Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii reveals the unique properties of a chloroplastic CGFS-type glutaredoxin.

Glutaredoxins (GRXs) are small ubiquitous disulfide oxidoreductases known to use GSH as electron donor. In photosynthetic organisms, little is known about the biochemical properties of GRXs despite the existence of approximately 30 different isoforms in higher plants. We report here the biochemical characterization of Chlamydomonas GRX1 and GRX3, the major cytosolic and chloroplastic isoforms, respectively. Glutaredoxins are classified on the basis of the amino acid sequence of the active site. GRX1 is a typical CPYC-type GRX, which is reduced by GSH and exhibits disulfide reductase, dehydroascorbate reductase, and deglutathionylation activities. In contrast, GRX3 exhibits unique properties. This chloroplastic CGFS-type GRX is not reduced by GSH and has an atypically low redox potential (-323 +/- 4 mV at pH 7.9). Remarkably, GRX3 can be reduced in the light by photoreduced ferredoxin and ferredoxin-thioredoxin reductase. Both GRXs proved to be very efficient catalysts of A(4)-glyceraldehyde-3-phosphate dehydrogenase deglutathionylation, whereas cytosolic and chloroplastic thioredoxins were inefficient. Glutathionylated A(4)-glyceraldehyde-3-phosphate dehydrogenase is the first physiological substrate identified for a CGFS-type GRX

framework of the ESPON 2013 Programme, partly financed by the European Regional Development Fund.

consists of the EU Commission and the Member States of the EU27, plus Iceland, Liechtenstein, Norway and Switzerland. Each partner is represented in the ESPON Monitoring Committee. This report does not necessarily reflect the opinion of the members of the Monitoring Committee. Information on the ESPON Programme and projects can be found on www.espon.eu The web site provides the possibility to download and examine the most recent documents produced by finalised and ongoing ESPON projects. This basic report exists only in an electroni

Development Fund.

framework of the ESPON 2013 Programme, partly financed by the European Regiona

Upregulation of PPARβ/δ Is Associated with Structural and Functional Changes in the Type I Diabetes Rat Diaphragm

Diabetes mellitus is associated with alterations in peripheral striated muscles and cardiomyopathy. We examined diaphragmatic function and fiber composition and identified the role of peroxisome proliferator-activated receptors (PPAR alpha and beta/delta) as a factor involved in diaphragm muscle plasticity in response to type I diabetes.Streptozotocin-treated rats were studied after 8 weeks and compared with their controls. Diaphragmatic strips were stimulated in vitro and mechanical and energetic variables were measured, cross bridge kinetics assessed, and the effects of fatigue and hypoxia evaluated. Morphometry, myosin heavy chain isoforms, PPAR alpha and beta/delta gene and protein expression were also assessed. Diabetes induced a decrease in maximum velocity of shortening (-14%, P<0.05) associated with a decrease in myosin ATPase activity (-49%, P<0.05), and an increase in force (+20%, P<0.05) associated with an increase in the number of cross bridges (+14%, P<0.05). These modifications were in agreement with a shift towards slow myosin heavy chain fibers and were associated with an upregulation of PPARbeta/delta (+314% increase in gene and +190% increase in protein expression, P<0.05). In addition, greater resistances to fatigue and hypoxia were observed in diabetic rats.Type I diabetes induced complex mechanical and energetic changes in the rat diaphragm and was associated with an up-regulation of PPARbeta/delta that could improve resistance to fatigue and hypoxia and favour the shift towards slow myosin heavy chain isoforms

Rôle des glutarédoxines et de la glutathionylation chez les végétaux

Le glutathion, principal thiol libre soluble de faible masse moléculaire chez la plupart des organismes, a été récemment impliqué dans une modification post-traductionnelle réversible appelée glutathionylation, pouvant permettre la régulation d activités enzymatiques, et/ou la protection des thiols libres et réactifs des protéines contre une oxydation irréversible. Tout d abord, nous avons étudié la glutathionylation in vitro des thiorédoxines (TRX) chloroplastiques d Arabidopsis et de Chlamydomonas. Seules les TRX f sont glutathionylables, ce qui affecte leur réduction. De même, l activité de la glycéraldéhyde-3-phosphate déshydrogénase A4 (GAPDH-A4), une enzyme du cycle de Calvin, est inhibée par glutathionylation. Nous avons ensuite développé une approche protéomique afin d identifier des protéines glutathionylées in vivo chez Chlamydomonas. 25 cibles ont été identifiées, dont 18 chloroplastiques, surtout impliquées dans les processus de photosynthèse et de réponse au stress oxydatif. 3 cibles ont été confirmées in vitro : l HSP70B, la peroxyrédoxine 2-cys chloroplastique, l isocitrate lyase. Chez les organismes non-photosynthétiques, les glutarédoxines (GRX) peuvent catalyser efficacement la déglutathionylation. Nous avons caractérisé biochimiquement 2 GRX de Chlamydomonas. La GRX1, GRX CPYC cytosolique, possède les activités GRX classiques, alors que la GRX3, GRX CGFS chloroplastique, est inactive dans ces tests. Par ailleurs, la GRX3 possède un potentiel électrochimique particulièrement bas, proche de celui des TRX. Le système chloroplastique de réduction des TRX permet de réduire la GRX3 qui peut alors déglutathionyler très efficacement la GAPDH-A4.Glutathione, the main soluble low molecular weight free thiol in most organisms, has been recently involved in a reversible post-translational reaction called glutathionylation, which can allow regulation of enzyme activity, and/or protection of protein reactive free thiols from oxidation. We first studied in vitro glutathionylation of chloroplastic thioredoxins (TRX) from Arabidopsis and Chlamydomonas. Only TRX f underwent glutathionylation, leading to impaired reduction of the protein. A4-glyceraldehyde-3-phosphate dehydrogenase (A4-GAPDH), a Calvin cycle enzyme, was also found to undergo glutathionylation in vitro with a subsequent inhibition of its activity. In order to determine the role and importance of glutathionylation in photosynthetic organisms, we developed a method based on proteomics to identify glutathionylated proteins in vivo in Chlamydomonas. 25 targets have been identified, mainly chloroplastic, mostly involved in photosynthesis and in response to oxidative stress. 3 targets were confirmed in vitro: HSP70B, chloroplastic 2-cys peroxiredoxin and isocitrate lyase. In non-photosynthetic organisms, it has been demonstrated that glutaredoxins (GRX) can catalyze efficiently deglutathionylation. Thus, we biochemically characterized 2 GRX from Chlamydomonas. GRX1, a cytosolic CPYC GRX, exhibits classical GRX activities, whereas GRX3, a chloroplastic CGFS GRX, exhibits no activity in these assays. Further analysis demonstrated that GRX3 has an atypically low redox potential close to that of TRX. The chloroplastic system of TRX reduction was able to reduce GRX3, which then can catalyze very efficiently A4-GAPDH deglutathionylation.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Les premiers peuplements du nord du Pérou (l'apport de la technologie lithique à la définition des ensembles culturels et de leurs relations, à la fin du Pléistocène et à l'Holocène ancien et moyen)

D'après nos connaissances actuelles, les premiers groupes humains à avoir peuplé le nord du Pérou sont arrivés vers 13 5OO cal BP: ils ont laissé des traces nombreuses au cours des millénaires suivants (période appelée Précéramique ancien ), traces dont les interprétations divergent encore. Dans ce travail, nous nous sommes attachée à étudier les industries lithiques de ces peuples, afin de mieux les caractériser. Il s'agit d'une étude transversale, parallèle et contrastée, prenant en compte la bande côtière et la cordillère des Andes, tandis que l'Amazonie apparaît encore à ce jour comme un territoire désert. La côte est relativement bien connue, mais dans les Andes les sites sont rares et les études ont été peu nombreuses. Dans ces espaces, les vestiges témoignent d'ensembles culturels divers, comprenant pointes en queue de poisson, Paijanien, industries unifaciales et assemblages montagnards encore mal définis. C'est au travers d'études typo-technologiques des industries lithiques exhumées (de l'ensemble des chaînes opératoires en présence) et d'une étude raisonnée de leurs contextes que nous avons tenté d'éclaircir les chronologies, les étendues géographiques et les relations potentielles existant entre ces complexes et traditions. Ces aspects ont été abordés tout en prenant en compte les dernières données disponibles sur l'évolution climatique et environnementale régionale depuis la fin du Pléistocène. La mise en parallèle de ces données avec d'autres informations extérieures à notre région d'étude, dans les Andes et au-delà, nous a finalement permis d'aborder la question du peuplement des Andes septentrionales et d'en proposer une modélisation.PARIS1-BU Pierre Mendès-France (751132102) / SudocSudocFranceF

Reactive oxygen intermediates produced by photosynthetic electron transport are enhanced in short-day grown plants

AbstractLeaves of tobacco plants grown in short days (8h light) generate more reactive oxygen species in the light than leaves of plants grown in long days (16h light). A two fold higher level of superoxide production was observed even in isolated thylakoids from short day plants. By using specific inhibitors of photosystem II and of the cytochrome b6f complex, the site of O2 reduction could be assigned to photosystem I. The higher rate of O2 reduction led to the formation of a higher proton gradient in thylakoids from short day plants. In the presence of an uncoupler, the differences in O2 reduction between thylakoids from short day and long day plants were abolished. The pigment content and the protein content of the major protein complexes of the photosynthetic electron transport chain were unaffected by the growth condition. Addition of NADPH, but not of NADH, to coupled thylakoids from long day plants raised the level of superoxide production to the same level as observed in thylakoids from short day plants. The hypothesis is put forward that the binding of an unknown protein permits the higher rate of pseudocyclic electron flow in thylakoids from short-day grown plants and that this putative protein plays an important role in changing the proportions of linear, cyclic and pseudocyclic electron transport in favour of pseudocyclic electron transport. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Articifical

Logical Thinking and Critical Understanding in Translation

International audienceINTRODUCTION: The objective of this study was to assess natural microbial agents, history and risk factors for total joint arthroplasty (TJA) infections in patients receiving tumor necrosis factor (TNF)α-blockers, through the French RATIO registry and a case-control study. METHODS: Cases were TJA infections during TNFα-blocker treatments. Each case was compared to two controls (with TJA and TNFα-blocker therapy, but without TJA infection) matched on age (±15 years), TJA localization, type of rheumatic disorder and disease duration (±15 years). Statistical analyses included univariate and multivariate analyses with conditional logistic regression. RESULTS: In the 20 cases (18 rheumatoid arthritis), TJA infection concerned principally the knee (n = 12, 60%) and the hip (n = 5, 25%). Staphylococcus was the more frequent microorganism involved (n = 15, 75%). Four patients (20%) were hospitalized in an intensive care unit and two died from infection. Eight cases (40%) versus 5 controls (13%) had undergone primary TJA or TJA revision for the joint subsequently infected during the last year (P = 0.03). Of these procedures, 5 cases versus 1 control were performed without withdrawing TNFα-blockers (P = 0.08). In multivariate analysis, predictors of infection were primary TJA or TJA revision for the joint subsequently infected within the last year (odds ratio, OR = 88.3; 95%CI 1.1-7,071.6; P = 0.04) and increased daily steroid intake (OR = 5.0 per 5 mg/d increase; 1.1-21.6; P = 0.03). Case-control comparisons showed similar distribution between TNFα-blockers (P = 0.70). CONCLUSIONS: In patients receiving TNFα-blockers, TJA infection is rare but potentially severe. Important risk factors are primary TJA or TJA revision within the last year, particularly when TNFα-blockers are not interrupted before surgery, and the daily steroid intake

Crystal Structure of Chloroplastic Thioredoxin f2 from <i>Chlamydomonas reinhardtii</i> Reveals Distinct Surface Properties

Protein disulfide reduction by thioredoxins (TRXs) controls the conformation of enzyme active sites and their multimeric complex formation. TRXs are small oxidoreductases that are broadly conserved in all living organisms. In photosynthetic eukaryotes, TRXs form a large multigenic family, and they have been classified in different types: f, m, x, y, and z types are chloroplastic, while o and h types are located in mitochondria and cytosol. In the model unicellular alga Chlamydomonas reinhardtii, the TRX family contains seven types, with f- and h-types represented by two isozymes. Type-f TRXs interact specifically with targets in the chloroplast, controlling photosynthetic carbon fixation by the Calvin&#8315;Benson cycle. We solved the crystal structures of TRX f2 and TRX h1 from C. reinhardtii. The systematic comparison of their atomic features revealed a specific conserved electropositive crown around the active site of TRX f, complementary to the electronegative surface of their targets. We postulate that this surface provides specificity to each type of TRX

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii: kinetics and specificity in deglutathionylation reactions.

International audienceProtein deglutathionylation is mainly catalyzed by glutaredoxins (GRXs). We have analyzed the biochemical properties of four of the six different GRXs of Chlamydomonas reinhardtii. Kinetic parameters were determined for disulfide and dehydroascorbate reduction but also for deglutathionylation of artificial and protein substrates. The results indicate that GRXs exhibit striking differences in their catalytic properties, mainly linked to the class of GRX considered but also to the pK(a) of the N-terminal catalytic cysteine. Furthermore, glutathionylated proteins were found to exhibit distinct reactivities with GRXs. These results suggest that glutathionylation may allow a fine tuning of cell metabolism under stress conditions