Dynamin- and Rab5-Dependent Endocytosis of a Ca²⁺-Activated K⁺ Channel, KCa2.3

Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca2+-activated K+ channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane. © 2012 Gao et al

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

Apexificación utilizando el hidróxido de calcio como primera alternativa

When a pulp necrosis it's established in young teeth that have not developed an apical seal or the incomplete development of the root, an apexitication is the election treatment, because it inducts the formation of a calcified barrier that obliterates the apical foramen or allows the complete radicular development. The mixture of Calcium Hydroxide with physiological serum is the most simple technique of inducting apexification. The case report presents a 10 year old male, with necrotic pulp in O.D 36, in the x-ray can be observed radiolucent zone in apex and furcation, pulp retraction and lack of apical seal; in this teeth apexification was performed with calcium hydroxide, and it shows a correct evolution in decrasing of lesion in furcation and seal in apex; that permits the obturation or radicular system and finally the rehabilitation with a steel crown.Cuando una necrosis pulpar se instala en dientes jóvenes que aún no han completado el cierre apical o no han terminado el desarrollo radicular, la apexificación es el tratamiento indicado, el cual induce la información de una barra calcificada que oblitere el orificio apical o que permita el desarrollo radicular completo. La mezcla del hidróxido de calcio Ca(OH)2 con suero fisiológico es la forma más deseable y sencilla de introducir la apexificación con pronóstico exitoso. El caso clínico que se presenta es un paciente masculino de 10 años de edad, al cual se diagnostica necrosis pulpar en O.D.36, radiográficamente con zona radiolúcida en ápices y furca, retracción pulpar y falta de cierre apical al cual se realiza el tratamiento apexificación con hidróxido de calcio, mostrando en el control radiográfico disminución de la lesión en furca y ápices, lográndose el cierre apical permitiendo el tratamiento de endodoncia con gutapercha y finalmente la rehabilitación con corona de acero cromo

Rehabilitación oral en niños, con enfoque preventivo y psicológico: reporte de un caso

It is important to maintain the integrity of the primary dentition until the moment of its exfoliation, since it plays an essential role in the development of the child, and it may be affected adversely by factors among which the most common is the tooth decay, that can lead to alterations in the physiological, psychological and social development of the child. In cases of lost tooth by tooth decay, the pediatric dentistry proposes the use of removable dental prosthesis that allows the restoration of the fundamental functions of teeth: Chewing, phonation, aesthetics, occlusion and preservation of the dental arches, all of them necessary for the correct physiological and emotional development of the patient. The objective of this work is to present the treatment of oral rehabilitation performed on a 5 year, 7 months old male patient which was diagnosis with early childhood caries. The treatment done consisted of dental extractions, composite resin restoration, silver amalgam, steel-chrome crown and removable dental prosthesis. Obtaining this way restore the essential functions of the teeth and the oral cavity. Psychological and socially the dentures influenced in a positive way because there was a clear change in the patient' personality.Es importante mantener la integridad de la dentición primaria hasta el momento de su exfoliación, puesto que juega un papel esencial en el desarrollo del niño y puede verse afectada adversamente por factores entre los cuales el más frecuente es la caries dental, ya que puede conducir a alteraciones en el desarrollo fisiológico, psicológico y social del niño. En casos de pérdida dentaria por caries la Odontopediatría propone la utilización de prótesis removibles que permiten el restablecimiento de las funciones fundamentales de los dientes: masticación, fonación, estética, oclusión y preservación de los arcos dentarios, todas ellas necesarias para el correcto desarrollo fisiológico y emocional del paciente. El objetivo de este trabajo es presentar el tratamiento de rehabilitación oral realizado a un paciente masculino de 5 años 7 meses al cual se le diagnosticó caries temprana de la infancia. Se realizaron exodoncias, restauraciones con resina compuesta, amalgama de plata, corona de acero-cromo y prótesis removibles. Obteniendo de esta manera el restablecimiento de las funciones esenciales de los dientes y por consecuencia de la cavidad bucal. De manera psicológica y social las prótesis influyeron de manera positiva ya que hubo un cambio evidente en la personalidad del paciente

The Increased Expression of Regulator of G-Protein Signaling 2 (RGS2) Inhibits Insulin-Induced Akt Phosphorylation and Is Associated with Uncontrolled Glycemia in Patients with Type 2 Diabetes

Experimental evidence in mice models has demonstrated that a high regulator of G-protein signaling 2 (RSG2) protein levels precede an insulin resistance state. In the same context, a diet rich in saturated fatty acids induces an increase in RGS2 protein expression, which has been associated with decreased basal metabolism in mice; however, the above has not yet been analyzed in humans. For this reason, in the present study, we examined the association between RGS2 expression and insulin resistance state. The incubation with palmitic acid (PA), which inhibits insulin-mediated Akt Ser473 phosphorylation, resulted in the increased RGS2 expression in human umbilical vein endothelial-CS (HUVEC-CS) cells. The RGS2 overexpression without PA was enough to inhibit insulin-mediated Akt Ser473 phosphorylation in HUVEC-CS cells. Remarkably, the platelet RGS2 expression levels were higher in type 2 diabetes mellitus (T2DM) patients than in healthy donors. Moreover, an unbiased principal component analysis (PCA) revealed that RGS2 expression level positively correlated with glycated hemoglobin (HbA1c) and negatively with age and high-density lipoprotein cholesterol (HDL) in T2DM patients. Furthermore, PCA showed that healthy subjects segregated from T2DM patients by having lower levels of HbA1c and RGS2. These results demonstrate that RGS2 overexpression leads to decreased insulin signaling in a human endothelial cell line and is associated with poorly controlled diabetes

The Increased Expression of Regulator of G-Protein Signaling 2 (RGS2) Inhibits Insulin-Induced Akt Phosphorylation and Is Associated with Uncontrolled Glycemia in Patients with Type 2 Diabetes

Experimental evidence in mice models has demonstrated that a high regulator of G-protein signaling 2 (RSG2) protein levels precede an insulin resistance state. In the same context, a diet rich in saturated fatty acids induces an increase in RGS2 protein expression, which has been associated with decreased basal metabolism in mice; however, the above has not yet been analyzed in humans. For this reason, in the present study, we examined the association between RGS2 expression and insulin resistance state. The incubation with palmitic acid (PA), which inhibits insulin-mediated Akt Ser473 phosphorylation, resulted in the increased RGS2 expression in human umbilical vein endothelial-CS (HUVEC-CS) cells. The RGS2 overexpression without PA was enough to inhibit insulin-mediated Akt Ser473 phosphorylation in HUVEC-CS cells. Remarkably, the platelet RGS2 expression levels were higher in type 2 diabetes mellitus (T2DM) patients than in healthy donors. Moreover, an unbiased principal component analysis (PCA) revealed that RGS2 expression level positively correlated with glycated hemoglobin (HbA1c) and negatively with age and high-density lipoprotein cholesterol (HDL) in T2DM patients. Furthermore, PCA showed that healthy subjects segregated from T2DM patients by having lower levels of HbA1c and RGS2. These results demonstrate that RGS2 overexpression leads to decreased insulin signaling in a human endothelial cell line and is associated with poorly controlled diabetes