c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

The antiviral adaptor proteins Cardif and Trif are processed and inactivated by caspases

The outcome of a viral infection depends on the interplay between the host's capacity to trigger potent antiviral responses and viral mechanisms that counteract them. Although Toll-like receptor (TLR)-3, which recognizes virally derived double-stranded (ds) RNA, transmits downstream antiviral signaling through the TIR adaptor Trif (TICAM-1), viral RNA-sensing RIG-like helicases (RLHs) use the mitochondrial-bound CARD protein Cardif (IPS-1/MAVS/VISA). The importance of these two antiviral signaling pathways is reflected by the fact that both adaptors are inhibited through specific cleavage triggered by the hepatitis C virus serine protease NS3-4A. Here, we show that inactivation can also occur through cellular caspases activated by various pro-apoptotic signals. Upon caspase-dependent cleavage both adaptors loose their capacity to activate the transcription factors interferon regulatory factors (IRF) and NF-kappaB. Importantly, poliovirus infection triggers a caspase-dependent cleavage of Cardif, suggesting that some viruses may activate caspases not only as a mean to facilitate shedding and replication, but also to impair antiviral responses

Putative adverse outcome pathways relevant to neurotoxicity.

The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

Role of peroxynitrite in the redox regulation of cell signal transduction pathways.

Peroxynitrite is a potent oxidant and nitrating species formed from the reaction between the free radicals nitric oxide and superoxide. An excessive formation of peroxynitrite represents an important mechanism contributing to cell death and dysfunction in multiple cardiovascular pathologies, such as myocardial infarction, heart failure and atherosclerosis. Whereas initial works focused on direct oxidative biomolecular damage as the main route of peroxynitrite toxicity, more recent evidence, mainly obtained in vitro, indicates that peroxynitrite also behaves as a potent modulator of various cell signal transduction pathways. Due to its ability to nitrate tyrosine residues, peroxynitrite affects cellular processes dependent on tyrosine phosphorylation. Peroxynitrite also exerts complex effects on the activity of various kinases and phosphatases, resulting in the up- or downregulation of signalling cascades, in a concentration- and cell-dependent manner. Such roles of peroxynitrite in the redox regulation of key signalling pathways for cardiovascular homeostasis, including protein kinase B and C, the MAP kinases, Nuclear Factor Kappa B, as well as signalling dependent on insulin and the sympatho-adrenergic system are presented in detail in this review.

Deriving the hard thermal loops of QCD from classical transport theory

Classical transport theory is employed to analyze the hot quark-gluon plasma at the leading order in the coupling constant. A condition on the (covariantly conserved) color current is obtained. From this condition, the generating functional of hard thermal loops with an arbitrary number of soft external bosonic legs can be derived. Our approach, besides being more direct than alternative ones, shows that hard thermal loops are essentially classical.

Runoff and sediment transport in a degraded area

Gully erosion occurs by the combined action of splash, sheetwash and rill-wash (interrill and rill erosion). These erosion processes have a great capacity for both sediment production and sediment transport. The objectives of this experiment were to evaluate hydrological and sediment transport in a degraded area, severely dissected by gullies; to assess the hydraulic flow characteristics and their aggregate transport capacity; and to measure the initial splash erosion rate. In the study area in Guarapuava, State of Paraná, Brazil (lat 25º 24' S; long 51º24' W; 1034 m asl), the soil was classified as Cambissolo Húmico alumínico, with the following particle-size composition: sand 0.116 kg kg-1; silt 0.180 kg kg-1; and clay 0.704 kg kg-1. The approach of this research was based on microcatchments formed in the ground, to study the hydrological response and sediment transport. A total of eight rill systems were simulated with dry and wet soil. An average rainfall of 33.7 ± 4.0 mm was produced for 35 to 54 min by a rainfall simulator. The equipment was installed, and a trough was placed at the end of the rill to collect sediments and water. During the simulation, the following variables were measured: time to runoff, time to ponding, time of recession, flow velocity, depth, ratio of the initial splash and grain size. The rainsplash of dry topsoil was more than twice as high as under moist conditions (5 g m-2 min-1 and 2 g m-2 min-1, respectively). The characteristics of the flow hydraulics indicate transition from laminar to turbulent flow [Re (Reynolds number) 1000-2000]. In addition, it was observed that a flow velocity of 0.12 m s-1 was the threshold for turbulent flow (Re > 2000), especially at the end of the rainfall simulation. The rill flow tended to be subcritical [Fr (Froude Number) < 1.0]. The variation in hydrological attributes (infiltration and runoff) was lower, while the sediment yield was variable. The erosion in the rill systems was characterized as limited transport, although the degraded area generated an average of 394 g m-2 of sediment in each simulation.

Evaluation of uptake and transport of cationic and anionic ultrasmall iron oxide nanoparticles by human colon cells.

Nanoparticles (NPs) are in clinical use or under development for therapeutic imaging and drug delivery. However, relatively little information exists concerning the uptake and transport of NPs across human colon cell layers, or their potential to invade three-dimensional models of human colon cells that better mimic the tissue structures of normal and tumoral colon. In order to gain such information, the interactions of biocompatible ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) (iron oxide core 9-10 nm) coated with either cationic polyvinylamine (aminoPVA) or anionic oleic acid with human HT-29 and Caco-2 colon cells was determined. The uptake of the cationic USPIO NPs was much higher than the uptake of the anionic USPIO NPs. The intracellular localization of aminoPVA USPIO NPs was confirmed in HT-29 cells by transmission electron microscopy that detected the iron oxide core. AminoPVA USPIO NPs invaded three-dimensional spheroids of both HT-29 and Caco-2 cells, whereas oleic acid-coated USPIO NPs could only invade Caco-2 spheroids. Neither cationic aminoPVA USPIO NPs nor anionic oleic acid-coated USPIO NPs were transported at detectable levels across the tight CacoReady? intestinal barrier model or the more permeable mucus-secreting CacoGoblet? model.

Characterization of the expression, regulation and function of Sorting Nexin 9 and Sorting Nexin 18

Summary : Sorting nexin (SNX) family members play important roles in intracellular protein and membrane trafficking, The membrane-tubulating SNX9 protein has been shown to interact with multiple components of the endocytic machinery and to participate in clathrin-mediated endocytosis of cell surface receptors. It has not been investigated if SNX9 may also participate in other protein sorting pathways that involve vesicular transport, specifically the biogenesis of lysosome-related organelles (LROs). Closely related to SNX9 is SNXl8, whose function is largely unknown. In this work, we have characterized the expression of SNX9 and SNXl8 in LRO-containing cells and investigated their role in protein trafficking during the formation of LROs. Our results indicate that SNX9 and SNXl8 are not essential for the formation of LROs, nor for the sorting of melanosomal proteins. We investigated how the level of intracellular SNX9 protein is regulated and found that it is a substrate of the ubiquitin ligase Itch, a member of the NEDD4 family of E3 ubiquitin ligases. Itch ubiquitylates SNX9 and regulates SNX9 levels by enhancing its degradation. Using ? truncated proteins we found that the interaction with SNX9 is mediated by the proline-rich domain of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. We further showed that Itch binding is not affected by tyrosine phosphorylation of SNX9. Using lentivector-mediated siRNA techniques, we found that Itch regulates the level of melanosomal proteins, while knock-down of SNX9 does not alter their level. Interestingly, we revealed that silencing of SNXIS affects the amount of the melanosomal protein Melan-A, but also of SNX9, and that SNXl8 can interact with SNX9. Taken together, our results highlight that the pool of substrates of NEDD4 family E3 ligases extends to proteins containing SH3 domains and provide insight into the potential functions of SNXI8. Résumé : Les membres de la famille des Sorting Nexins (SNX) jouent des rôles importants dans le trafic intracellulaire de protéines et membranes. Il a été démontré que la protéine SNX9, qui génère les tubules membranaires, interagit avec plusieurs composants de la machinerie d'endocytose et participe à l'endocytose des récepteurs de surface mediée par la clathrine. Aucune étude n'a investigué si SNX9 pourrait aussi participer à d'autres voies de trafic de protéines tel que le transport vésiculaire, et plus particulièrement la biogenèse des organites lysosomaux ("lysosome-related organelles", LR©s). SNXl8 est similaire à SNX9, mais sa fonction est largement inconnue. Dans ce travail, nous avons caractérisé l'expression de SNX9 et SNX18 dans des cellules contenants des LROs et investigué leur rôle dans le trafic de protéines pendant la formation des LROS. Nos résultats indiquent que SNX9 et SNXI8 ne sont essentiels ni pour la formation des LR©s, ni pour le trafic de protéines mélanosomales. Nous avons examiné la régulation du niveau intracellulaire de la protéine SNX9 et avons trouvé qu'elle est un substrat de l'ubiquitine ligase Itch, un membre de la famille NEDD4 des ubiquitine ligases E3. Itch ubiquitine SNX9 et régule les niveaux de SNX9 en augmentant sa dégradation. En utilisant des protéines mutées nous avons découvert que l'interaction avec SNX9 est médiée par le domaine riche en proline de Itch, qui est différent du domaine conventionnel de reconnaissance WW, et par le domaine SH3 de SNX9. L'interaction avec le domaine riche en proline de Itch est essentielle pour l'ubiquitination et la dégradation de SNX9. De plus, nous avons montré que cette liaison n'est pas affectée par la phosphorylation des résidus tyrosine de SNX9. En utilisant des vecteurs lentiviraux exprimant des siARN, nous avons trouvé que Itch régule les niveaux de protéines mélanosomales, alors que l'extinction de l'expression de SNX9 ne change pas leurs niveaux. En autre, nous avons révélé que la diminution de SNXl8 affecte le niveau de la protéine mélanosomale Melan-A et de SNX9, et aussi que SNXl8 peut interagir avec SNX9. En résumé, nos résultats démontrent que l'ensemble des substrats de la famille NEDD4 des ubiquitine ligases E3 s'élargit aux protéines contenant des domaines SH3 et ouvrent des perspectives sur les fonctions potentielles de SNXl8.

Dispersionless transport in a washboard potential

We study and characterize a new dynamical regime of underdamped particles in a tilted washboard potential. We find that for small friction in a finite range of forces the particles move essentially nondispersively, that is, coherently, over long intervals of time. The associated distribution of the particle positions moves at an essentially constant velocity and is far from Gaussian-like. This new regime is complementary to, and entirely different from, well-known nonlinear response and large dispersion regimes observed for other values of the external force.

Ventral and dorsal pathways of speech perception: An intracerebral ERP study.

Recent theory of physiology of language suggests a dual stream dorsal/ventral organization of speech perception. Using intra-cerebral Event-related potentials (ERPs) during pre-surgical assessment of twelve drug-resistant epileptic patients, we aimed to single out electrophysiological patterns during both lexical-semantic and phonological monitoring tasks involving ventral and dorsal regions respectively. Phonological information processing predominantly occurred in the left supra-marginal gyrus (dorsal stream) and lexico-semantic information occurred in anterior/middle temporal and fusiform gyri (ventral stream). Similar latencies were identified in response to phonological and lexico-semantic tasks, suggesting parallel processing. Typical ERP components were strongly left lateralized since no evoked responses were recorded in homologous right structures. Finally, ERP patterns suggested the inferior frontal gyrus as the likely final common pathway of both dorsal and ventral streams. These results brought out detailed evidence of the spatial-temporal information processing in the dual pathways involved in speech perception.

Transport of the organic cation N1-methylnicotinamide by the rabbit proximal tubule. I. Accumulation in the isolated nonperfused tubule.

Isolated nonperfused rabbit renal proximal tubules were used to investigate the basolateral step of transport of the organic cation N1-methylnicotinamide (NMN). NMN accumulation was highest and saturable in S2 and S3 segments, but lowest and nonsaturable in S1 segments. In S1 segments, accumulation of [3H]-NMN (0.5-8 microM in the bath) resulted in an average tubular water/medium concentration ratio (T/M) of 8.2, whereas in S2 and S3 segments T/M averaged 19.5 and 18.6, respectively. At these concentrations, about 30% of the label was attached in all segments to a metabolite comigrating with nicotinamide. KCN (10(-2) M) or ouabain (10(-4) M) reduced T/M to about 8 for all segments. NMN accumulation was inhibited (to a T/M of about 3 with mepiperphenidol) by other organic cations (10(-5)-10(-3) M) with the potency sequence mepiperphenidol greater than tetraethylammonium = quinine greater than morphine, these organic cations having no effect on p-aminohippurate accumulation, except for the highest concentration of quinine (10(-3) M). After correction for metabolism, NMN accumulation could be accounted for by simple electrochemical equilibrium across the basolateral membrane. The basolateral step of NMN transport appears therefore to be a carrier-mediated diffusion, in opposition to the active basolateral accumulation described for tetraethylammonium.