Le Peuple : organe quotidien du syndicalisme

29 juillet 19341934/07/29 (A14,N4940)-1934/07/29

Impact of GSK1016790A on cell proliferation/survival.

<p>A) Upper panel on left: Concentration-dependent reduction of cell proliferation/survival of A375 as measured by Janus Green-Assay. Upper panel on right: Note that half-maximal inhibition was achieved at ca. 1 nM GSK1016790A for all time intervals, except day-1. Lower panel on left: HC067047 antagonized the response to GSK1016790A. Lower panel on right: The negative gating-modulator of KCa3.1, the 13b derivate, RA-2 (10 μM) reduced cell proliferation/survival and potentiated the response to GSK1016790A. The positive-gating modulator of KCa3.1, SKA-121, had no effects. Data points are means ± SEM (n = 6–36 from n = 2–6 independent experiments). B) GSK1016790A impaired proliferation/survival of HaCaT cells. HC067047 partially antagonized the response. The negative and positive KCa3.1-gating modulators, RA-2 and SKA-121, respectively, did not modulate the response. Data points are means ± SEM (n = 18; number of independent experiments, n = 3). *P<0.05 vs. DMSO, #P<0.05 vs. GSK1016790A; Student’s T test.</p

GSK1016790A-induced TRPV4-currents and inhibition by HC067047 in the melanoma lines, MKTBR and SK-MEL-28, and the human non-cancer keratinocyte line, HaCaT.

<p>Data points are means ± SEM (cells, n = 4–6 each). Panel on right: Quantitative RT-PCR analysis of TRPV4 and KCa3.1 gene expression in HaCaT as percentage of GAPDH expression (replicates, n = 3). Data points are means ± SEM.</p

Alterations of cell morphology, cell detachment and cell death induced by GSK1016790A.

<p>A) Upper two panels: Exemplary light microscopic images illustrating time course of cell retraction, membrane blebbing (indicated by arrows), and cell detachment during the first hour of exposure to GSK1016790A (1 μM). HC067047 (1 μM) prevented visibly GSK1016790A-induced changes. HC067047 or vehicle (DMSO) had no visible effect. Lower panel: Giemsa-stained A375 cells after 1 h exposure to GSK1016790A, in combination with HC067047, or DMSO. Note the densification of nuclei (dark-grey dots indicated by arrows) in GSK1016790A-treated cells. B) Counts of non-viable, “death” cells in supernatant. Data points are means ± SEM (number of independent experiments, n = 3). *P<0.05 vs. DMSO, #P <0.05 vs. GSK1016790A; Student’s T test.</p

Characterization of TRPV4 channels in A375 melanoma cells.

<p>A) Upper panel: Exemplary whole-cell recordings showing activation of TRPV4 channels by GSK1016790A (200 nM) and inhibition of currents by HC067047 (1 μM). The arrow indicates a positive reversal potential of GSK1016790A-activated currents. Baseline currents were not considerable inhibited by HC067047. Lower panel: Co-activation of K_Ca-currents. The right arrow indicates a negative reversal potential (E_rev) of ca. -35 mV of the mixed TRPV4 and K_Ca current and the left arrow indicates an E_rev of ca. -75 mV of the isolated K_Ca-current after inhibition of TRPV4 currents by HC067047. The K_Ca-current was fully blocked by the negative-gating modulator of KCa3.1 channels, 13b (1 μM). B) Upper panel: Mean normalized currents at clamp potentials of -80 and +80 mV before and after addition GSK1016790A (n = 8, experiments) and after addition of HC067047 (n = 8). Lower panel: Mean mixed TRPV4/KCa3.1 currents at a clamp potential of 0 mV after addition of GSK1016790A (n = 5) and inhibition of TRPV4 currents by HC067047 (n = 4) and of KCa3.1 currents by 13b (n = 5). C) Quantitative RT-PCR analysis of TRPV4 and KCa3.1 gene expression as percentage of GAPDH expression (replicates, n = 3). Data points are means ± SEM.</p

FACS analysis of apoptosis.

<p>A) Representative flow cytometry dot plots with double Annexin V-FITC/PI staining for control cells (DMSO 0,2%), cells exposed to GSK1016790A (10 nM), and cells exposed to GSK1016790A and HC067047 (1 μM) at 1 h, 24 h, and 72 h. B) Summary data. C) Induction of apoptosis in HaCaT cells and protective effects of HC067047. D) Summary data. *P<0.05 vs. Control, #P<0.05 vs. GSK1016790A, ANOVA, n = 3). Data are means ± SEM (number of independent experiments, n = 3).</p

Improved Methods for Processing Optical Mapping Signals From Human Left Ventricular Tissues at Baseline and Following Adrenergic Stimulation

Optical mapping (OM) allows ex vivo measurement of electrophysiological signals at high spatio-temporal resolution, but the signal-to-roise ratio is commonly low. A variety of software options have been proposed to extract relevant information from OM recordings, being ElectroMap the most advanced tool currently available. In this study, improved methods are presented for processing OM signals of cardiac transmembrane voltage. A software called OMap is developed that incorporates novel techniques into ElectroMap for improved baseline drift removal, spatiotemporal filtering and characterization of action potential duration (APD) maps. In synthetically generated signals contaminated with baseline wander, white noise and the combination of both, the errors in APD maps between noisy and clean signals are remarkably lower for OMap than for ElectroMap, particularly for high noise levels. In OM signals recorded from human ventricular tissue specimens, OMap allows to clearly characterize the APD shortening effect induced by ß-adrenergic stimulation, whereas ElectroMap renders highly overlapped APD distributions for baseline and ß-adrenergic stimulation. In conclusion, improved methods are proposed and tested to characterize human ventricular electrophysiology from noisy OM recordings.Fil: Perez Zabalza, Maria. Universidad de Zaragoza; EspañaFil: Diez, Emiliano Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Rhyins, Julia. Northeastern University; Estados UnidosFil: Mountris, Kostantinos A.. Universidad de Zaragoza; EspañaFil: Vallejo Gil, Jose M.. Hospital Miguel Servet; EspañaFil: Fresneda Roldan, Pedro C.. Hospital Miguel Servet; EspañaFil: Fananas-Mastral, Javier. Hospital Miguel Servet; EspañaFil: Matamal Adell, Marta. Hospital Miguel Servet; EspañaFil: Sorribas Berjon, Fernando. Hospital Miguel Servet; EspañaFil: Vazquez Sancho, Manuel. Hospital Miguel Servet; EspañaFil: Ballester Cuenca, Carlos. Hospital Miguel Servet; EspañaFil: Segovia Roldan, Margarita. Universidad de Zaragoza; EspañaFil: Olivan Viguera, Aida. Universidad de Zaragoza; EspañaFil: Pueyo, Esther. Universidad de Zaragoza; Españ