Effect of Continuous Phorbol Ester Treatment on Muscarinic Receptor-Mediated Calmodulin Redistribution in SK-N-SH Neuroblastoma Cells

Stimulation of muscarinic receptors by carbachol and activation of protein kinase C elicits the translocation of calmodulin (CaM) from membranes to cytosol in the human neuroblastoma cell line SK-N-SH. Our previous studies have suggested a role for protein kinase C in the regulation of CaM redistribution. To explore further the role of protein kinase C in carbachol-induced calmodulin translocation, we treated cells for 17 h with 12- O -tetradecanoylphorbol 13-acetate (TPA) to down-regulate protein kinase C isozymes or 72 h to differentiate the cells. Treatment of SK-N-SH cells for 17 h with 70 n M TPA nearly abolished the effect of carbachol on CaM redistribution. After 72 h of TPA, however, the cells appeared differentiated, and the ability of carbachol to increase cytosolic CaM levels was restored. In untreated control cells, the carbachol-mediated increase in cytosolic CaM content was mimicked by TPA and blocked by pretreatment with the selective protein kinase C inhibitor Ro 31-8220 at 10 µ M . In the 72-h TPA-treated cells, however, the ability of TPA to increase cytosolic CaM levels was significantly reduced, and the action of carbachol was no longer blocked by Ro 31-8220. The effect of prolonged TPA treatment on select protein kinase C isozymes was examined by immunoblotting. Treatment of cells for either 17 or 72 h abolished the Α-isozyme in the cytosol and reduced (17 h) or abolished (72 h) the content in the membranes. In both 17- and 72-h TPA-treated cells, the ε-isozyme was nearly abolished in the cytosol and slightly reduced in the membranes. Some protein kinase C activity may have been maintained during TPA treatment because the basal level of phosphorylation of the protein kinase C substrate myristoylated alanine-rich C kinase substrate was enhanced in cells treated for either 17 or 72 h with TPA. The potential dissociation of carbachol and protein kinase C in eliciting increases in cytosolic CaM content was a function of prolonged TPA treatment and not differentiation per se because carbachol-mediated increases in cytosolic CaM levels were inhibited by Ro 31-8220 in retinoic acid-differentiated SK-N-SH cells. This study demonstrates that continuous TPA treatment, although initially down-regulating the protein kinase C-mediated effect of carbachol on CaM redistribution, uncouples carbachol and protein kinase C at longer times.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65656/1/j.1471-4159.1997.68010040.x.pd

Prediction of Load in Reverse Extrusion Process of Hollow Parts using Modern Artificial Intelligence Approaches

Extrusion is one of the important processes to manufacture and produce military and industrial components. Designing its tools is usually associated with trial and error and needs great expertise and adequate experience. Reverse extrusion process is known as one of the common processes for production of hollow parts with closed ends. The significant load required in formation of a workpiece is one of the existing constraints for the reverse extrusion process. This issue becomes rather difficult especially for the parts having thin walls since its analysis using finite element softwares is exposed to some limitations. In this regard, application of artificial intelligence for prediction of load in the reverse extrusion process will not only save time and money, but also improve quality features of the product. Based on the existing data and methods suggested for variations of punching force through the reverse extrusion process, the system is trained and then performance of the system is evaluated using the test data in this paper. Efficiency of the proposed method is also assessed via comparison with the results of others.DOI:http://dx.doi.org/10.11591/ijece.v4i3.535

Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca 2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca 2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca 2+ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000- g cytosol and membrane fractions. The influx of extracellular Ca 2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca 2+ channel blocker Ni 2+ . Blocking the influx of extracellular Ca 2+ had no effect on carbachol-mediated increases in cytosolic CaM (168 18% of control values for carbachol treatment alone vs. 163 28% for Ni 2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca 2+ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca 2+ by pretreatment with the cell-permeant Ca 2+ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 37% of control without BAPTA/AM vs. 136 13% with BAPTA/AM). The effect of direct entry of extracellular Ca 2+ into the cell by K + depolarization was assessed. Incubation of SK-N-SH cells with 60 m M K + elicited an immediate and persistent increase in intracellular free Ca 2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca 2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca 2+ , which releases Ca 2+ from intracellular stores, induced an increase in cytosolic CaM (203 30% of control). The mechanism for the CaM release may involve activation of the isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K + depolarization. These data demonstrate that release of Ca 2+ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66213/1/j.1471-4159.1998.70010139.x.pd

Modélisation biomécanique du diaphragme humain : du CT-4D au modèle du mouvement

Session "Articles"National audienceL'hadronthérapie est une technique avancée de traitement du cancer par radiothérapie. Elle offre une balistique d'irradiation bien supérieure à la radiothérapie conventionnelle. Lorsque la tumeur se trouve sur un organe en mouvement, la difficulté majeure est de pouvoir la cibler pendant le traitement. En ce qui concerne la tumeur pulmonaire, le diaphragme joue un rôle majeur et prépondérant dans le mouvement tumoral. Le diaphragme est une membrane musculo-tendineuse en forme de dôme qui sépare le thorax de l'abdomen. Dans ce travail nous présentons un modèle biomécanique permettant de modéliser les mouvements du diaphragme pendant la respiration. Dans cette démarche nous simulons le mouvement du diaphragme entre l'inspiration et l'expiration, à partir d'un modèle de contraction musculaire. Pour cela, un modèle biomécanique 3D personnalisé du diaphragme, basé sur la méthode des éléments finis, a été développé à partir de données expérimentales (4D CT-scan) d'un patient. Les résultats de notre modélisation montrent une bonne concordance entre la simulation et les données expérimentales