Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway.

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.

THE CXCR7/CXCR4/CXCL12 AXIS IN HUMAN NEUROBLASTOMA: INVOLVEMENT IN MALIGNANT PROGRESSION

Le neuroblastome (NB) est la tumeur maligne solide extra-crânienne la plus fréquente chez le jeune enfant. L'évolution clinique est très hétérogène, et les NBs de haut risque échappent encore aux traitements les plus agressifs. Diverses études ont montré que les chimiokines et leurs récepteurs, particulièrement l'axe CXCR4/CXCL12, sont impliqués dans la progression tumorale. Dans le NB, l'expression de CXCR4 est corrélée à un pronostic défavorable. De récentes études ont identifié l'expression d'un autre récepteur, CXCR7, présentant une forte affinité pour le ligand CXCL12. Cependant, son implication potentielle dans l'agressivité des NBs reste encore inconnue. Notre étude a pour objectif d'analyser le rôle de CXCR7 dans le comportement malin du NB, et son influence sur la fonctionnalité de l'axe CXCR4/CXCL12. Les profils d'expression de CXCR7 et CXCL12 ont d'abord été évalués sur un large échantillonnage de tissus de NB, incluant des tissus de tumeurs primaires et de métastases, provenant de 156 patients. CXCL12 est fortement détecté dans les vaisseaux et le stroma des tumeurs. Contrairement à CXCR4, CXCR7 n'est que très faiblement exprimé par les tumeurs indifférenciées. Néanmoins, l'expression de CXCR7 augmente dans les tumeurs matures, et se trouve spécifiquement associée aux cellules neurales différentiées, telles que les cellules ganglionnaires. L'expression de CXCR7 est faiblement détectée dans un nombre réduit de lignées de NB, mais peut-être induite suite à des traitements avec des agents de différenciation in vitro. La surexpression de CXCR7, CXCR4 et une combinaison des deux récepteurs dans les lignées IGR-NB8 et SH-SY5Y a permis l'analyse de leur fonction respective. En réponse à leur ligand commun, chaque récepteur induit l'activation de la voie ERK 1/2, mais pas celle de la voie Akt. Contrairement à CXCR4, l'expression exogène de CXCR7 réduit fortement la prolifération des cellules de NB in vitro, et in vivo dans un modèle d'injection sous-cutanée de. souris immunodéprimées. CXCR7 altère également la migration des cellules induite par l'axe CXCR4/CXCL12. De plus, l'utilisation d'un modèle orthotopique murin a démontré que la croissance tumorale induite par CXCR4 peut être fortement retardée lorsque les deux récepteurs sont co-exprimés dans les cellules de NB. Aucune induction de métastases n'a pu être observée dans ce modèle. Cette étude a permis d'identifier un profil d'expression opposé et des rôles distincts pour CXCR7 et CXCR4 dans le NB. En effet, contrairement à CXCR4, CXCR7 présente des propriétés non tumorigéniques et peut être associé au processus de différenciation du NB. De plus, nos analyses suggèrent que CXCR7 peut réguler les mécanismes induits par CXCR4. Ces données ouvrent donc de nouvelles perspectives de recherche quant au rôle de l'axe CXCR7/CXCR4/CXCL12 dans la biologie des NBs. - Neuroblastoma (NB) is a typical childhood and heterogeneous neoplasm for which efficient targeted therapy for high-risk tumours is not yet identified. The chemokine CXCL12, and its receptors CXCR4 and CXCR7 have been involved in tumour progression and dissemination in various cancer models. In the context of NB, CXCR4 expression is associated to undifferentiated tumours and poor prognosis, while the role of CXCR7, the recently identified second CXCL12 receptor, has not yet been elucidated. In this report, CXCR7 and CXCL12 expression were evaluated using a tissue micro-array (TMA) including 156 primary and 56 metastatic NB tissues. CXCL12 was found to be highly associated to NB vascular and stromal structures. In opposite to the CXCR4 expression pattern, the neural-associated CXCR7 expression was extremely low in undifferentiated tumours, while its expression increased in maturated tissues and was specifically associated to the differentiated neural tumour cells. As determined by RT-PCR, CXCR7 expression was only found in a minority of NB cell lines. Moreover, its expression in two CXCR7-negative NB cell lines was further induce upon treatment with differentiation agents in vitro. The relative roles of the two CXCL12 receptors was further assessed by overexpressing individual CXCR7 or CXCR4 receptors, or a combination of both, in the IGR-NB8 and SH-SY5Y NB cell lines. In vitro functional analyses indicated that, in response to their common ligand, both receptors induced activation of ERK 1/2 cascade, but not Akt signaling pathway. CXCR7 strongly reduced in vitro growth, in contrast to CXCR4. Sub-cutaneous implantations of CXCR7-expressing NB cells showed that CXCR7 also drastically reduced in vivo growth. Moreover, CXCR7 impaired CXCR4-mediated chemotaxis, and altered CXCR4-mediated growth when CXCR4/CXCR7-expressing NB cells were engrafted orthotopically in mouse adrenal gland, a CXCL12-producing environment. In such model, CXCR7 alone, or in association with CXCR4, did not induce NB cell metastatic dissemination. In conclusion, the CXCL12 receptors, CXCR7 and CXCR4, revealed opposite expression patterns and distinct functional roles in NB. While CXCR4 favours NB growth and chemotaxis, CXCR7 elicits anti-tumorigenic properties and may be associated with NB differentiation. Importantly, CXCR7 may act as a negative modulator of CXCR4 signaling, further opening new research perspectives for the role of the global CXCR7/CXCR4/CXCL12 axis in NB.

Capsaicin mimics mechanical load-induced intracellular signaling events: involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy.

Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca ( 2+) ]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca ( 2+) ]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

Involvement of microglia-neuron interactions in the tumor necrosis factor-alpha release, microglial activation, and neurodegeneration induced by trimethyltin.

Trimethyltin (TMT) is a neurotoxicant known to induce early microglial activation. The present study was undertaken to investigate the role played by these microglial cells in the TMT-induced neurotoxicity. The effects of TMT were investigated in monolayer cultures of isolated microglia or in neuron-enriched cultures and in neuron-microglia and astrocyte-microglia cocultures. The end points used were morphological criteria; evaluation of cell death and cell proliferation; and measurements of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) release in culture supernatant. The results showed that, in cultures of microglia, TMT (10(-6) M) caused, after a 5-day treatment, an increased release of TNF-alpha, without affecting microglial shape or cell viability. When microglia were cocultured with astrocytes, TNF-alpha release was decreased to undetectable levels. In contrast, in neuron-microglia cocultures, TNF-alpha levels were found to increase at lower concentrations of TMT (i.e., 10(-8) M). Moreover, at 10(-6) M of TMT, microglia displayed further morphological activation, as suggested by process retraction and by decrease in cell size. No morphological activation was observed in cultures of isolated microglial cells and in astrocyte-microglia cocultures. With regard to neurons, 10(-6) M of TMT induced about 30% of cell death, when applied to neuron-enriched cultures, whereas close to 100% of neuronal death was observed in neuron-microglia cocultures. In conclusion, whereas astrocytes may rather dampen the microglial activation by decreasing microglial TNF-alpha production, neuronal-microglial interactions lead to enhanced microglial activation. This microglial activation, in turn, exacerbates the neurotoxic effects of TMT. TNF-alpha may play a major role in such cell-cell communications.

Documenting a time-bound, circular view of hierarchies: a microanalysis of parent-infant dyadic interaction.

This article presents a new theory that separates the levels of communication and relates them circularly, namely, by separating time from space/meaning variables. Documenting this proposition requires sequential microdescriptions--a far-out project in the field of family therapy. In an extensive study of clinical and nonclinical families, starting with available microanalytic data on nonverbal parent-infant dialogue, distinct time organizations have been found to modify the degree of circularity between the levels of interaction according to the observed types of engagement, that is, consensual, conflictual, and paradoxical. The double description of the dyad as a totality versus the dyad as a framing/developing organization imparts crucial information on how development proceeds in dyadic, co-evolutive systems, and presumably in larger ones too. In this perspective, a model is elaborated and then applied to a case description in our therapeutic consultation.

The influence of sentinel lymph node tumour burden on additional lymph node involvement and disease-free survival in cutaneous melanoma: a retrospective analysis of 392 cases

Twenty per cent of sentinel lymph node (SLN)-positive melanoma patients have positive non-SLN lymph nodes in completion lymph node dissection (CLND). We investigated SLN tumour load, non-sentinel positivity and disease-free survival (DFS) to assess whether certain patients could be spared CLND. Sentinel lymph node biopsy was performed on 392 patients between 1999 and 2005. Median observation period was 38.8 months. Sentinel lymph node tumour load did not predict non-SLN positivity: 30.8% of patients with SLN macrometastases (> or =2 mm) and 16.4% with micrometastases (< or =2 mm) had non-SLN positivity (P=0.09). Tumour recurrences after positive SLNs were more than twice as frequent for SLN macrometastases (51.3%) than for micrometastases (24.6%) (P=0.005). For patients with SLN micrometastases, the DFS analysis was worse (P=0.003) when comparing those with positive non-SLNs (60% recurrences) to those without (17.6% recurrences). This difference did not translate into significant differences in DFS: patients with SLN micrometastasis, either with (P=0.022) or without additional positive non-SLNs (P<0.0001), fared worse than patients with tumour-free SLNs. The 2-mm cutoff for SLN tumour load accurately predicts differences in DFS. Non-SLN positivity in CLND, however, cannot be predicted. Therefore, contrary to other studies, no recommendations concerning discontinuation of CLND based on SLN tumour load can be deduced.

Acid-sensing ion channels : modulation by serine-proteases and involvement in acid-induced action potential generation in hippocampal neurons

SUMMARY Acid-sensing ion channels (ASICs) are non-voltage gated sodium channels. They are activated by rapid extracellular acidification and generate an inactivating inward current. Four ASIC genes have been cloned: ASIC1, 2, 3 and 4, with variants a and b for ASIC1and AS1C2. ASICs are expressed in neurons of the central (CNS) and peripheral nervous system (PNS). In the CNS, ASICs have a role in learning, memory, as well as in neuronal death in ischemia. In the PNS, ASICs are involved in the perception of acid-induced pain, as well as in mechanoperception. In one part of my thesis project, we addressed the question of the mechanism of regulation of ASIC1 a by the serine protease trypsin at the molecular level. Trypsin modifies the function of ASIC1 a but not of ASIC1b. In order to identify the channel region responsible for this effect, we created chimeras between ASIC1 a and 1b. Subsequently, to identify the exact trypsin target(s), we mutated predicted trypsin sites in the region identified by the chimera. In the second part of a project, we investigated the role of ASICs at the cellular level, in neuronal signaling. Using the whole-cell patch clamp in hippocampal neuronal culture, we studied the potential involvement of ASICs in action potential (AP) generation. In the first part of the thesis work, we showed that trypsin modifies ASIC1a function: it shifts the pH activation and the steady-state inactivation curve towards more acidic values and accelerates the time course of the channel recovery from inactivation. We also showed that trypsin cleaves ASIC1a and that the functional effect and a channel cleavage correlate. In the inactivated state, channels cannot be modified by trypsin. Cleavage occurs in a channel region that is also important for inactivation of all ASICs; a part of this region is critical for the inhibition of ASIC1 a by the spider toxin Psalmotoxin1. In the second part of the thesis work, we showed that ASIC activity can modulate AP generation. ASIC activity by itself can induce trains of APs. In situations in which this activity by itself is not sufficient to induce APs, it can contribute to AP generation. During high neuronal activity, ASIC activity can block already existing trains of APs. In conclusion, depending on the activity of neuron in a particular moment, ASICs can differently modulate AP generation; they can induce, facilitate or inhibit APs. We also showed that trypsin changes the capability of ASICs to modulate AP generation by shifting the pH dependence to more acidic values, which adapts channel gating to pH conditions which may occur in pathological conditions such as ischemia. Our finding that trypsin modifies ASIC1 a function identifies a novel pharmacological tool, and proposes a mechanism of ASIC1a regulation that may have a physiological importance. The identification of the exact site of trypsin action gives insight to the molecular mechanisms of ASIC regulation. This work proposes a role in modulation of AP generation for ASICs in the CNS. RESUME Les canaux ASIC sont les canaux ioniques activés par l'acidification rapide extracellulaire. Activés, ils génèrent un courant entrant qui inactive en présence de stimulus acide. Quatre gènes ASIC ont été clonés, ASIC1, 2, 3 et 4, avec les variants a et b pour ASIC1 et 2. Les ASICs sont exprimés dans les neurones du système nerveux central (SNC) et périphérique (SNP). Dans le SNC, les ASIC ont un rôle dans le mémoire, apprentissage et la mort neuronale dans t'ischémie. Dans le SNP, ils ont un rôle dans la perception de la douleur et méchanosensation. Dans une partie de mon projet de thèse, nous avons étudié les mécanismes de la régulation d'ASIC1a par la sérine-protéase trypsine au niveau moléculaire. La trypsine modifie la fonction d'ASIC1a et pas ASIC1b. Nous avons créé les chimères entre ASIC1 a et 1 b, afin d'identifier la région du canal responsable pour l'effet. Pour identifier le(s) site(s) exactes de l'action de la trypsine, nous avons muté les sites potentiels de la trypsine dans la région identifiée par les chimères. Dans la deuxième partie du projet, nous avons étudié le rôle des ASICs au niveau cellulaire. En utilisant la technique du patch clamp dans les cultures des neurones de l'hippocampe, nous avons étudié l'implication des ASICs dans la génération des potentiels d'action (PA). Nous avons montré que la trypsine agit sur le canal ASIC1a ; elle décale l'activation et « steady-state » inactivation vers les valeurs plus acides, et elle raccourcit le temps du « recovery » du canal. La trypsine coupe ASIC1a sur le résidu K145 et l'effet fonctionnel et la coupure corrèlent. Nous avons identifié la région du canal responsable pour l'inactivation de tous les ASICs ; une partie de cette région est responsable pour ['inhibition d'ASIC1 a par la Psalmotoxinel . Nous avons montré que les ASICs peuvent moduler la génération des PAs. L'activité des ASICs peut induire les trains des PAs. Quand l'activité des ASICs n'est pas suffisante pour induire le PA, elle peut contribuer à sa génération. Pendant l'activité neuronale forte, l'activité des ASICs peut bloquer les trains des PAs qui existent déjà. En conclusion, dépendant de l'activité neuronale, les ASICs peuvent moduler la génération des PAs différemment ; ils peuvent induire, faciliter ou inhiber les PAs. La trypsine change la capacité des ASICs de moduler les PAs. Après l'action de la trypsine, les ASICs peuvent moduler la génération des PAs dans les conditions légèrement acides, suivies par les fluctuations du pH acide, qui peuvent exister dans l'ischémie. Le fait que la trypsine agit sur ASIC1a définit l'outil pharmacologique et propose le mécanisme de la régulation d'ASICI a qui pourrait avoir l'importance physiologique. L'identification du site de l'action de la trypsine éclaircit les mécanismes moléculaires de la régulation des ASICs. Cette étude propose un rôle des ASICs dans la modulation de la génération des PAs. Résumé pour le public large Les neurones sont les cellules de système nerveux dont la fonction est la signalisation. Comme toutes les autres cellules, les neurones ont une membrane qui sépare l'intérieur du milieu extérieur. Cette membrane est imperméable pour des particules chargées (ions). Dans cette membrane existent les protéines spécifiques, « canaux », qui permettent le transport des ions d'un côté de la membrane à l'autre, comme réponse aux stimuli différents. Ce transport des ions à travers la membrane génère un courant, qu'on peut mesurer. Ce courant est la base de la communication entre les neurones, ou, ce qu'on appelle la signalisation neuronale. Quand ce courant est suffisamment grand, il permet la génération du potentiel d'action, qui est le message principal de communication neuronale. Les canaux ASIC (acid-sensing ion channel), que nous étudions dans le laboratoire, sont activés par les acides. Les acides sont relâchés dans beaucoup de situations dans le système nerveux. Les ASIC ont été découverts récemment (en 1996), et nous ne connaissons pas encore très bien toutes les fonctions de ces canaux. Nous savons qu'ils ont un rôle dans le mémoire, apprentissage, la sensation de la douleur et l'infarctus cérébral. Dans la première partie de ce projet de thèse, nous avons voulu mieux comprendre comment fonctionnent ces canaux. Pour faire ça, nous avons étudié la régulation des ASICs par une protéine, trypsine, qui coupe le canal ASIC. Nous avons étudié ou exactement la trypsine coupe le canal et quels effets ça produit sur la fonction du canal. Dans la deuxième partie du projet de thèse, nous avons voulu mieux connaître comment le canal fonctionne au niveau de la cellule, comment il interagit avec les autres canaux et si il a un rôle dans la génération des potentiels d'action. Nous avons pu montrer que la trypsine change la fonction du canal, ce qui lui permet de fonctionner différemment. Nous avons aussi déterminé ou exactement ta trypsine coupe le canal. Au niveau de la cellule, nous avons montré que les ASIC peuvent moduler la génération des potentiels d'action, étant, dépendant de l'activité du neurone, soit activateurs, soit inhibiteurs. La trypsine est une molécule qui peut être libérée dans le système nerveux pendant certaines conditions, comme l'infarctus cérébral. A cause de ça, les connaissances que la trypsine agit sur le anal ASIC pourraient être important physiologiquement. La connaissance de l'endroit exacte ou la trypsine coupe le canal nous aide à mieux comprendre la relation structure-fonction du canal. La modulation de la génération des potentiels d'actions par les ASIC indique que ces canaux peuvent avoir un rôle important dans la signalisation neuronale.

Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior.

The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor alpha (PPARalpha) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARalpha helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARalpha helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARalpha transcription activation potential. Finally, we disclose important functions of residues in PPARalpha AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARalpha constitutive activity, without changing PPARalpha ligand-dependent activation potential.

Effectiveness of a pediatric palliative home care team as experienced by parents and health care professionals.

OBJECTIVES: Little data are available on palliative home care for children. The objective of this study was to evaluate the effectiveness of a specialized pediatric palliative home care team (PPHCT) as experienced by parents and health care professionals (HCPs). METHODS: Parents and HCPs involved in the care of terminally ill children who died and whom the PPHCT was in charge of were surveyed with questionnaires focusing on satisfaction with the PPHCT, satisfaction with the course of the dying phase, and the development of anxiety, depression, and prolonged grief disorder. RESULTS: Forty-three parent dyads participated (return rate, 88%). Satisfaction with the PPHCT scored a median of 10 (numeric rating scale, 0-10). The child's death was predominantly experienced as very peaceful (median, 9); 71% died at home. According to parents, involvement of the PPHCT led to highly significant (p<0.001) improvements in the children's symptoms and quality of life, as well as in aspects of communication and administrative barrier reduction. Anxiety was detected in 25% of parents, depression in 19%, and prolonged grief disorder in 13%. HCPs (return rate, 83%) evaluated all investigated care domains (particularly cooperation/communication/family support) as being significantly improved (p<0.001). Thirty-five percent of HCPs felt uncertain concerning pediatric palliative care; 79% would welcome specific training opportunities. CONCLUSIONS: Involvement of a PPHCT is experienced as a substantial improvement of care by parents and HCPs. Coordination of palliative care during the last phase of life appears to be an important quality factor for the home care of dying children and their families.

Primary motor cortex involvement in Alzheimer disease.

In Alzheimer disease (AD) the involvement of entorhinal cortex, hippocampus, and associative cortical areas is well established. Regarding the involvement of the primary motor cortex the reported data are contradictory. In order to determine whether the primary motor cortex is involved in AD, the brains of 29 autopsy cases were studied, including, 17 cases with severe cortical AD-type changes with definite diagnoses of AD, 7 age-matched cases with discrete to moderate cortical AD-type changes, and 5 control cases without any AD-type cortical changes. Morphometric analysis of the cortical surface occupied by senile plaques (SPs) on beta-amyloid-immunostained sections and quantitative analysis of neurofibrillary tangles (NFTs) on Gallyas-stained sections was performed in 5 different cortical areas including the primary motor cortex. The percentage of cortical surface occupied by SPs was similar in all cortical areas, without significant difference and corresponded to 16.7% in entorhinal cortex, 21.3% in frontal associative, 16% in parietal associative, and 15.8% in primary motor cortex. The number of NFTs in the entorhinal cortex was significantly higher (41 per 0.4 mm2), compared with those in other cortical areas (20.5 in frontal, 17.9 in parietal and 11.5 in the primary motor cortex). Our findings indicate that the primary motor cortex is significantly involved in AD and suggest the appearance of motor dysfunction in late and terminal stages of the disease.