Muscle cells and motoneurons differentially remove mutant SOD1 causing familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disease which occurs in sporadic or familial forms, clinically indistinguishable. About 15% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene that may induce misfolding in the coded protein, exerting neurotoxicity to motoneurons. However, other cell types might be target of SOD1 toxicity, because muscle-restricted expression of mutant SOD1 correlates with muscle atrophy and motoneurons death. We analysed the molecular behaviour of mutant SOD1 in motoneuronal NSC34 and muscle C2C12 cells. We found that misfolded mutant SOD1 clearance is much more efficient in muscle C2C12 than in motoneuronal NSC34 cells. Mutant SOD1 forms aggregates and impairs the proteasome only in motoneuronal NSC34 cells. Interestingly, NSC34 cells expressing mutant SOD1 are more sensitive to a superoxide-induced oxidative stress. Moreover, in muscle C2C12 cells mutant SOD1 remains soluble even when proteasome is inhibited with MG132. The higher mutant SOD1 clearance in muscle cells correlates with a more efficient proteasome activity, combined with a robust autophagy activation. Therefore, muscle cells seem to better manage misfolded SOD1 species, not because of an intrinsic property of the mutant protein, but in function of the cell environment, indicating also that the SOD1 toxicity at muscle level may not directly depend on its aggregation rate.     

A host-specific, temperature-sensitive translation defect determines the attenuation phenotype of a human rhinovirus/poliovirus chimera, PV1(RIPO)

By using a rhinosvirus/poliovirus type 1 chimera, PV1(RIPO), with the cognate internal ribosome entry site (IRES) of human rhinovirus type 2 (HRV2), we set out to shed light on the mechanism by which this variant expresses its attenuated phenotype in poliovirus-sensitive, CD155 transgenic (tg) mice and cynomolgus monkeys. Here we report that replication of PV1(RIPO) is restricted not only in human cells of neuronal origin, as was reported previously, but also in cells of murine origin at physiological temperature. This block in replication was enhanced at 39.5°C but, remarkably, it was absent at 33°C. PV1(RIPO) variants that overcame the replication block were derived by serial passage under restrictive conditions in either mouse cells or human neuronal cells. All adapting mutations mapped to the 5'-nontranslated region of PV1(RIPO). Variants selected in mouse cells, but not in human neuronal cells, exhibited increased mouse neurovirulence in vivo. The observed strong mouse-specific defect of PV1(RIPO) at nonpermissive temperature correlated with the translational activity of the HRV2 IRES in this chimeric virus. These unexpected results must be kept in mind when poliovirus variants are tested in CD155 tg mice for their neurovirulent potential, particularly in assays of live attenuated oral poliovirus vaccine lots. Virulence may be masked by adverse species-specific conditions in mouse cells that may not allow accurate prediction of neurovirulence in the human host. Thus, novel poliovirus variants in line for possible development of human vaccines must be tested in nonhuman primates.     

Inhibitory effect of combinations of digoxin and endogenous cardiotonic steroids on Na+/K+-ATPase activity in human kidney membrane preparation

AimsCardiac glycosides have been extensively used in the treatment of congestive heart failure for more than 200 years. Recently, cardenolides and bufadienolides were isolated from mammalian tissue and are considered as a new class of steroidal hormones. The aim of the present work was to characterize the interaction between the most clinical used cardiac glycoside digoxin and the cardiac glycosides known to exist endogenously, i.e., ouabain, marinobufagin and telocinobufagin, on human kidney Na⁺/K⁺-ATPase.Main methodsInhibition of Na⁺/K⁺-ATPase activity from crude membrane preparations of human kidney was performed using increasing concentrations of the drugs alone or mixtures of ouabain:digoxin, telocinobufagin:digoxin and marinobufagin:digoxin in a fixed ratio 1:4, 2:3 and 3:2, respectively. The colorimetric method of Fiske and Subbarow was used to measure the inorganic phosphate released.Key findingsAnalyses of inhibition curves showed that the experimental curves for all combinations were superimposed on the theoretical additive curves indicating that an additive effect occurs among distinct cardenolides and bufadienolides combinations on the human α1β1 Na⁺/K⁺-ATPase protomer.SignificanceConsidering the extensive use of digoxin in the treatment of heart failure and the recent findings that endogenous cardiac glycosides may have altered levels in many diseases, including heart failure, the demonstration of additive effect between cardiac glycosides can help in the understanding of recent clinical observations, including that lower than usual doses of cardiac glycosides are necessary for decreasing mortality in these patients.     

Investigation of adipose tissues in Zucker rats using in vivo and ex vivo magnetic resonance spectroscopy

In vivo single-voxel magnetic resonance spectroscopy (MRS) at 4.7T and ex vivo high-resolution proton magnetic resonance spectroscopy (HR-NMR) at 500 MHz were used to study the composition of adipose tissues in Zucker obese and Zucker lean rats. Lipid composition was characterized by unsaturation and polyunsaturation indexes and mean chain lengths. In vitro experiments were conducted in known mixtures of triglycerides and oils in order to validate the method. To avoid inaccuracies due to partial peak overlapping in MRS, peak quantification was performed after fitting of spectral peaks by using the QUEST algorithm. The intensity of different spectral lines was also corrected for T2 relaxation. Albeit with different sensitivity and accuracy, both techniques revealed that white adipose tissue is characterized by lower unsaturation and polyunsaturation indexes in obese rats compared with controls. HR-NMR revealed similar differences in brown adipose tissue. The present findings confirm the hypothesis that obese and lean Zucker rats have different adipose tissue composition.     

Genetic and pharmacological inhibition of PDK1 in cancer cells: characterization of a selective allosteric kinase inhibitor

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.     

The tumor suppressor hamartin enhances Dbl protein transforming activity through interaction with ezrin

The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between β1 and β2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.     

A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase

Maintenance of stable E-cadherin-dependent adhesion is essential for epithelial function. The small GTPase Rac is activated by initial cadherin clustering, but the precise mechanisms underlying Rac-dependent junction stabilization are not well understood. Ajuba, a LIM domain protein, colocalizes with cadherins, yet Ajuba function at junctions is unknown. We show that, in Ajuba-depleted cells, Rac activation and actin accumulation at cadherin receptors was impaired, and junctions did not sustain mechanical stress. The Rac effector PAK1 was also transiently activated upon cell-cell adhesion and directly phosphorylated Ajuba (Thr172). Interestingly, similar to Ajuba depletion, blocking PAK1 activation perturbed junction maintenance and actin recruitment. Expression of phosphomimetic Ajuba rescued the effects of PAK1 inhibition. Ajuba bound directly to Rac·GDP or Rac·GTP, but phosphorylated Ajuba interacted preferentially with active Rac. Rather than facilitating Rac recruitment to junctions, Ajuba modulated Rac dynamics at contacts depending on its phosphorylation status. Thus, a Rac-PAK1-Ajuba feedback loop integrates spatiotemporal signaling with actin remodeling at cell-cell contacts and stabilizes preassembled cadherin complexes.     

The chemokine receptor, CCR5

The chemokine receptor, CCR5, is a G protein coupled receptor responsible for some of the effects of the chemokines CCL3, CCL4 and CCL5. It is also one of the co-receptors for the entry of human immunodeficiency virus-1 (HIV-1) into cells. Regulation of CCR5 number on cells is, therefore, important for determining the infection rate by HIV-1.     

Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic cancer

Despite recognition of the devastating malignant potential of the pancreatic ductal cancer, the exact pathophysiological events contributing to tumor growth remain to be elucidated. Expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were found to be frequently elevated in several types of human cancer and have also been directly linked to carcinogenesis. The purpose of this study was to determine the expression of COX-1, COX-2 and iNOS in human pancreatic cancer and matched normal adjacent tissue by the Western blot assay. Marked COX-2 expression was observed in cancer tissue compared with the normal surrounding tissue. The iNOS protein was markedly expressed only in pancreatic cancer while the expression of COX-1 was similar in both normal and cancerous tissue. Our findings indicate that COX-2 up-regulation and the expression of iNOS in pancreatic cancer, not seen in normal tissue, may play a role in the pathogenesis of human pancreatic adenocarcinomas. These observations suggest that COX-2 and iNOS may be a target for prevention or treatment of pancreatic carcinomas.