Photoaffinity labeling of the azidoatrazine receptor site in reaction centers of Rhodopseudomonas sphaeroides

AbstractPhotoaffinity labeling of photosynthetic reaction centers of Rhodopseudomonas sphaeroides by the herbicide inhibitor, azido[14C]atrazine, occurs principally on the L-subunit. The specificity of labeling is greater at 77 than at 295 K. Kinetic studies of charge recombination in reaction centers indicate competition between azidoatrazine and ubiquinone-1 (Q-1) for binding to the reaction center. This competition occurs through the L-subunit binding site, as increasing concentrations of Q-1 decrease azido[14C]atrazine labeling of this site. It is proposed that the inhibitor binding site, predominantly on the L-subunit, and the secondary quinone binding site on the M-subunit, are adjacent so that there is partial overlap by one molecule of the domain occupied by the other

Biogenesis of cytochrome b6 in photosynthetic membranes

In chloroplasts, binding of a c′-heme to cytochrome b6 on the stromal side of the thylakoid membranes requires a specific mechanism distinct from the one at work for c-heme binding to cytochromes f and c6 on the lumenal side of membranes. Here, we show that the major protein components of this pathway, the CCBs, are bona fide transmembrane proteins. We demonstrate their association in a series of hetero-oligomeric complexes, some of which interact transiently with cytochrome b6 in the process of heme delivery to the apoprotein. In addition, we provide preliminary evidence for functional assembly of cytochrome b6f complexes even in the absence of c′-heme binding to cytochrome b6. Finally, we present a sequential model for apo- to holo-cytochrome b6 maturation integrated within the assembly pathway of b6f complexes in the thylakoid membranes

Canadian guidelines for clinical practice: an analysis of their quality and relevance to the care of adults with comorbidity

<p>Abstract</p> <p>Background</p> <p>Clinical guidelines have been the subject of much criticism in primary care literature partly due to potential conflicts in their implementation among patients with multiple chronic conditions. We assessed the relevance of selected Canadian clinical guidelines on chronic diseases for patients with comorbidity and examined their quality.</p> <p>Methods</p> <p>We selected 16 chronic medical conditions according to their frequency of occurrence, complexity of treatment, and pertinence to primary care. Recent Canadian clinical guidelines (2004 - 2009) on these conditions, published in English or French, were retrieved. We assessed guideline relevance to the care of patients with comorbidity with a tool developed by Boyd and colleagues. Quality was assessed using the Appraisal of Guidelines Research and Evaluation (AGREE) instrument.</p> <p>Results</p> <p>Regarding relevance, 56.2% of guidelines addressed treatment for patients with multiple chronic conditions and 18.8% addressed the issue for older patients. Fifteen guidelines (93.8%) included specific recommendations for patients with one concurrent condition; only three guidelines (18.8%) addressed specific recommendations for patients with two comorbid conditions and one for more than two concurrent comorbid conditions. Quality of the evaluated guidelines was good to very good in four out of the six domains measured using the AGREE instrument. The domains with lower mean scores were Stakeholder Involvement and Applicability.</p> <p>Conclusions</p> <p>The quality of the Canadian guidelines examined is generally good, yet their relevance for patients with two or more chronic conditions is very limited and there is room for improvement in this respect.</p

Light-driven processes: key players of the functional biodiversity in microalgae

International audienc

Generic pregabalin : current situation and implications for health authorities, generics and biosimilars manufacturers in the future

The manufacturer of pregabalin has a second use patent covering prescribing for neuropathic pain: its principal indication. The manufacturer has threatened legal action in the UK if generic pregabalin rather than Lyrica is prescribed for this indication. No problems exist for practitioners who prescribe pregabalin for epilepsy or generalized anxiety disorder. This has serious implications for health authorities. In Germany, however, generics could be legally prescribed for any approved indication once one indication loses its patent. We aim to establish the current situation with pregabalin among principally European countries. Personnel from 33 regional and national health authorities mainly from Europe, and nine from universities across Europe working as advisers to health authorities or with insight into their activities, were surveyed regarding four specific questions via email to shed light on the current situation with Lyrica and pregabalin in their country. The information collated from each country was subsequently checked for accuracy with each co-author by email and face-to-face contact and collated into five tables. The scenarios ranged from extending the patent life of Lyrica (e.g. France), endorsing the prescribing of Lyrica for neuropathic pain (e.g. Catalonia and South Korea), and current prescribing of pregabablin for all indications (e.g. Serbia and Germany). Little activity has taken place in European countries in which generic pregabalin is not yet reimbursed. The availability of generic pregabalin has prompted a number of different activities to be undertaken among the 33 countries and regions surveyed. The situation in Serbia and the historic situation in Germany provide examples of ways to maximize savings once a product loses its patent for at least one indication

Une approche génétique de recherche de suppresseur pour l étude de la biogenèse, du contrôle qualité et de la fonction des complexes photosynthétiques chez Chlamydomonas reinhardtii

Le cytochrome b6f est un complexe majeur de la chaîne photosynthétique oxygénique de par son activité quinol:plastocyanine oxydoréductase, qui contribue à la formation d ATP via un transfert d électrons couplé à un transfert de protons. La présence d un hème c particulier lié par une seule liaison covalente, l hème ci, au sein du site de réduction de quinone Qi du cytochrome b6f constitue une différence notable en comparaison avec son homologue de la chaîne respiratoire, le cytochrome bc1. Un cytochrome b6f dépourvu d hème ci est dégradé, sa faible accumulation ne permet pas une croissance photosynthétique. Cette observation a donné lieu à une recherche de suppresseurs permettant une plus grande accumulation de cytochrome b6f dont la fonction même altérée, serait suffisante pour assurer une croissance photosynthétique. Cette approche génétique de recherche de suppresseur a été entreprise chez Chlamydomonas reinhardtii. Ce travail de thèse a permis l isolation et la caractérisation d un mutant de la protéase FtsH1 (mutation R420C qui affecterait l activité ATPasique). Le mutant ftsh1-1 s est révélé être un outil puissant pour l étude fonctionnelle de complexes mutés autrement dégradés. Une approche multidisciplinaire combinant expériences de génétique, biochimie, physiologie et biophysique a démontré notamment que : (i) le mutant QiKO, dont le complexe b6f est dépourvu des hèmes bh et ci, peut pousser de manière phototrophique malgré un Q-cycle cassé, (ii) l absence d hème ci lié covalemment, pour le mutant Rccb2, génère une photosensibilité exacerbée en présence d oxygène, ce qui sous-tend un rôle pour l hème ci dans un environnement riche en oxygène, (iii) la protéase FtsH exerce un contrôle qualité global des complexes majeurs photosynthétiques.Central in oxygenic photosynthesis, the cytochrome b6f complex, couples electron transfer to proton translocation across the thylakoid membrane via its quinol:plastocyanin oxidoreductase activity, contributing to ATP formation. Cytochrome b6f complex differs from its respiratory homolog, the bc1 complex, by the presence of an additional heme, heme ci located within the quinone reduction site Qi and attached by a unique thioether bond. Mutants lacking heme ci show low accumulation of partially functional b6f complex and, hence, cannot grow phototrophically. This grounded a screen for suppressor mutations that would restore higher accumulation of b6f complexes whose function, even if compromised, would sustain phototrophic growth.The genetic suppressor approach undertook in Chlamydomonas reinhardtii during this PhD thesis led to the isolation and characterisation of the ftsh1-1 protease mutant (mutation R420C which should affect ATP hydrolysis). The mutant ftsh1-1 proved to be a versatile tool for the functional study of otherwise degraded proteins. The combination of genetic, biochemical, physiological and biophysical experiments demonstrated notably that: (i) a QiKO mutant, whose b6f complexes are devoid of both bh and ci hemes, can grow phototrophically despite a broken Q-cycle, (ii) the absence of covalently bound heme ci, in the Rccb2 mutant, triggers photosensivity enhanced in the presence of O2 supporting a role for heme ci in oxygen rich environment, (iii) FtsH is involved in the maintenance of the main photosynthetic complexes.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Les protéines CCB définissent une nouvelle voie de biogenèse des cytochromes de type c

Les cythochromes C sont des hémoprotéines impliquées dans de nombreux processus biologiques et notamment dans le transfert d électrons au sein des membranes transductrices d énergie. La biogénèse des cytochromes C requiert la fixation convalente du cofacteur hémique qui, in vivo, est assistée par des ensembles de protéines formant au moins trois systèmes distincts suivant les organismes ou les compartiments subcellulaires étudiés. Le complexe cytochrome B6f transfère des électrons du transporteur hydrosoluble entre les deux photosystèmes, tout en construisant un gradient transmembranaire de protons par leur translocation depuis le côté négatif vers le côté positif de la membrane photosynthétique. L une de ses sous-unités , le cytochrome b6 contient trois cofacteurs hémiques dont un hème de type C , l hème Ci.. Chez Chlamydomonas, la biogenèse du cytochrome B6 requiert un ensemble de protéines nommées CCB, spécifiquement impliquées dans la liaison covalente de l hème Ci. Nous avons identifié les gènes et caractérisés les quatre protéines CCB connues à ce jour. Pour comprendre l implication du système IV dans la biogenèse du cytochrome B6, nous avons caractérisé les interactions entre les cinq protéines mis en jeu. L apocytochrome B6 est tout d abord partiellement replié suite à l insertion supposée spontanée de ces deux hèmes B. La protéine est ensuite prise en charge par CCBI, puis probablement transféré à CCB3 qui recrute un hétérodimère stable formé des deux protéines homologues CCB2 et CCB3. Ces trois derniers composants du système IV constituent probablement le complexe de liaison de l hème Ci sur le cytochrome.Cytochrome C are heme proteins involved in numerous biological processes, notably in electron transfer that occurs in energy transducing membranes. Cytochrome C biogenesis requires covalent attachment of the heme that, in vivo, is assisted by several groups of proteins classified in three different systems depending of the subcellular compartment under study. Cytochrome B6f complex transfers electrons from a lipid soluble transporter to a water soluble protein transporter between the two photosystems while building up a transmembrane proton gradient by translocating protons from the negative side to the positive of the photosynthetic membrane.Cytochrome B6 one the eight subunits of the complex contains tree hemes, two b-types and on c -type, the heme Ci. In Chlamydomonas, cytochrome B6 biogenesis requires a set of specialized proteins, named CCB to ensure the covalent binding of heme Ci. We identified the genes and characterized the four CCB proteins currently know. In order to understand the role of system IV in cytochrome B6 biogenesis, we have characterized the interactions between the four CCB and their substrat. Cythochrome B6 is first partially folded by the spontaneous insertion of the two b-type hemes. The proteins is then chaperoned by CCB1, probably transferred to CCb3, which recruits the stable heterodimer formed by CCB2 and CCB4. These latter three proteins represent probably the heme Ci ligation complex of the system IV.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Thylakoid targeting of Tat passenger proteins shows no ΔpH dependence in vivo

The Tat pathway is a major route for protein export in prokaryotes and for protein targeting to thylakoids in chloroplasts. Based on in vitro studies, protein translocation through this pathway is thought to be strictly dependent on a transmembrane ΔpH. In this paper, we assess the ΔpH sensitivity of the Tat pathway in vivo. Using Chlamydomonas reinhardtii, we observed changes in the efficiency of thylakoid targeting in vivo by mutating the Tat signal of the Rieske protein. We then employed two endogenous pH probes located on the lumen side of the thylakoid membranes to estimate spectroscopically the ΔpH in vivo. Using experimental conditions in which the trans-thylakoid ΔpH was almost zero, we found no evidence for a ΔpH dependence of the Tat pathway in vivo. We confirmed this observation in higher plants using attached barley leaves. We conclude that the Tat pathway does not require a ΔpH under physiological conditions, but becomes ΔpH sensitive when probed in vitro/in organello because of the loss of some critical intracellular factors