-field by dust devils.•Dust devil induced E-field is linearly related to vortex ...pressure drop and speed.•Dust devils and dust storms show the same relation E-field/lifted dust grains.•Terrestrial and Martian dust devils show similar pressure drops distributions.
Dust devils are one of the most effective phenomena able to inject dust grains into the atmosphere. On Mars, they play an important role to maintain the haze and can significantly affect the global dust loading, especially outside the dust storm season. Despite dust devils having been studied for a century and a half, many open questions regarding their physics still exist. In particular, the nature of the dust lifting mechanisms inside the vortices, the development of the induced electric field and the exact contribution to the global atmospheric dust budget are still debated topics. In this paper, we analyze the dust devil activity observed in the Moroccan Sahara desert during a 2014 field campaign. We have acquired the most comprehensive field data set presently available for the dust devils: including meteorological, atmospheric electric field and lifted dust concentration measurements. We focus our attention on the electric field induced by vortices, using this as the principal detection parameter. We present, for the first time, the statistical distribution of dust devil electric field and its relationships with the pressure drop, the horizontal and vertical vortex velocity and the total dust mass lifted. We also compare the pressure drop distribution of our sample with the ones observed on the martian surface showing the similarity of the dust devils samples and the usefulness of this study for the next martian surface missions.
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