Evidence That Ca2+ within the Microdomain of the L-Type Voltage Gated Ca2+ Channel Activates ERK in MIN6 Cells in Response to Glucagon-Like Peptide-1

Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic β-cells potentiating glucose-stimulated insulin secretion and stimulating β-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca2+ through L-type voltage gated Ca2+ channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic β-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca2+ concentration ([Ca2+]i) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca2+]i or Ca2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca2+]i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca2+ signalling to ERK, we provide evidence that a sustained increase in Ca2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation

Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics.

We describe practical aspects of photobleaching fluorescence energy transfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are present. Measuring differences in donor photobleaching rates on cells labeled with donor only (fluorescein isothiocyanate-conjugated proteins) and with both donor and acceptor (tetramethylrhodamine-conjugated proteins) allows calculation of the fluorescence energy transfer efficiency. We assess possible methods of data analysis in light of the underlying processes of photobleaching and energy transfer and suggest optimum strategies for this purpose. Single murine B lymphocytes binding various ratios of donor and acceptor conjugates of tetravalent concanavalin A (Con A) and divalent succinyl Con A were examined for interlectin energy transfer by these methods. For Con A, a maximum transfer efficiency of 0.49 +/- 0.02 was observed. Under similar conditions flow cytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transfer determinations, we present examples of individual cell data and kinetic analyses, population rate constant distributions, and error estimates for the various quantities involved

Chapter: 'The Booth: Bringing Out the Super Hero in You' from book: Adaptive and Adaptable Learning: 11th European Conference on Technology Enhanced Learning, EC-TEL 2016, Lyon, France, September 13-16, 2016, Proceedings

The acquisition of knowledge is a key aspect for learners. However, in moments of stress the cognitive capacities of learners decrease considerably, making it very difficult for learners to get access and use their already acquired knowledge. Therefore we developed The Booth, a prototypical toolkit designed to support learners to prepare for key situations that are foreseen to be stressful. It guides the learner through a series of lectures helping them to gain personal power, and become in touch with their sincere-self. This paper presents the prototype, including the description of its technical aspects and the theory behind its lectures

A determination of the diffusion length of thermal neutrons in the x-10 standard graphite pile /

Mode of access: Internet

Un nouveau système d’imagerie gamma portable dédié à la détection et à la visualisation des points chauds dans les centrales nucléaires

International audienceA new portable gamma imaging system dedicated to the detection and the direct visualisation of hot spots in nuclear power plants. In a nuclear power plant (NPP), the localization of radioactive hot spots is a major issue, as they are responsible for a significant part of the doses received by the workers, especially during maintenance operations. Since 2010, EDF and CEA havebeen adapting a new generation caméra gamma, GAMPIX, to have a dedicated portable tool available for this use. This system enables to superimpose a gamma image with a visible image, in order to locate radioactive sources in a given area. Onsitemeasurements at the Tricastin EDF NPP, combined with specific developments at CEA, have been carried out in order to meet the particular environmental and radiological conditions experienced in such industrial facilities: the relatively high energy of cobalt-60 (1.17 and 1.33 MeV) and the existence of an ubiquitous background. This article will present the specifications required, the corresponding improvements and the main results obtained during the on-site measurements campaigns. Even if they were dedicated to the qualification of the camera, the tests carried out showed useful results like the leakage lines along a lead protection and the displacement of a hot spot in a pipe (compared to its identified position). At the end, further developments are presented.Dans une centrale nucléaire (CNPE), la localisation des points chauds est une préoccupation importante, car ils participent à la dose que reçoivent les intervenants, en particulier lors des opérations de maintenance. Depuis 2010, EDF et le CEA ont entrepris d’adapter une nouvelle génération de caméra gamma, GAMPIX, pour disposer d’un instrument portable dédié à cet usage. Ce système permet de superposer une image gamma à une image visible, afin de localiser les sources radioactives pré-sentes dans un environnement. Des essais sur site au CNPE de Tricastin ont été associés aux développements spécifiques réalisés par le CEA, afin d’appréhender les conditions particulières rencontrées en CNPE, à la fois environnementales et radiologiques : l’énergie relativement élevée des gamma du cobalt 60 (1,17 et 1,33 MeV) et la présence d’un bruit de fond ambiant permanent. Cet article présente les principaux axes du cahier des charges initial, les développements réalisés pour y répondre, et les résultats obtenus lors des campagnes d’essais. Même s’ils étaient uniquement dédiés à la qualification de la caméra gamma, les tests réalisés ont montré des résultats très prometteurs, par exemple la mise en évidence de lignes de fuite le long d’une protection biologique ou le mouvement d’un point chaud dans une tuyauterie (par rapport à sa position identifiée). En dernier lieu, les développements futurs sont évoqués