Synthèse chimio-enzymatique de dérivés de laminari-oligosaccharides,<br />et leur utilisation biochimique

?-(1,3)-glucans are found in a wide range of plants and have been recognised for their immunostimulating activities in mammals, as well as for their role in plant defence system. The first chapter of this manuscript is a short review on glycoside-hydrolases and, particularly on glycosynthases. Glucan-binding proteins, enzymes implied in ?-glucan recognition, are also introduced, as well as the knowledge acquired on ?-(1,3)-glucans and ?-(1,3)-gluco-oligosaccharides. The second chapter describes the work done in the CERMAV (Grenoble), in order to obtain the necessary tools to study ?-(1,3)-glucans and ?-(1,3)-gluco-oligosaccharides action modes. The synthesis of linear ?-(1,3)-gluco-oligosaccharides, of controled DP, using the ?-(1,3)-glucosynthase GII E231G, and the synthesis of a fluorogenic substrate for endo-?-(1,3)-glucanase activity study, are described. The third chapter is about ?-glucan-binding proteins, and describes the work done in the botanical institute of the LMU (Munich), for the european research project SACC-SIG-NET (n°HPRN-CT-2202-00251). This research deals, on one hand, with the study of the Glycine max ?-glucan-binding protein, which allowed to determined for the first time the hydrolysis molecular mechanism of a glycoside-hydrolase belonging to the GH-81 family. On the other hand, a cloning work on the Gbps gene family of Medicago truncatula, a model plant for the Fabaceae, is presented.Les ?-(1,3)-glucanes se rencontrent chez diverses espèces végétales et ont été reconnus pour leurs activités immunostimulantes chez les mammifères, ainsi que leur rôle dans le système de défense des plantes. Le premier chapitre de ce manuscrit est un rappel bibliographique sur les glycoside-hydrolases et, particulièrement sur les glycosynthases, et présente également une introduction aux "glucan-binding proteins", enzymes impliquées dans la reconnaissance des ?-glucanes, ainsi qu'un état des lieux des connaissances acquises sur les ?-(1,3)-glucanes et ?-(1,3)-gluco-oligosaccharides. Le deuxième chapitre décrit le travail réalisé au CERMAV (Grenoble), afin de disposer des outils nécessaires à l'étude des mécanismes d'action des ?-(1,3)-glucanes et des ?-(1,3)-gluco-oligosaccharides. Une méthode de synthèse de ?-(1,3)-gluco-oligosaccharides linéaires, de DP contrôlé, à l'aide de la ?-(1,3)-glucosynthase GII E231G, ainsi que la synthèse d'un substrat fluorescent destiné au dosage d'activité endo-?-(1,3)-glucanase, ont été mises au point. Le troisième chapitre traite des ?-glucan-binding proteins, et décrit le travail effectué à l'institut botanique de la LMU (Munich), dans le cadre du projet de recherche européen SACC-SIG-NET (n°HPRN-CT-2202-00251). Cette recherche concerne d'une part, l'étude de la ?-glucan-binding protein de Glycine max, et a permis de déterminer pour la première fois le mécanisme moléculaire d'hydrolyse d'une glycoside-hydrolase appartenant à la famille GH-81. D'autre part, un travail de clonage de la famille de gènes Gbps de Medicago truncatula, plante modèle pour les Fabacées, a été réalisé

Synthèse chimio-enzymatique de dérivés de laminari-oligosaccharides, et leur utilisation biochimique

Les b-(1,3)-glucanes se rencontrent chez diverses espèces végétales et ont été reconnus pour leurs activités immunostimulantes chez les mammifères, ainsi que leur rôle dans le système de défense deb plantes. Le premier chapitre de ce manuscrit est un rappel bibliographique sur les glycosidehydrolases et, particulièrement sur les glycosynthases, et présente également une introduction aux "glucan-binding proteins", enzymes impliquées dans la reconnaissance des b-glucanes, ainsi qu'un état des lieux des connaissances acquises sur les b-(1,3)-glucanes et b-(1,3)-gluco-oligosaccharides. Le deuxième chapitre décrit le travail réalisé au CERMA V (Grenoble), afin de disposer des outils nécessaires à l'étude des mécanismes d'action des b-(1,3)-glucanes et des b-(1,3)-glucooligosaccharides. Une méthode de synthèse de b-(1,3)-gluco-oligosaccharides linéaires, de DP contrôlé, à l'aide de la b-(1,3)-glucosynthase GU E231G, ainsi que la synthèse d'un substrat fluorescent destiné au dosage d'activité endo-b-(1,3)-glucanase, ont été mises au point. Le troisième chapitre traite des b-glucan-binding proteins, et décrit le travail effectué à l'institut botanique de la LMU (Munich), dans le cadre du projet de recherche européen SACC-SIG-NET (nHPRN-CT-2202 -00251). Cette recherche concerne d'une part, l'étude de la b-glucan-binding protein de Glycine max, et a permis de déterminer pour la première fois le mécanisme moléculaire d'hydrolyse d'une glycoside-hydrolase appartenant à la famille GH-81. D'autre part, un travail de clonage de la famille de gènes Gbps de Medicago truncatula, plante modèle pour les Fabacées, a été réalisé.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Reconstitution de communautés microbiennes complexes pour l'inhibition de Listeria monocytogenes à la surface de fromages à pâte pressée non cuite

L'objectif était de déterminer si la diversité des espèces microbiennes peut contribuer à la maîtrise de Listeria monocytogenes à la surface de fromage au lait cru. La stratégie reposait sur le criblage de communautés microbiennes de croûtes de fromage St Nectaire fermiers puis la reconstitution de communautés de composition plus simplifiée. Dix consortiums microbiens naturellement présents à la surface de ces fromages au lait cru sur trente quatre testés protégeaient contre L. monocytogenes. Le consortium de croûte le plus inhibiteur, composé de 8 espèces de bactéries lactiques dont Brochothrix thermosphacta, Marinilactobacillus psychrotolerans et Carnobacterium mobile peu fréquentes dans les produits laitiers, 12 espèces de bactéries à Gram positif et catalase positive, 10 espèces de bactéries à Gram négatif, 4 espèces de levures et 3 moisissures, était difficile à reconstituer. Par méthodes cultures dépendantes et indépendantes (Single Strand Conformation Polymorphism) il a été montré qu'au cours d'affinage les profils bactériens du consortium naturel était plus divers que ceux des consortiums reconstitués. Néanmoins, un consortium simplifié composés de flores cultivables sur un milieu "Brain Heart Infusion" et caractérisé par la présence de bactéries à Gram négatif et la dominance, notamment en fin d'affinage, d'espèces halophiles Brochotrix, Carnobacterium et Marinilactibacillus, était presque aussi inhibiteur que le consortium complexe. L'inhibition par ce consortium serait essentiellement associée à la production d'acide lactique en début d'affinage et d'acide acétique en fin d'affinage. Elle pourrait être contrecarrée par une consommation de lastate par les levuresCLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

Low-temperature alteration of monazite: Fluid mediated coupled dissolution-precipitation, irradiation damage, and disturbance of the U-Pb and Th-Pb chronometers

International audienceLow-temperature alteration of monazite is documented in three centimeter-sized monazite crystals from Norway (Arendal), Madagascar (Ambato), and Sri Lanka. The three crystals have different chemical compositions, especially in their U, Th, Y and Pb contents and have 208Pb/232Th ages ranging from 491 to 900 Ma. Optical microscope (OM), Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) images and electron microprobe analyses (EPMA) show that all three preserve a similar patchy texture. This texture is interpreted as resulting from an alteration reaction in which unaltered monazite (Mnz1) reacts to form a secondary, Th-U(Y)-depleted, high-Th/U, monazite (Mnz2) accompanied by thorite/huttonite (ThSiO4), thorianite (ThO2) and xenotime (YPO4), the proportions of which are dependent upon the initial composition of the monazite (Mnz1). Images reveal variably intense internal fracturing, with cracks filled with Th-rich ± Fe-rich phases. Monazite-xenotime thermometry demonstrates that the pristine monazites (Mnz1) interacted with a low-temperature fluid. The alteration process is interpreted to follow a mechanism of fluid-mediated coupled dissolution-precipitation. Chemical dating with the electron microprobe shows no Th-U-Pb age differences between primary and secondary monazites, except in the case of the Ambato monazite, in which altered domains yield older (750 Ma) apparent ages than the pristine Mnz1 domains. U-Pb and Th-Pb isotope dating using LA-ICP-MS yields ages consistent with electron probe dates for pristine Mnz1 zones. However, disturbance of these systems in the altered monazite domains leads to variable age results for these, depending on sample. In the case of Sri Lanka and Arendal, only 208Pb/232Th dates provide a reasonable estimate of the age of alteration, which are constrained to be 450 and 864 Ma, respectively. U/Pb systems are disturbed due to common Pb contamination (up to 40%) and U fractionation relative to Th during alteration, responsible for depletion of U in altered monazites (and increase of Th/U). In contrast, for the Ambato monazite, both the U-Pb and Th-Pb systems were affected and yield inconsistent older dates for altered zones. This is attributed to significant common Pb contamination (up to 80%), which affects all Pb isotopes and explains why electron probe ages are erroneous. Th-U-silicate contamination during measurement, resulting from the presence of a numerous nano-phases and nano-fractures filled with Th-U-silicates that are visible only under TEM, also contributes to the anomalously old ages for these disturbed (Mnz2) domains. These results demonstrate the important role of radiation damage effects, in particular swelling-induced fracturing, and the essential role of porosity and cracks, which allow fluid (charged with elements) migration through monazite during low-temperature alteration

Milk fat composition modifies the texture and appearance of Cantal-type cheeses but not their flavor

In Press, Corrected ProofAlthough the effects of cow diet on cheese sensory properties have been well documented, the putative interactions between the biochemical and microbial milk components and their respective roles in the development of the sensory properties of cheeses have yet to be explored in depth. The aim of this study was to evaluate the specific contribution of milk fat composition to the formation of cheese sensory properties. Two creams with different fat compositions were obtained from cows fed either pasture or maize silage. Cheeses were manufactured from the same skim milk (identical chemical and microbial composition) with either the pasture- or maize silage-origin pasteurized cream added. The gross composition and microbial composition of milks did not vary with cream origin. In milks and cheeses, the fatty acid (FA) profiles were modified by the origin of the cream. The concentrations of C18:0 and unsaturated FA such as cis-9 C18:1, trans-11 C18:1, C18:3n-3, total conjugated linoleic acids, and mono- and polyunsaturated FA were higher in milks and cheeses with the pasture-origin cream than in those with the maize-origin cream. In contrast, the maize milks and cheeses had higher concentrations of short- and medium-chain saturated FA, C16:0, and C18:2n-6. The level of lipolysis was 11% in the cheese rind and only 0.30% in the cheese core. The rind of pasture cheeses had a higher concentration of free C18:0 and C18:3n-3 and a lower concentration of free C14:0 and free C16:0 than the rind of maize cheeses. The levels of major microbial groups were similar in pasture and maize cheeses at different stages of ripening. The pasture cheeses had a more elastic and creamier texture, a yellower color, and a thinner rind than the maize cheeses, but the odor and aroma of cheeses were not affected by the origin of the cream, despite a few modifications in the balance of volatile compounds from FA catabolism. Based on these results, we conclude that milk fat composition modulated by cow diet had a direct role in the texture of the cheese but no effect on flavor. The high degree of lipolysis in cheese rind, along with the higher concentration of long-chain unsaturated free FA in pasture cheeses may be responsible for antimicrobial activity, which could explain differences in the appearance of cheese rind

Catalytic properties of the bifunctional soybean β-glucan-binding protein, a member of family 81 glycoside hydrolases

International audienc

A chemoenzymatic route to conjugatable beta(1 -&gt; 3)-glucan oligosaccharides

3II-O-Allyl-α-laminaribiosyl fluoride was prepared as a key synthon for the enzymatic synthesis of β(1→3)-glucan oligosaccharides, catalyzed by a mutated β(1→3)-glucanase (E231G) from barley (Hordeum vulgare L.). A strategy was developed for enzymatic elongation of the β(1→3)-glucan chain from the reducing end, using a single glucoside acceptor. When β-glucoside phenyl disulfide was used as the acceptor, this methodology generated laminari-oligosaccharides conjugatable at both their reducing and non-reducing ends.Emilie Montel, Maria Hrmova, Geoffrey B. Fincher, Hugues Driguez and Sylvain Cotta

Endospanin-2 enhances skeletal muscle energy metabolism and running endurance capacity.

Metabolic stresses such as dietary energy restriction or physical activity exert beneficial metabolic effects. In the liver, endospanin-1 and endospanin-2 cooperatively modulate calorie restriction-mediated (CR-mediated) liver adaptations by controlling growth hormone sensitivity. Since we found CR to induce endospanin protein expression in skeletal muscle, we investigated their role in this tissue. In vivo and in vitro endospanin-2 triggers ERK phosphorylation in skeletal muscle through an autophagy-dependent pathway. Furthermore, endospanin-2, but not endospanin-1, overexpression decreases muscle mitochondrial ROS production, induces fast-to-slow fiber-type switch, increases skeletal muscle glycogen content, and improves glucose homeostasis, ultimately promoting running endurance capacity. In line, endospanin-2-/- mice display higher lipid peroxidation levels, increased mitochondrial ROS production under mitochondrial stress, decreased ERK phosphorylation, and reduced endurance capacity. In conclusion, our results identify endospanin-2 as a potentially novel player in skeletal muscle metabolism, plasticity, and function

p53 activation during ribosome biogenesis regulates normal erythroid differentiation

International audienceThe role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis shows that ribosome biogenesis is abruptly interrupted by the drop of rDNA transcription and the collapse of ribosomal protein neo-synthesis. Its premature arrest by RNA polI inhibitor, CX-5461 targets the proliferation of immature erythroblasts. We also show that p53 is activated spontaneously or in response to CX-5461 concomitantly to ribosome biogenesis arrest, and drives a transcriptional program in which genes involved in cell cycle arrest, negative regulation of apoptosis and DNA damage response were upregulated. RNA polI transcriptional stress results in nucleolar disruption and activation of ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation are crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold of ribosome biogenesis down-regulation could be prematurely reached and together with pathological p53 activation prevents a normal expansion of erythroid progenitors