Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- And Rab8-Dependent and recycling endosome-independent

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the m1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells. © 2014 Bertuccio et al

A influência da variação da curvatura da base do braquete em uma união ortodôntica submetida a diferentes cargas, através do método dos elementos finitos The influence of the variation of the bracket base curvature in a bonded orthodontic attachment submitted by different load cases using the finite element method

Este trabalho procurou avaliar, através do Método dos Elementos Finitos, a resistência ao deslocamento de quatro diferentes bases de braquetes "straight-wire" (Morelli, Unitek, A-Company e Abzil-Lancer) na aplicação de forças de torção e cisalhamento, correlacionando essa resistência à melhor adaptação das bases à superfície vestibular dentária. As curvaturas das bases de braquetes utilizados no presente estudo foram descritas em um estudo prévio, enquanto o contorno dentário foi obtido a partir do escaneamento e cálculo da curvatura média vestibular do canino inferior de uma amostra de 30 indivíduos brasileiros. Empregou-se a análise tridimensional por Elementos Finitos da interface esmalte vestibular/cimento/base do braquete para cada marca testada. Forças de 1N, promovendo deslocamento lateral e torção, foram aplicadas no centro da base do braquete e observou-se a distribuição desses esforços sobre os modelos assim como as tensões normais e de cisalhamento geradas. Os resultados mostraram que a base que melhor se adaptou à superfície vestibular construída foi a da marca Unitek, seguida pela marca A-Company, Abzil-Lancer e Morelli. As deformações sofridas pelos braquetes foram inversamente proporcionais à adaptação à superfície dentária. Os maiores picos de tensão localizaram-se nas proximidades do ponto de aplicação da força. A interface adesivo/esmalte foi mais sujeita à falha na adesão que a interface braquete/adesivo. A força de cisalhamento demonstrou ser mais provável de causar falha na adesão quando comparada à força de torção.<br>The objective of this study was to evaluate the adaptability of the four straight-wire brackets bases (Morelli, Unitek, A-Company and Abzil-Lancer) to facial surface of a lower canine. The lower canine facial curvature data to be used in the three-dimensional finite element model were established by 30 Brazilian adult individuals and the brackets bases curvature used were described in a prior study. The three-dimensional finite element analysis of the interface enamel/cement/base of bracket for each tested mark was used. Loads of 1N promoting lateral displacement and torsion had been applied in the center of the base of brackets and the distribution of these efforts on the models was observed as well as the normal and shear tensions generated. The results had shown that the base that better fit to facial surface was the one from Unitek, followed by the A-Company, Abzil-Lancer and Morelli.The brackets deformations were inversely proportional to its adaptation.The biggest peaks of tensions had been situated near to the point of force application. The cement/enamel interface was more likely to fail in the adhesion that the bracket/cement interface. The torsion load was less likely to cause damage in the adhesion when compared with the shear load