CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation.

CARMA1 is a lymphocyte-specific member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which coordinate signaling pathways emanating from the plasma membrane. CARMA1 interacts with Bcl10 via its caspase-recruitment domain (CARD). Here we investigated the role of CARMA1 in T cell activation and found that T cell receptor (TCR) stimulation induced a physical association of CARMA1 with the TCR and Bcl10. We found that CARMA1 was constitutively associated with lipid rafts, whereas cytoplasmic Bcl10 translocated into lipid rafts upon TCR engagement. A CARMA1 mutant, defective for Bcl10 binding, had a dominant-negative (DN) effect on TCR-induced NF-kappa B activation and IL-2 production and on the c-Jun NH(2)-terminal kinase (Jnk) pathway when the TCR was coengaged with CD28. Together, our data show that CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation and CD28 costimulation-dependent Jnk activation.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [214-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.

Aldosterone paradox: differential regulation of ion transport in distal nephron.

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K(+) secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K(+) balance.

Purine transport and the cell cycle.

Background: Alliance evolutions, i.e. ruptures and resolutions over the course of psychotherapy, have been shown to be important descriptive features in different forms of psychotherapy, and in particular in psychodynamic psychotherapy. This case study of a client presenting elements of adjustment disorder undergoing short-term dynamic psychotherapy is drawn from a systematic naturalistic study and aims at illustrating, on a session-by-session-level, the processes of alliance ruptures and resolutions, by comparing both the client's and the therapist's perspectives. Method: Two episodes of alliance evolution were more fully studied, in relation to the evolution of transference, as well as the client's defensive functioning and core conflictual theme. These concepts were measured by means of valid, reliable observer-rater methods, based on session transcripts: the Defense Mechanisms Rating Scales (DMRS) for defensive functioning and the Core Conflictual Relationship Theme (CCRT) for the conflicts. Alliance was measured after each session using the Helping Alliance questionnaire (HAq-II). Results: The results indicated that these episodes of alliance rupture and resolutions may be understood as key moments of the whole therapeutic process reflecting the client's main relationship stakes. Illustrations are provided based on the client's in-session processes and related to the alliance development over the course of the entire therapy.

Biometal muscle to restore atrial transport function in a permanent atrial fibrillation animal model: a potential tool in the treatment of end-stage heart failure.

BACKGROUND: Half of the patients with end-stage heart failure suffer from persistent atrial fibrillation (AF). Atrial kick (AK) accounts for 10-15% of the ejection fraction. A device restoring AK should significantly improve cardiac output (CO) and possibly delay ventricular assist device (VAD) implantation. This study has been designed to assess the mechanical effects of a motorless pump on the right chambers of the heart in an animal model. METHODS: Atripump is a dome-shaped biometal actuator electrically driven by a pacemaker-like control unit. In eight sheep, the device was sutured onto the right atrium (RA). AF was simulated with rapid atrial pacing. RA ejection fraction (EF) was assessed with intracardiac ultrasound (ICUS) in baseline, AF and assisted-AF status. In two animals, the pump was left in place for 4 weeks and then explanted. Histology examination was carried out. The mean values for single measurement per animal with +/-SD were analysed. RESULTS: The contraction rate of the device was 60 per min. RA EF was 41% in baseline, 7% in AF and 21% in assisted-AF conditions. CO was 7+/-0.5 l min(-1) in baseline, 6.2+/-0.5 l min(-1) in AF and 6.7+/-0.5 l min(-1) in assisted-AF status (p<0.01). Histology of the atrium in the chronic group showed chronic tissue inflammation and no sign of tissue necrosis. CONCLUSIONS: The artificial muscle restores the AK and improves CO. In patients with end-stage cardiac failure and permanent AF, if implanted on both sides, it would improve CO and possibly delay or even avoid complex surgical treatment such as VAD implantation.

The interaction of human pathogenic arena viruses with the host cell

SummaryResearch projects presented in this thesis aimed to investigate two major aspects of the arenaviruses life cycle in the host cell: viral entry and the biosynthesis of the viral envelope glycoprotein.Old World arenaviruses (OWAV), such as Lassa virus (LASV) and lymphocytic choriomeningitis virus (LCMV), attach to the cell by binding to their receptor, alpha-dystroglycan. Virions are then internalized by a largely unknown pathway of endocytosis and delivered to the late endosome/lysosome where fusion occurs at low pH. In the major project of my thesis, we sought to identify cellular factors involved in OWAV cell entry. Our work indicates that OWAV cell entry requires microtubular transport and a functional multivesicular body (MVB) compartment. Infection indeed depends on phosphatidyl inositol 3-kinase (PI3K) activity and lysobisphosphatidic acid (LBPA), a lipid found in membranes of intraluminal vesicles (ILVs) of the MVB. We further found a requirement of factors that are part of the endosomal sorting complex required for transport (ESCRT), involved in the formation of ILVs. This suggests an ESCRT-mediated sorting of virus- receptor complex during the entry process.During viral replication, biosynthesis of viral glycoprotein takes place in the endoplasmic reticulum (ER) of the host cell. When protein load exceeds the folding capacity of the ER, the accumulation of unfolded proteins is sensed by three ER resident proteins, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and PKR-like ER kinase (PERK), whose signaling induces the cellular unfolded protein response (UPR). Our results indicate that acute LCMV infection transiently induces the activation of the ATF6 branch of the UPR, whereas the PERK, and IRE1 axis of UPR are neither triggered nor blocked during infection. Our data also demonstrate that activation of ATF6 pathway is required for optimal viral replication during acute infection.The formation of the mature, fusion-active form of arenaviruses glycoproteins requires proteolytic cleavage mediated by the cellular protease subtilisin kexin isozyme-1 (SKI-l)/site-l protease (SIP). We show that targeting the SKI-1/S1P enzymatic activity with specific inhibitors is a powerful strategy to block arenaviruses productive infection. Moreover, characterization of protease function highlights differences in processing between cellular and viral substrates, opening new possibilities in term of drug development against human pathogenic arenaviruses.RésuméLes projets de recherche présentés dans cette thèse visaient à étudier deux aspects du cycle de vie des arenavirus: l'entrée du virus dans la cellule hôte et la biosynthèse de la glycoprotéine durant la réplication virale.Les arenavirus du vieux monde (OWAV), tels que le virus de Lassa (LASV) et le virus de la chorioméningite lymphocytaire (LCMV) s'attachent à la cellule hôte en se liant à leur récepteur, l'alpha-dystroglycane. Les virions sont ensuite intemalisés par une voie d'endocytose inconnue et livrés à l'endosome tardif/lysosome, où le pH acide permet la fusion entre l'enveloppe virale et la membrane du compartiment. Le projet principal de ma thèse consistait à identifier les facteurs cellulaires impliqués dans l'entrée des OWAV dans la cellule hôte. Nos résultats indiquent que l'entrée des OWAV nécessite le transport microtubulaire et la présence d'un corps multivésiculaire (MVB) fonctionnel. L'infection dépend en effet de l'activité de phosphatidyl inositol 3-kinase (PI3K) et de lysobisphosphatidic acid (LBPA), un lipide présent dans les membranes des vésicules intraluminales (ILVs) du MVB. Nous avons également trouvé l'implication de facteurs constituant l'endosomal sorting complex required for sorting (ESCRT) qui joue un rôle dans la formation des ILVs. Ces donnés suggèrent l'incorporation du complexe virus-récepteur dans des ILVs durant le processus d'entrée.Lors de la réplication virale, la biosynthèse de la glycoprotéine virale a lieu dans le réticulum endoplasmique (ER) de la cellule hôte. Lorsque la charge de protéines nouvellement synthétisées excède la capacité de pliage des protéines dans le ER, l'accumulation de protéines mal pliées est détectée par trois facteurs: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) et PKR-like ER kinase (PERK). Leur signalisation constitue la réponse cellulaire face aux protéines mal pliées (UPR). Nos résultats montrent que l'infection aiguë avec LCMV induit transitoirement l'activation de la voie de signalisation ATF6 alors que les axes PERK et IRE1 de l'UPR ne sont ni induits ni bloqués pendant l'infection. Nos données prouvent également que l'activation de la voie ATF6 est nécessaire à une réplication virale optimale lors de l'infection aiguë avec LCMV.La maturation des glycoprotéines des arenavirus nécessite un clivage protéolytique par la protéase cellulaire subtilisin kexin isozyme-1 (SKI-l)/site-l protease (SIP). Nous avons démontré que le ciblage de l'activité enzymatique de SKI-1/SIΡ avec des inhibiteurs spécifiques est une stratégie prometteuse pour bloquer l'infection par les arenavirus. La caractérisation du mécanisme d'action de la protéase a, par ailleurs, révélé des différences au niveau du clivage entre les substrats cellulaires et viraux, ce qui ouvre de nouvelles perspectives en terme de développement de médicaments contre les arenavirus pathogènes pour l'homme.

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts.

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

Anàlisi del riu Llobregat com a via de transport del Baix Llobregat a l'època romana, amb eines SIG

Desenvolupament d'un Sistema d'Informació Geogràfica (SIG), que permeti analitzar les funcions dels jaciments romans de la zona del riu Llobregat i consultar els jaciments, emmagatzemats en una base de dades de forma espacial.

NF-kappaB inhibits sodium transport via down-regulation of SGK1 in renal collecting duct principal cells.

Tubulointerstitial inflammation is a common feature of renal diseases. We have investigated the relationship between inflammation and Na(+) transport in the collecting duct (CD) using the mCCD(cl1) and mpkCDD(cl4) principal cell models. Lipopolysaccharide (LPS) decreased basal and aldosterone-stimulated amiloride-sensitive transepithelial current in a time-dependent manner. This effect was associated with a decrease in serum and glucocorticoid-regulated kinase 1 (SGK1) mRNA and protein levels followed by a decrease in epithelial sodium channel (ENaC) alpha-subunit mRNA levels. The LPS-induced decrease in SGK1 expression was confirmed in isolated rat CD. This decreased expression of either SGK1 or the ENaC alpha-subunit was not due to enhanced degradation of mRNA. In contrast, LPS inhibited transcriptional activity of the SGK1 promoter measured by luciferase-reporter gene assay. The effect of LPS was not mediated by inhibition of mineralocorticoid or glucocorticoid receptor, because expression of both receptors was unchanged and blockade of either receptor by spironolactone or RU486, respectively, did not prevent the down-regulation of SGK1. The effect of LPS was mediated by the canonical NF-kappaB pathway, as overexpression of a constitutively active mutant, IKKbeta (inhibitor of nuclear factor kappaB kinase-beta) decreased SGK1 mRNA levels, and knockdown of p65 NF-kappaB subunit by small interfering RNA increased SGK1 mRNA levels. Chromatin immunoprecipitation showed that LPS increased p65 binding to two NF-kappaB sites along the SGK1 promoter. In conclusion, we show that activation of the NF-kappaB pathway down-regulates SGK1 expression, which might lead to decreased ENaC alpha-subunit expression, ultimately resulting in decreased Na(+) transport.

Imaging and quantifying salt-tracer transport in a riparian groundwater system by means of 3D ERT monitoring

Determining groundwater flow paths of infiltrated river water is necessary for studying biochemical processes in the riparian zone, but their characterization is complicated by strong temporal and spatial heterogeneity. We investigated to what extent repeat 3D surface electrical resistance tomography (ERT) can be used to monitor transport of a salt-tracer plume under close to natural gradient conditions. The aim is to estimate groundwater flow velocities and pathways at a site located within a riparian groundwater system adjacent to the perialpine Thur River in northeastern Switzerland. Our ERT time-lapse images provide constraints on the plume's shape, flow direction, and velocity. These images allow the movement of the plume to be followed for 35 m. Although the hydraulic gradient is only 1.43 parts per thousand, the ERT time-lapse images demonstrate that the plume's center of mass and its front propagate with velocities of 2x10(-4) m/s and 5x10(-4) m/s, respectively. These velocities are compatible with groundwater resistivity monitoring data in two observation wells 5 m from the injection well. Five additional sensors in the 5-30 m distance range did not detect the plume. Comparison of the ERT time-lapse images with a groundwater transport model and time-lapse inversions of synthetic ERT data indicate that the movement of the plume can be described for the first 6 h after injection by a uniform transport model. Subsurface heterogeneity causes a change of the plume's direction and velocity at later times. Our results demonstrate the effectiveness of using time-lapse 3D surface ERT to monitor flow pathways in a challenging perialpine environment over larger scales than is practically possible with crosshole 3D ERT.

Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand.

In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

Linking epigenetics to lipid metabolism: focus on histone deacetylases.

A number of recent studies revealed that epigenetic modifications play a central role in the regulation of lipid and of other metabolic pathways such as cholesterol homeostasis, bile acid synthesis, glucose and energy metabolism. Epigenetics refers to aspects of genome functions regulated in a DNA sequence-independent fashion. Chromatin structure is controlled by epigenetic mechanisms through DNA methylation and histone modifications. The main modifications are histone acetylation and deacetylation on specific lysine residues operated by two different classes of enzymes: Histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. The interaction between these enzymes and histones can activate or repress gene transcription: Histone acetylation opens and activates chromatin, while deacetylation of histones and DNA methylation compact chromatin making it transcriptionally silent. The new evidences on the importance of HDACs in the regulation of lipid and other metabolic pathways will open new perspectives in the comprehension of the pathophysiology of metabolic disorders.

Mechanisms of leukocyte lipid body formation and function in inflammation

An area of increasingly interest for the understanding of cell signaling are the spatio-temporal aspects of the different enzymes involved in lipid mediator generation (eicosanoid-forming enzymes, phospholipases and their regulatory kinases and phosphatases) and pools of lipid precursors. The compartmentalization of signaling components within discrete and dynamic sites in the cell is critical for specificity and efficiency of enzymatic reactions of phosphorilation, enzyme activation and function. We hypothesized that lipid bodies - inducible non-membrane bound cytoplasmic lipid domains - function as specialized intracellular sites of compartmentalization of signaling with major roles in lipid mediator formation within leukocytes engaged in inflammatory process. Over the past years substantial progresses have been made demonstrating that all enzymes involved in eicosanoid synthesis localize at lipid bodies and lipid bodies are distinct sites for eicosanoid generation. Here we will review our current knowledge on the mechanisms of formation and functions of lipid bodies pertinent to inflammation.

Electronic monitoring of compliance to lipid-lowering therapy in clinical practice

Nonadherence to treatment is a common problem in the clinical management of hypercholesterolemic patients. This study was carried out with the aim of monitoring the daily compliance to a 6-month course of lipid-lowering therapy, using a microelectronic device, the Medication Event Monitoring System (MEMS), versus pill count. Forty men with primary hypercholesterolemia were prescribed fluvastatin 1 x 40 mg daily, provided in a MEMS package to record the date and time of each opening of the pillbox. Thirty-nine of 40 patients (98%) completed the study. Total cholesterol and LDL cholesterol levels decreased significantly (18% and 25%, p < 0.001) during the 6-month therapy period. A high mean rate of compliance was achieved by MEMS using the following three indexes--compliance to total prescribed dose (88.8% +/- 13.5%), compliance to prescribed days (82.4% +/- 19.5%), and compliance to prescribed time of day (81.86% +/- 19.5%)--and by pill count (93.4% +/- 9.5%). In addition, the MEMS provided some patterns of nonadherence to medication, undetectable by pill count alone, such as a drug holiday in 38% of cases, a drug omission for more than 7 consecutive days in 9% of cases, and, conversely, use of more than the one prescribed daily dose in 47% of cases. A significant correlation between the rate of compliance and the decrease in LDL cholesterol was observed only when the compliance was assessed by MEMS. The results indicate that MEMS is a useful tool for monitoring compliance in clinical practice and may possibly increase adherence to long-term lipid-lowering therapy.