Concentration des lipides issus de Parachlorella kessleri en voie humide : effet de la destruction cellulaire sur la performance du procédé de fractionnement par membrane

International audienceLe bioraffinage de microalgues a pour but de produire des biomolécules (lipides, protéines, polysaccharides, pigments, etc.) pour divers secteurs industriels tels que l'alimentation, la cosmétique, la pharmacie, l'énergie ou la chimie verte. Le défi dans ce domaine consiste à développer une cascade de procédés doux et efficaces afin de garantir l'intégrité des molécules fragiles ainsi qu'une production économe en énergie et respectueuse de l'environnement. Pour la production à grande échelle, un bioraffinage en voie humide a été proposé afin de limiter les coûts de séchage, la dégradation de molécules thermolabiles et l'utilisation de solvant. Les différentes étapes proposées sont : la récolte de la biomasse, le broyage des cellules pour libérer les composés intracellulaires d'intérêts dans la phase aqueuse, puis leur fractionnement par procédé membranaire et leur purification. Le verrou au développement de ce procédé est le manque de maîtrise de l'impact des procédés de déconstruction cellulaire sur les procédés de fractionnement. Les travaux présentés ici ont pour but d'étudier l'impact du broyage à billes sur les performances du procédé de filtration membranaire, pour séparer les lipides (triglycérides, phospholipides et glycolipides) des composés hydrosolubles (protéines et sucres) produits lors d'une culture carencée de Parachlorella kessleri. La quantité de molécules cibles libérées (lipides, protéines, sucres) dans la phase aqueuse, l'état de ces molécules (possible dégradation des composés cibles en raison de l'activité enzymatique ou de l'oxydation) et leur organisation dans le mélange complexe dépendent fortement des conditions de broyage et de clarification de la suspension. Ces paramètres déterminent également la qualité de la séparation. Des filtrations membranaires de surnageants issus de suspensions obtenues dans diverses conditions de broyage de Parachlorella kessleri sont réalisées à l'aide d'une membrane préalablement sélectionnée. Les performances (flux, taux de rétention) sont comparées. Les conditions opératoires optimales sont enfin choisies, permettant le couplage entre le procédé de destruction cellulaire et le procédé de fractionnement par membrane, et la séparation des lipides et des composés hydrosolubles

Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP ‘core’. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions

Characterization of several biological activities of porcine interferon gamma

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Formation of tubules and giant vesicles from large multilamellar vesicles

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Control of Antagonistic Components of the Hedgehog Signaling Pathway by microRNAs in Drosophila

Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diverse cancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to gene transcription output. We previously showed that stability of antagonistic regulatory proteins, including the coreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected by Hh signaling activation. Here, we show that the level of these three proteins is also regulated by a microRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 3′-UTRs of smo, cos2, and fu mRNA decreases the levels of the three proteins and activates the pathway. Further, the loss of the microRNA cluster or of Dicer function modifies the 3′-UTR regulation of smo and cos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targets of microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates an hh-like phenotype, possibly due to dose compensation between the different antagonistic targets. This study reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs

Enzymatic hydrolysis studies of arabinogalactan-protein structure from Acacia gum: the self-similarity hypothesis of assembly from a common building block

Arabinogalactan (AG) and arabinogalactan-protein (AGP) fractions were treated enzymatically using several proteases in acidic (pH 4) and alkaline (pH 7) conditions in order to go deeper insight into the structure and conformations of the two main fractions of Acacia senegal gum. Endoproteinase Glu-C, pepsin and phosphatase acid were thus used in acidic conditions while subtilisin A, pronase, trypsin, papain and proteinase K were used in alkaline conditions to cleave protein moieties of the two fractions. Structures of AG and AGP were probed using HPSEC-MALLS, small angle neutron scattering and far-UV circular dichroism. Enzymes did not affect AG fraction structure whatever the pH conditions used, highlighting the inaccessibility of the peptide backbone and the remarkable stability of this fraction in acidic and alkaline conditions. This result was in agreement with the thin oblate ellipsoid model we previously identified for the AG fraction where the 43 amino-acid residues peptide sequence was supposed, based on spectroscopic methods, to be totally buried. Contrary to AG fraction, AGP protein component was therefore cleaved using enzymes in alkaline conditions, the absence of enzymatic efficiency in acidic conditions being probably ascribed to long range electrostatic repulsions occurring between negatively charged AGP and enzymes at pH 4. The decrease of AGP molecular weight after hydrolysis in alkaline conditions went from 1.79 x 10(6) gmol(-1) for control AGP to as low as 1.68 x 10(5) g mol(-1) for papain-treated AGP. The overall structure of the enzyme-treated AGPs was found to be surprisingly quite similar whatever the enzyme used and close, with however some subtle differences, to AG unit. A tri-axial ellipsoid conformation was found in enzyme-treated AGPs and the two main preferential distances identified in the pair distance distribution function would claim in favor of rod-like or elongated particles or alternatively would indicate the presence of two particles differing in dimensions. The secondary structures content of control and enzyme-treated AGPs were similar, highlighting both the high rigidity of the protein backbone and the overall symmetry of AGP. This conclusion was reinforced by the more compact structures found when AGP was intact compare to the more elongated structures found when AGP was enzymatically cleaved. Finally, the structural similarities found in enzyme-treated AGP together with the theoretical calculations to analytically probe the type of branching would suggest that AGP would be made of a self-similar assembly of two types of building blocks, the second being a five-fold repetition of the first one, for which palindromic amino acid sequence would ensure a self-ordering of carbohydrate moieties along the polypeptide chains. The cleavage would therefore lead to hydrolysed building blocks with similar secondary structures and conformations whatever the enzyme used

Bead milling disruption kinetics of microalgae: Process modeling, optimization and application to biomolecules recovery from Chlorella sorokiniana

International audienceIndustrial development of microalgae biomass valorization relies on process optimization and controlled scale-up. Both need robust modeling: (i) for biomass production and (ii) for integrated processes in the downstream processing (DSP). Cell disruption and primary fractionation are key steps in DSP. In this study, a kinetic model, including microalgal cell size distribution, was developed for Chlorella sorokiniana disruption in continuous bead milling. Glass beads of 0.4 mm size at impeller tip velocity of 14 m.s(-1) were used as optimal conditions for efficient cell disruption. These conditions allowed faster disruption of big cells than small ones. A modified expression of the Stress Number, including cell size effect, was then proposed and validated. Separation of starch, proteins and chlorophyll by mild centrifugation was studied as function of the disruption parameters. Low energy consumption conditions led to extreme comminution. An intermediate zone drew attention for allowing moderate energy consumption and efficient metabolites separation by centrifugation

Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog.

International audienceThe mechanisms involved in transduction of the Hedgehog (Hh) signal are of considerable interest to developmental and cancer biologists. Stabilization of the integral membrane protein Smoothened (Smo) at the plasma membrane is a crucial step in Hh signalling but the molecular events immediately downstream of Smo remain to be elucidated. We have shown previously that the transcriptional mediator Cubitus interruptus (Ci) is associated in a protein complex with at least two other proteins, the kinesin-like Costal2 (Cos2) and the serine-threonine kinase Fused (Fu). This protein complex governs the access of Ci to the nucleus. Here we show that, consequent on the stabilization of Smo, Cos2 and Fu are destabilized. Moreover, we find that the Cos2-Fu-Ci protein complex is associated with Smo in membrane fractions both in vitro and in vivo. We also show that Cos2 binding on Smo is necessary for the Hh-dependent dissociation of Ci from this complex. We propose that the association of the Cos2 protein complex with Smo at the plasma membrane controls the stability of the complex and allows Ci activation, eliciting its nuclear translocation

Dally and Notum regulate the switch between low and high level Hedgehog pathway signalling.

International audienceDuring development, secreted morphogens, such as Hedgehog (Hh), control cell fate and proliferation. Precise sensing of morphogen levels and dynamic cellular responses are required for morphogen-directed morphogenesis, yet the molecular mechanisms responsible are poorly understood. Several recent studies have suggested the involvement of a multi-protein Hh reception complex, and have hinted at an understated complexity in Hh sensing at the cell surface. We show here that the expression of the proteoglycan Dally in Hh-receiving cells in Drosophila is necessary for high but not low level pathway activity, independent of its requirement in Hh-producing cells. We demonstrate that Dally is necessary to sequester Hh at the cell surface and to promote Hh internalisation with its receptor. This internalisation depends on both the activity of the hydrolase Notum and the glycosyl-phosphatidyl-inositol (GPI) moiety of Dally, and indicates a departure from the role of the second glypican Dally-like in Hh signalling. Our data suggest that hydrolysis of the Dally-GPI by Notum provides a switch from low to high level signalling by promoting internalisation of the Hh-Patched ligand-receptor complex