Correlation between local cell membrane displacements and filterability of human red blood cells

AbstractLocal mechanical fluctuations of the cell membrane of human erythrocytes were shown to involve MgATP- and Mg2+-driven fast membrane displacements. We propose that these local bending deformations of the cell membrane are important for cell passage through capillaries. In order to verify this hypothesis, we examined cell membrane fluctuations and filterability of erythrocytes over a wide range of medium osmolalities (180–675 mosmol/kg H2O). The results indicate the existence of a positive correlation between the amplitude of local cell membrane displacements and cell filterability. We suggest that the occurrence of metabolically driven membrane displacements on the side surface of the red blood cell diminishes its bending stiffness and enables it to fold more efficiently upon entrance into blood capillaries. Thus, local cell membrane displacements seem to play an important role in microcirculation

The ubiquitin E3 ligase POSH regulates calcium homeostasis through spatial control of Herp

The ubiquitin (Ub) domain protein Herp plays a crucial role in the maintenance of calcium homeostasis during endoplasmic reticulum (ER) stress. We now show that Herp is a substrate as well as an activator of the E3 Ub ligase POSH. Herp-mediated POSH activation requires the Ubl domain and exclusively promotes lysine-63–linked polyubiquitination. Confocal microscopy demonstrates that Herp resides mostly in the trans-Golgi network, but, shortly after calcium perturbation by thapsigargin (Tpg), it appears mainly in the ER. Substitution of all lysine residues within the Ubl domain abolishes lysine-63–linked polyubiquitination of Herp in vitro and calcium-induced Herp relocalization that is also abrogated by the overexpression of a dominant-negative POSHV14A. A correlation exists between the kinetics of Tpg-induced Herp relocalization and POSH-dependent polyubiquitination. Finally, the overexpression of POSH attenuates, whereas the inhibition of POSH by the expression of POSHV14A or by RNA interference enhances Tpg-induced calcium burst. Altogether, these results establish a critical role for POSH-mediated ubiquitination in the maintenance of calcium homeostasis through the spatial control of Herp

Intracoronary Injection of In Situ Forming Alginate Hydrogel Reverses Left Ventricular Remodeling After Myocardial Infarction in Swine

ObjectivesThis study sought to determine whether alginate biomaterial can be delivered effectively into the infarcted myocardium by intracoronary injection to prevent left ventricular (LV) remodeling early after myocardial infarction (MI).BackgroundAlthough injectable biomaterials can improve infarct healing and repair, the feasibility and effectiveness of intracoronary injection have not been studied.MethodsWe prepared a calcium cross-linked alginate solution that undergoes liquid to gel phase transition after deposition in infarcted myocardium. Anterior MI was induced in swine by transient balloon occlusion of left anterior descending coronary artery. At 4 days after MI, either alginate solution (2 or 4 ml) or saline was injected selectively into the infarct-related coronary artery. An additional group (n = 19) was treated with incremental volumes of biomaterial (1, 2, and 4 ml) or 2 ml saline and underwent serial echocardiography studies.ResultsExamination of hearts harvested after injection showed that the alginate crossed the infarcted leaky vessels and was deposited as hydrogel in the infarcted tissue. At 60 days, control swine experienced an increase in left ventricular (LV) diastolic area by 44%, LV systolic area by 45%, and LV mass by 35%. In contrast, intracoronary injection of alginate (2 and 4 ml) prevented and even reversed LV enlargement (p < 0.01). Post-mortem analysis showed that the biomaterial (2 ml) increased scar thickness by 53% compared with control (2.9 ± 0.1 mm vs. 1.9 ± 0.3 mm; p < 0.01) and was replaced by myofibroblasts and collagen.ConclusionsIntracoronary injection of alginate biomaterial is feasible, safe, and effective. Our findings suggest a new percutaneous intervention to improve infarct repair and prevent adverse remodeling after reperfused MI

The E3 Ubiquitin-Ligase Bmi1/Ring1A Controls the Proteasomal Degradation of Top2α Cleavage Complex – A Potentially New Drug Target

The topoisomerases Top1, Top2alpha and Top2beta are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2beta are subject to proteasomal degradation, this phenomena was not demonstrated for Top2alpha.We show here that Top2alpha is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2beta. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2alpha degradation, increases the persistence of Top2alpha-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2alpha in-vitro and cellular overexpression of Bmi1 increases drug induced Top2alpha ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2alpha ubiquitination and drug-induced Top2alpha degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner.The discovery that poisoned Top2alpha is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1