GREBP, un nouveau facteur de transcription contrôlant l’expression de la guanylate cyclase A, récepteur de l’ANP, via l’élément de réponse au cGMP

La découverte du système des peptides natriurétiques (NP), au début des années 80, fut une découverte majeure qui révéla le rôle endocrinien du cœur. Les connaissances sur la relaxation vasculaire, la diurèse et la natriurèse provoquées par ce système ont évolué vers un niveau de complexité insoupçonné à cette époque. Nous savons à présent que les NP sont impliqués dans plusieurs autres mécanismes dont la prolifération cellulaire, l’apoptose, l’inhibition du système rénine-angiotensine-aldostérone (RAAS) et le métabolisme des adipocytes. Le métabolisme des lipides est maintenant devenu une cible de choix dans la lutte contre l’obésité. Cette condition aux proportions pandémiques est un facteur de risque majeur dans l’apparition de l’hypertension et du syndrome métabolique (MetS). La compréhension des mécanismes et des défauts de la voie des NP pourrait avoir un impact positif sur le contrôle du MetS et de l’hypertension. L’expression du récepteur des peptides natriuretiques de type 1 (NPR1/GCA) est contrôlée par plusieurs agents incluant son propre ligand, le peptide natriurétique de l’oreillette (ANP). La découverte d’une boucle de retro-inhibition, dans les années 90, a été un événement majeur dans le domaine des NP. En effet, suite à une stimulation à l’ANP, le NPR1/GCA peut inhiber l’activité transcriptionnelle de son propre gène par un mécanisme dépendant du cGMP. Notre groupe a identifié un élément cis-régulateur responsable de cette sensibilité au cGMP et mon projet consistait à identifier la ou les protéine(s) liant cet élément de réponse au cGMP (cGMP-RE). Nous avons identifié un clone liant le cGMP-RE en utilisant la technique du simple hybride chez la levure et une banque d’ADN complémentaire (ADNc) de rein humain. Ce clone provient d’un ADNc de 1083-bp dont le gène est localisé sur le chromosome 1 humain (1p33.36) et codant pour une protéine dont la fonction était inconnue jusqu’ici. Nous avons nommé cette nouvelle protéine GREBP en raison de sa fonction de cGMP Response Element Binding Protein. Des essais de liaison à l’ADN ont montré que cette protéine possède une affinité 18 fois plus élevée pour le cGMP-RE que le contrôle, tandis que des expériences de retard sur gel (EMSA) ont confirmé la spécificité des interactions protéine-ADN. De plus, l’immuno-précipitation de la chromatine (ChIP) a prouvé que GREBP lie le cGMP-RE dans des conditions physiologiques. La liaison de GREBP au cGMP-RE inhibe l’expression du gène rapporteur luciférase sous contrôle du promoteur de npr1/gca. L’inhibition de GREBP à l’aide d’ARN interférant active le promoteur de npr1/gca. Dans les cellules NCI-H295R, l’ANP stimule l’expression de grebp de 60% après seulement 3 heures et inhibe l’expression de npr1/gca de 30%. GREBP est une protéine nucléaire surtout exprimée dans le cœur et ayant le facteur eIF3F comme partenaire. Les variations nucléotidiques du gène sont plus fréquentes chez les patients hypertendus que chez des patients normotendus ou hypertendus souffrant de MetS. Nous rapportons ici l’existence d’un gène spécifique à l’humain qui agit comme répresseur transcriptionnel de npr1/gca et potentiellement impliqué dans le développement de l’hypertension.The natriuretic peptide (NP) system was a milestone discovery that revealed the endocrine role of the heart for the first time in the early 1980s. From its vasodilatory, natriuretic and diuretic actions, knowledge about this system has evolved to a degree of complexity unsuspected at that time. Now, through cGMP generation, NPs are involved in several other mechanisms, such as cell proliferation, apoptosis, renin-angiotensine-aldosterone system (RAAS) inhibition, and fat cell function. The latter point is of growing interest in lipid metabolism and has become an important issue in the fight against obesity. This pandemic condition is one of the main risk factors leading to hypertension development and metabolic syndrome (MetS) progression. Thus, understanding, at least in part, the lipid mobilization pathways controlled by NPs could have a positive impact in MetS management. As with hypertension, identifying defects in signaling pathways will certainly help to identify mechanisms implicated in lost sensitivity of the NP system. Natriuretic peptide receptor 1 (npr1/gca) expression is controlled by several agents including its own ligand, the atrial natriuretic peptide (ANP). A major finding in NPs field occured in the mid-90s when a mechanism involving a retro-inhibition loop was described. Indeed, after ANP stimulation, NPR1/GCA down-regulates the transcriptional activity of its gene via a cGMP-dependent mechanism. Since our group previously identified a cis-acting element responsible for this cGMP sensitivity, I proceeded to explore novel putative protein binding to the cGMP-response element (cGMP-RE). Using the yeast-one-hybrid technique with a human kidney cDNA library, we identified a strongly positive clone able to bind cGMP-RE. The clone was derived from a 1083-bp long cDNA of a gene of yet unknown function localized on human chromosome 1 (1p33.36). We named this new protein GREBP for cGMP-Response Element-Binding Protein. DNA-binding assays showed 18-fold higher cGMP-RE-binding capacity than the controls while electromobility shift assay (EMSA) indicated a specific binding for the cGMP-RE and chromatin immuno-precipitation (ChIP) confirmed the binding of GREBP to the element under physiological conditions. By acting on cGMP-RE, GREBP inhibited the activity of a luciferase-coupled NPR1 promoter construct. In H295R cells, ANP heightened GREBP expression by 60% after just 3 hours of treatment while inhibiting npr1/gca expression by 30%. Silencing GREBP with specific small interfering RNA increased the activity of the luciferase-coupled NPR1/GCA promoter and NPR1/GCA mRNA levels. GREBP is a nuclear protein mainly expressed in the heart and has the eIF3F factor as partner. Its nucleotide variations are more frequent in non-obese hypertensive patients than normotensive subjects or hypertensive patients suffering from MetS. We report here the existence of a human specific gene acting as a transcriptional repressor of npr1/gca gene that could be implicated in hypertension development

Large scale composting model

One way to treat the organic wastes accordingly to the environmental policies is to develop biological treatment like composting. Nevertheless, this development largely relies on the quality of the final product and as a consequence on the quality of the biological activity during the treatment. Favourable conditions (oxygen concentration, temperature and moisture content) in the waste bed largely contribute to the establishment of a good aerobic biological activity and guarantee the organic matter stabilisation with limitation and control of odorous and greenhouse effect gaseous emissions. Several approaches (0D biochemical reducing, see Pommier et al. 2007, effective 1D modelling coupling transport and biochemical) have been made to understand the behaviour of such systems. In this paper we will present a 2D numerical model using Darcy scale equations for heat and mass transport coupled with a biochemical reactive scheme. Then, we will solve that system (using experimental measurements on reactivity and transport coefficients) with a commercial code (COMSOL TM). The model described here is based on the biological model presented in Trémier et al 2005 coupled with an upscale transport model detailed in Hénon 2008 which takes into account the major components of the gas phase: N₂, O₂, CO₂ and also H₂O. This is a crucial point because of: - The reaction rate, depending on the moisture content (humidity comes from the initial condition of the sludge but also from the reactive scheme because reactions produce water), - heat content, very sensitive to the evaporation rate in the sludge. It has been shown in Pujol et al 2011 that the impact of drying could be important on the reactivity but also that the pseudo component air could not be sufficient to represent the drying in the sludge. The process studied was a closed reactor composting process (180 m³ rectangular box) with positive forced aeration. The air was blown from the bottom of the reactor, via two ventilation pipes. In the upper part of the reactor, air was sucked and led to a biofilter treatment system. The treated waste was a mixture of sewage sludge and bulking agent that was composted during four weeks without turning. Several informations were recorded during the treatment like temperature evolutions at different locations (see Henon et al. 2009 for more details about the temperature recording). We have validated this code by comparing the temperatures obtained through the simulations with those recorded during the experiments. After this step of validation and a discussion on final composition of the organic matter in the experiments compared to the ones estimated by simulations, we have used this numerical model as an optimization tool. Modifying the initial, boundary and operating conditions we have been able to determine the best conditions to this particular composting process. A whole set of conditions is discussed in the paper

Guide de lutilisation pédagogique des médias sociaux

Titre de l'écran-titre (visionné le 24 jan. 2013)Cette publication est mise à disposition selon les termes de la licence "Créative Commons". Vous êtes libres de l'utiliser, en tout ou en partie en citant l'auteur, vous ne pouvez pas l'utiliser pour un usage commercial et vous n'êtes pas autorisés à la modifier

F.A.R.O.G. FORUM, Vol. 2 No. 9

https://digitalcommons.library.umaine.edu/francoamericain_forum/1008/thumbnail.jp

France’s proposal for Guidelines about setting Maximum Residue Limits in honey

contribution to session I Regulatory issues Background: Honey is produced in an environment potentially polluted by different sources of contamination, so it is necessary to set Maximum Residue Limits (MRLs). These MRLs should be fixed as low as possible in relation to Good Agricultural Practices (GAPs). The guidance provided in this Draft Working Document gives advice on: • when and for what kind of active substance a MRL has to be set in honey • how to propose a temporary MRL for a given active substance • how to design, prepare and realise supervised residue trials when necessary Results: The proposed approach is based on using the available data before an active substance or product is registered, and is divided into several successive steps, represented in a global decision-making scheme. The MRL will be set depending on the results obtained at each different step. Besides, the applicants will have the choice between different methods to set a provisional MRL in preregistration. Conclusion: The initial proposal was a protocol on field residue trials proposed by Germany. The approach used in this guidance document proposes also other possibilities for fixing MRL without conducting systematically field trials. This proposition will be discussed at European level. Keywords: Regulation 396/2005, MRL, honey, plant protection produc