Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

International audienceHeterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2-GDPNP-tRNA complex. © The Author(s) 2012. Published by Oxford University Press

Hypercalcemia iin an adult patient aggravated by vitamin D administration and revealing a heterozygous composite mutation CYP24A1: response and long-term treatment with rifampicin.

Cas clinique : un patient de 61 ans est hospitalisé avec une perte de poids (10kg) et une insuffisance rénale aigue. Il prenait de la vitamine D 800 UI/j depuis quelques mois. A l’admission : Calcium : 4.2 mmol/L (2.1-2.5), PTH : 5ng/ml, PTH-Rp <20 pg/ml, 25-hydroxy-vitD3(cholécalciférol) : 76 ng/ml (<60), PO4 : 1.1 mmol/L (0.8-1.45), ACE <25, normo calciurie, pas de lithiases rénales. On note une ostéoporose lombaire et une ostéopénie fémorale sévère. Le PET scan corps entier exclu une lésion néoplasique. Une fibrose pulmonaire interstitielle idiopathique est décelée. Des prélèvements ganglionnaires excluent une sarcoïdose. L’hydratation, la calcitonine, les corticoïdes et l’acide zoledronique ne normalisent pas la calcémie. La 1,25-dihydroxy-vitD3(calcitriol) : 161 ng/ml (29-82) et le FGF23 : 284 pg/ml (23-95) font suspecter un déficit fonctionnel de la CYP24A1, enzyme responsable de la dégradation du cholécalciférol et du calcitriol. Une analyse génétique par NSG confirme une mutation hétérozygote de CYP24A1 combinant : c.62del (p.Pro21ArFs 8) et c.428_430 del (p.Glu143del), mutations décrites de classe V. Depuis 1 an, l’administration de rifampicine 300 mgx2/J permet de normaliser le bilan phosphocalcique : calcium 2.6 mmol/L, PTH 15 ng/ml, 1-25 vitD 46 pg/ml. Conclusions : Les mutations inactivantes de CYP24A1 sont une cause rare d’hypercalcémie : elles contre indiquent la supplémentation en vitamine D et l’exposition solaire. La rifampicine, inducteur enzymatique stimulant la CYP3A4 et favorisant le catabolisme des métabolites de vitamine D, est un traitement efficace et bien toléré pour cette affection (Hawkes & al. J ClinEndocMetab 2017)

A virtual classroom can elicit teachers’ speech characteristics: Evidence from acoustic measurements during in vivo and in virtuo lessons, compared to a free speech control situation

peer reviewedTo achieve pedagogic goals and deal with environmental constraints such as noise when lecturing, teachers adapt their speech production in terms of frequency, intensity, and temporal aspects. The mastery of appropriate vocal skills is key to teachers’ speech intelligibility, health, and educational effectiveness. This project tests the relevance of virtual reality (VR) for training teachers’ vocal skills by simulating a lesson in a realistic VR environment characterized by adjustable constraints such as background noise and fidgety children. The VR environment depicts an elementary school classroom with 16 pupils aged 9 to 12 years old animated with typical childlike actions. To validate this virtual classroom in terms of speech characteristics, we conducted acoustic analyses on the speech productions of 30 female teachers in three conditions: (1) giving a free speech while facing the experimenter (control), (2) teaching in their usual classroom (in vivo), and (3) teaching the same lesson in a virtual classroom (in virtuo). The background noise in the VR setting was adjusted for each talker so it was similar to the level measured in vivo. Repeated measures ANOVAs showed that teachers significantly increased their voice frequency, intensity, and intonation, and made longer pauses while speaking in vivo and in virtuo, compared to the control condition (p < .001). These voice and speech adaptations (partly related to background noise), the strong feeling of presence and the lack of side effects suggest that the virtual classroom may facilitate voice training and rehabilitation for teachers.VirtuVo

PECAM-1 engagement counteracts ICAM-1-induced signaling in brain vascular endothelial cells

Interactions between leukocytes and vascular endothelial cells are mediated by a complex set of membrane adhesion molecules which transduce bi-directional signals in both cell types. Endothelium of the cerebral blood vessels, which constitute the blood–brain barrier, strictly controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells, we previously documented the role of ICAM-1 in activation of the tyrosine phosphorylation of several actin-binding proteins and subsequent rearrangements of the actin cytoskeleton. In the present study, we show that, whereas PECAM-1 is known to control positively the trans-endothelial migration of leukocytes via homophilic interactions between leukocytes and endothelial cells, PECAM-1 engagement on brain endothelial surface unexpectedly counteracts the ICAM-1-induced tyrosine phosphorylation of cortactin and rearrangements of the actin cytoskeleton. We present evidence that the PECAM-1-associated tyrosine phosphatase SHP-2 is required for ICAM-1 signaling, suggesting that its activity might crucially contribute to the regulation of ICAM-1 signaling by PECAM-1. Our findings reveal a novel activity for PECAM-1 which, by counteracting ICAM-1-induced activation, could directly contribute to limit activation and maintain integrity of brain vascular endothelium

Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

<p>Abstract</p> <p>Background</p> <p>Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including <it>Coffea arabica</it>.</p> <p>Results</p> <p>We identified the conditions required for successful long-term proliferation of embryogenic cultures in <it>C. arabica </it>and designed a highly efficient and reliable <it>Agrobacterium tumefaciens</it>-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD₆₀₀= 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.</p> <p>Conclusion</p> <p>Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make <it>Agrobacterium</it>-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.</p