A myosin-Va tail fragment sequesters dynein light chains leading to apoptosis in melanoma cells

Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Here, we demonstrated that over expression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Immobilized MVaf interacted directly with DLCs, but complexed MVaf/DLCs did not interact with Bmf. Overexpression of DLC2 attenuated MVaf-induced apoptosis. Thus, we suggest that, MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. Murine embryonic fibroblasts (MEFs) lacking Bim and Bmf or Bax and Bak were less sensitive to apoptosis caused by MVaf expression than wild-type MEFs, strengthening the putative role of the intrinsic apoptotic pathway in this response. Finally, MVaf expression attenuated B16-F10 solid tumor growth in mice, suggesting that this peptide may be useful as an apoptosis-inducing tool for basic and translational studies

Formation of Very Large Conductance Channels by Bacillus cereus Nhe in Vero and GH4 Cells Identifies NheA + B as the Inherent Pore-Forming Structure

The nonhemolytic enterotoxin (Nhe) produced by Bacillus cereus is a pore-forming toxin consisting of three components, NheA, -B and -C. We have studied effects of Nhe on primate epithelial cells (Vero) and rodent pituitary cells (GH4) by measuring release of lactate dehydrogenase (LDH), K+ efflux and the cytosolic Ca2+ concentration ([Ca2+]i). Plasma membrane channel events were monitored by patch-clamp recordings. Using strains of B. cereus lacking either NheA or -C, we examined the functional role of the various components. In both cell types, NheA + B + C induced release of LDH and K+ as well as Ca2+ influx. A specific monoclonal antibody against NheB abolished LDH release and elevation of [Ca2+]i. Exposure to NheA + B caused a similar K+ efflux and elevation of [Ca2+]i as NheA + B + C in GH4 cells, whereas in Vero cells the rate of K+ efflux was reduced by 50% and [Ca2+]i was unaffected. NheB + C had no effect on either cell type. Exposure to NheA + B + C induced large-conductance steps in both cell types, and similar channel insertions were observed in GH4 cells exposed to NheA + B. In Vero cells, NheA + B induced channels of much smaller conductance. NheB + C failed to insert membrane channels. The conductance of the large channels in GH4 cells was about 10 nS. This is the largest channel conductance reported in cell membranes under quasi-physiological conditions. In conclusion, NheA and NheB are necessary and sufficient for formation of large-conductance channels in GH4 cells, whereas in Vero cells such large-conductance channels are in addition dependent on NheC

Systematic meta-analyses and field synopsis of genetic association studies of violence and aggression

A large number of candidate gene studies for aggression and violence have been conducted. Successful identification of associations between genetic markers and aggression would contribute to understanding the neurobiology of antisocial behavior and potentially provide useful tools for risk prediction and therapeutic targets for high-risk groups of patients and offenders. We systematically reviewed the literature and assessed the evidence on genetic association studies of aggression and related outcomes in order to provide a field synopsis. We searched PubMed and Huge Navigator databases and sought additional data through reviewing reference lists and correspondence with investigators. Genetic association studies were included if outcome data on aggression or violent behavior either as a binary outcome or as a quantitative trait were provided. From 1331 potentially relevant investigations, 185 studies constituting 277 independent associations on 31 genes fulfilled the predetermined selection criteria. Data from variants investigated in three or more samples were combined in meta-analyses and potential sources of heterogeneity were investigated using subgroup analyses. In the primary analyses, which used relaxed inclusion criteria, we found no association between any polymorphism analyzed and aggression at the 5% level of significance. Subgroup analyses, including by severity of outcome, age group, characteristics of the sample and ethnicity, did not demonstrate any consistent findings. Current evidence does not support the use of such genes to predict dangerousness or as markers for therapeutic interventions

The role of reactive oxygen species in apoptosis of the diabetic kidney

Increased levels of reactive oxygen species (ROS) by hyperglycemia can induce apoptosis of renal cells and diabetic nephropathy. The redox balance in the renal cell seems, therefore, of the utmost importance. ROS-mediated apoptosis may be further aggravated by an inadequate cytoprotective response against ROS. When there are insufficient cytoprotective and ROS scavenging molecules, ROS lead to considerable cellular damage and to a point of no return in apoptosis. Induction of cytoprotective proteins may prevent or attenuate apoptosis, renal cell injury, and finally diabetic nephropathy. Here, we discuss some mechanisms of apoptosis and several strategies that have been probed to ameliorate, or to prevent apoptosis in the diabetic kidney

TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance

Abstract Ligands addressed to the mitochondrial Translocator Protein (TSPO) have been suggested as cell death/life and steroidogenesis modulators. Thus, TSPO ligands have been proposed as drug candidates in several diseases; nevertheless, a correlation between their binding affinity and in vitro efficacy has not been demonstrated yet, questioning the specificity of the observed effects. Since drug-target residence time is an emerging parameter able to influence drug pharmacological features, herein, the interaction between TSPO and irDE-MPIGA, a covalent TSPO ligand, was investigated in order to explore TSPO control on death/life processes in a standardized glioblastoma cell setting. After 90 min irDE-MPIGA cell treatment, 25 nM ligand concentration saturated irreversibly all TSPO binding sites; after 24 h, TSPO de-novo synthesis occurred and about 40 % TSPO binding sites resulted covalently bound to irDE-MPIGA. During cell culture treatments, several dynamic events were observed: (a) early apoptotic markers appeared, such as mitochondrial membrane potential collapse (at 3 h) and externalization of phosphatidylserine (at 6 h); (b) cell viability was reduced (at 6 h), without cell cycle arrest. After digitonin-permeabilized cell suspension treatment, a modulation of mitochondrial permeability transition pore was evidenced. Similar effects were elicited by the reversible TSPO ligand PIGA only when applied at micromolar dose. Interestingly, after 6 h, irDE-MPIGA cell exposure restored cell survival parameters. These results highlighted the ligand-target residence time and the cellular setting are crucial parameters that should be taken into account to understand the drug binding affinity and efficacy correlation and, above all, to translate efficiently cellular drug responses from bench to bedside

Differential regulation of cell death pathways by the microenvironment correlates with chemoresistance and survival in leukaemia

Glucocorticoids (GCs) and topoisomerase II inhibitors are used to treat acute lymphoblastic leukaemia (ALL) as they induce death in lymphoid cells through the glucocorticoid receptor (GR) and p53 respectively. Mechanisms underlying ALL cell death and the contribution of the bone marrow microenvironment to drug response/resistance remain unclear. The role of the microenvironment and the identification of chemoresistance determinants were studied by transcriptomic analysis in ALL cells treated with Dexamethasone (Dex), and Etoposide (Etop) grown in the presence or absence of bone marrow conditioned media (CM). The necroptotic (RIPK1) and the apoptotic (caspase-8/3) markers were downregulated by CM, whereas the inhibitory effects of chemotherapy on the autophagy marker Beclin-1 (BECN1) were reduced suggesting CM exerts cytoprotective effects. GCs upregulated the RIPK1 ubiquitinating factor BIRC3 (cIAP2), in GC-sensitive (CEM-C7-14) but not in resistant (CEM-C1-15) cells. In addition, CM selectively affected GR phosphorylation in a site and cell-specific manner. GR is recruited to RIPK1, BECN1 and BIRC3 promoters in the sensitive but not in the resistant cells with phosphorylated GR forms being generally less recruited in the presence of hormone. FACS analysis and caspase-8 assays demonstrated that CM promoted a pro-survival trend. High molecular weight proteins reacting with the RIPK1 antibody were modified upon incubation with the BIRC3 inhibitor AT406 in CEM-C7-14 cells suggesting that they represent ubiquitinated forms of RIPK1. Our data suggest that there is a correlation between microenvironment-induced ALL proliferation and altered response to chemotherapy