Etude de l'implication de la carnitine dans la tolérance aux stress abiotiques chez Arabidopsis thaliana

COMPIEGNE-BU (601592101) / SudocSudocFranceF

Effet de l'exposition in utero au diazépam sur la fonction respiratoire et la réponse à l'hypoxie du rat nouveau-né (impact sur les systémes GABAergiques et adénosinergiques)

Le diazépam (DZP) est une benzodiazépine thérapeutique qui potentialise l effet inhibiteur du GABA endogène lié aux récepteurs GABAA et qui peut être administrée à la femme enceinte. Le comportement du nouveau-né est perturbé par l exposition in utero au DZP. Notre objectif a été d apprécier les conséquences de cette exposition sur la fonction respiratoire et de déterminer les mécanismes impliqués chez le rat âgé de 0-2 jours. Des enregistrements in vivo par pléthysmographie chez l animal non contraint montrent que l exposition in utero au DZP modifie le pattern respiratoire en eupnée (diminution de la fréquence et augmentation du volume courant) mais pas la ventilation. Elle majore l effet dépresseur de l hypoxie alvéolaire sur le volume courant. Elle induit une augmentation de la fréquence de la commande respiratoire centrale, évaluée in vitro sur des préparations de tronc cérébral. Elle atténue les effets dépresseurs de l hypoxie tissulaire sur cette commande. La PCR quantitative en temps réel et une étude pharmacologique montrent selon les régions une régulation négative de l expression des gènes codant pour les sous-unités a1 et a2 du récepteur GABAA et/ou à une diminution de la réponse de ces récepteurs au DZP. De plus, l expression des récepteurs de l adénosine de type A1 et A2A et la réponse à leurs agonistes sont modifiées par l exposition au DZP. Enfin, l HPLC suggère une altération de la biosynthèse du glutamate et du GABA dans le tronc cérébral. Ces données montrent que l exposition prénatale au diazépam affecte des systèmes de neuromodulation qui ont un rôle majeur dans le contrôle respiratoire et que ses conséquences à plus long terme méritent d être évaluées.AMIENS-BU Santé (800212102) / SudocSudocFranceF

Consequences of prenatal exposure to diazepam on the respiratory parameters, respiratory network activity and gene expression of alpha1 and alpha2 subunits of GABA(A) receptor in newborn rat.

International audienceDiazepam (DZP) enhances GABA action at GABA(A) receptor. Chronic prenatal administration of DZP delays the appearance of neonatal reflexes. We examined whether maternal intake of DZP might affect respiratory control system in newborn rats (0-3 day-old). This study was conducted on unrestrained animals and medulla-spinal cord preparations. In addition, the level of expression of the genes encoding for the alpha1 and alpha2 subunits of the GABA(A) receptor was assessed by quantitative real-time RT-PCR. In rats exposed to DZP, the respiratory frequency was significantly lower and the tidal volume higher than in controls with no significant alteration of the minute ventilation. The recovery from moderate hypoxia was delayed compared to controls. The respiratory-like frequency of medullary spinal cord preparation from DZP-exposed neonates was higher than in the control group. Acute applications of DZP (1 microM) to these preparations increased respiratory-like frequency in both groups, but this facilitation was attenuated following prenatal DZP exposure. The present data indicate that prenatal exposure to DZP alters both eupneic breathing and the respiratory response to hypoxia. These effects might partly be ascribed to the down-regulation of the expression of genes encoding GABA(A) receptor subunits. On the other hand, the effects of DZP exposure on reduced preparations suggested changes in the GABA(A) receptor efficiency and/or disruption of the normal development of the medullary respiratory network

Consequences of prenatal exposure to diazepam on the respiratory parameters, respiratory network activity and gene expression of alpha1 and alpha2 subunits of GABA(A) receptor in newborn rat.

International audienceDiazepam (DZP) enhances GABA action at GABA(A) receptor. Chronic prenatal administration of DZP delays the appearance of neonatal reflexes. We examined whether maternal intake of DZP might affect respiratory control system in newborn rats (0-3 day-old). This study was conducted on unrestrained animals and medulla-spinal cord preparations. In addition, the level of expression of the genes encoding for the alpha1 and alpha2 subunits of the GABA(A) receptor was assessed by quantitative real-time RT-PCR. In rats exposed to DZP, the respiratory frequency was significantly lower and the tidal volume higher than in controls with no significant alteration of the minute ventilation. The recovery from moderate hypoxia was delayed compared to controls. The respiratory-like frequency of medullary spinal cord preparation from DZP-exposed neonates was higher than in the control group. Acute applications of DZP (1 microM) to these preparations increased respiratory-like frequency in both groups, but this facilitation was attenuated following prenatal DZP exposure. The present data indicate that prenatal exposure to DZP alters both eupneic breathing and the respiratory response to hypoxia. These effects might partly be ascribed to the down-regulation of the expression of genes encoding GABA(A) receptor subunits. On the other hand, the effects of DZP exposure on reduced preparations suggested changes in the GABA(A) receptor efficiency and/or disruption of the normal development of the medullary respiratory network

Cloning and molecular characterization of three lysophosphatidic acid acyltransferases expressed in flax seeds

International audienc

Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy

Field pea (Pisum sativum L.) appears well suited for the production of high-value molecules such as recombinant antibodies, with well-established agricultural practices world-wide and seeds that are easily stored and distributed. In order to evaluate the suitability of this grain legume for the production of biologically active antibodies, we transformed peas with a cDNA encoding the single-chain Fv fragment scFvT84.66. This scFv is derived from the monoclonal antibody T84.66, which recognises the well-characterised tumour-associated carcinoembryonic antigen. The antibody is useful for in vitro immunodiagnosis and in vivo imaging of human cancers. We expressed scFvT84.66 cDNA under the control of the seed-specific legumin A promoter. We targeted the antibody to the endoplasmic reticulum for better stability and high accumulation. Transgenic plants produced up to 9 mu g per gram fresh weight of functional scFvT84.66 in their seeds. The transgene was stably inherited and expressed in the progeny, and the antibody remained active after storage in dried transgenic seeds for two months at room temperature. Our results demonstrate the suitability of grain legume seeds to procedure biologically active recombinant antibodies, and the utility of field pea seeds as production vehicles for recombinant pharmaceutical macromolecules

Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies

This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 mg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries

Native and Artificial Reticuloplasmins Co-Accumulate in Distinct Domains of the Endoplasmic Reticulum and in Post-Endoplasmic Reticulum Compartments

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER