FABP4 Dynamics in Obesity: Discrepancies in Adipose Tissue and Liver Expression Regarding Circulating Plasma Levels.

BACKGROUND FABP4 is predominantly expressed in adipose tissue, and its circulating levels are linked with obesity and a poor atherogenic profile. OBJECTIVE In patients with a wide BMI range, we analyze FABP4 expression in adipose and hepatic tissues in the settings of obesity and insulin resistance. Associations between FABP4 expression in adipose tissue and the FABP4 plasma level as well as the main adipogenic and lipolytic genes expressed in adipose tissue were also analyzed. METHODS The expression of several lipogenic, lipolytic, PPAR family and FABP family genes was analyzed by real time PCR. FABP4 protein expression in total adipose tissues and its fractions were determined by western blot. RESULTS In obesity FABP4 expression was down-regulated (at both mRNA and protein levels), with its levels mainly predicted by ATGL and inversely by the HOMA-IR index. The BMI appeared as the only determinant of the FABP4 variation in both adipose tissue depots. FABP4 plasma levels showed a significant progressive increase according to BMI but no association was detected between FABP4 circulating levels and SAT or VAT FABP4 gene expression. The gene expression of FABP1, FABP4 and FABP5 in hepatic tissue was significantly higher in tissue from the obese IR patients compared to the non-IR group. CONCLUSION The inverse pattern in FABP4 expression between adipose and hepatic tissue observed in morbid obese patients, regarding the IR context, suggests that both tissues may act in a balanced manner. These differences may help us to understand the discrepancies between circulating plasma levels and adipose tissue expression in obesity.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

MicroRNA-200 family modulation in distinct breast cancer phenotypes.

The epithelial to mesenchymal transition (EMT) contributes to tumor invasion and metastasis in a variety of cancer types. In human breast cancer, gene expression studies have determined that basal-B/claudin-low and metaplastic cancers exhibit EMT-related characteristics, but the molecular mechanisms underlying this observation are unknown. As the family of miR-200 microRNAs has been shown to regulate EMT in normal tissues and cancer, here we evaluated whether the expression of the miR-200 family (miR-200f) and their epigenetic state correlate with EMT features in human breast carcinomas. We analyzed by qRT-PCR the expression of miR-200f members and various EMT-transcriptional inducers in a series of 70 breast cancers comprising an array of phenotypic subtypes: estrogen receptor positive (ER+), HER2 positive (HER2+), and triple negative (TN), including a subset of metaplastic breast carcinomas (MBCs) with sarcomatous (homologous or heterologous) differentiation. No MBCs with squamous differentiation were included. The DNA methylation status of miR-200f loci in tumor samples were inspected using Sequenom MassArray® MALDI-TOF platform. We also used two non-tumorigenic breast basal cell lines that spontaneously undergo EMT to study the modulation of miR-200f expression during EMT in vitro. We demonstrate that miR-200f is strongly decreased in MBCs compared with other cancer types. TN and HER2+ breast cancers also exhibited lower miR-200f expression than ER+ tumors. Significantly, the decreased miR-200f expression found in MBCs is accompanied by an increase in the expression levels of EMT-transcriptional inducers, and hypermethylation of the miR-200c-141 locus. Similar to tumor samples, we demonstrated that downregulation of miR-200f and hypermethylation of the miR-200c-141 locus, together with upregulation of EMT-transcriptional inducers also occur in an in vitro cellular model of spontaneous EMT. Thus, the expression and methylation status of miR-200f could be used as hypothetical biomarkers to assess the occurrence of EMT in breast cancer.

The classification of esterases: an important gene family involved in insecticide resistance - A review

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.

Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period.

PPARs are a family of nuclear hormone receptors involved in various processes that could influence ovarian function. We investigated the cellular localization and expression of PPARs during follicular development in ovarian tissue collected from rats 0, 6, 12, 24, and 48 h post-PMSG. A second group of animals received human CG (hCG) 48 h post-PMSG. Their ovaries were removed 0, 4, 8, 12, and 24 h post-hCG to study the periovulatory period. mRNAs corresponding to the PPAR isotypes (alpha, delta, and gamma) were localized by in situ hybridization. Changes in the levels of mRNA for the PPARs were determined by ribonuclease protection assays. PPAR gamma mRNA was localized primarily to granulosa cells, and levels of expression did not change during follicular development. Four hours post-hCG, levels of mRNA for PPAR gamma decreased (P < 0.05) but not uniformly in all follicles. At 24 h post-hCG, levels of PPAR gamma mRNA were reduced 64%, but some follicles maintained high expression. In contrast, mRNAs for PPAR alpha and delta were located primarily in theca and stroma, and their levels did not change during the intervals studied. To investigate the physiologic significance of PPAR gamma in the ovary, granulosa cells from PMSG-primed rats were cultured for 48 h with prostaglandin J(2) (PGJ(2)) and ciglitazone, PPAR gamma activators. Both compounds increased progesterone and E2 secretion (P < 0.05). These data suggest that PPAR gamma is involved in follicular development, has a negative influence on the luteinization of granulosa cells, and/or regulates the periovulatory shift in steroid production. The more general and steady expression of PPARs alpha and delta indicate that they may play a role in basal ovarian function.

Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca2+- and calcineurin-mediated NFAT2 activation.

NFAT (nuclear factors of activated T cells) proteins constitute a family of transcription factors involved in mediating signal transduction. The presence of NFAT isoforms has been described in all cell types of the immune system, with the exception of neutrophils. In the present work we report for the first time the expression in human neutrophils of NFAT2 mRNA and protein. We also report that specific antigens were able to promote NFAT2 protein translocation to the nucleus, an effect that was mimicked by the treatment of neutrophils with anti-immunoglobulin E (anti-IgE) or anti-Fcepsilon-receptor antibodies. Antigens, anti-IgE and anti-FcepsilonRs also increased Ca2+ release and the intracellular activity of calcineurin, which was able to interact physically with NFAT2, in parallel to eliciting an enhanced NFAT2 DNA-binding activity. In addition, specific chemical inhibitors of the NFAT pathway, such as cyclosporin A and VIVIT peptide, abolished antigen and anti-IgE-induced cyclooxygenase-2 (COX2) gene upregulation and prostaglandin (PGE(2)) release, suggesting that this process is through NFAT. Our results provide evidence that NFAT2 is constitutively expressed in human neutrophils, and after IgE-dependent activation operates as a transcription factor in the modulation of genes, such as COX2, during allergic inflammation.

Regulation of the cardiac voltage-gated sodium channel Nav1.5 : regulation of Nav1.5 by ubiquitin-protein ligases of the Nedd4/Nedd4-like family and characterisation of two novel mutations in the gene encoding Nav1.5 (SCN5A) implicated in the Brugada syndrome triggered by fever

Abstract: The genesis of the cardiac action potential, which accounts for the cardiac contraction, is due to the sodium current INa mediated by the voltage-gated sodium channel Nav1.5. Several cardiac arrhythmias such as the Brugada syndrome are known te be caused by mutations in SCN5A, the gene encoding Nav1.5. Studies of these mutations allowed a better understanding of biophysical and functional properties of Nav1.5. However, only few investigations have been performed in order to understand the regulation of Nav1.5. During my thesis, I investigated different mechanisms of regulation of Nav1.5 using a heterologous expression system, HEK293 cells, coupled with a technique of sodium current recording: the patch clamp in whole cell configuration. In previous studies it has been shown that an enzyme of the Nedd4 family (Nedd4-2) regulates an epithelial sodium channel via the interaction with PY-motifs present in the latter. Interestingly, Nav1.5 contains a similar PY-motif, which motivated us to study the role of Nedd4-2 expressed in heart for the regulation of Nav1.5. In a second study, we investigated the implication of two Nav1.5 mutants, which were either less functional or net functional (Nav1.5 R535X and Nav1.5 L325R respectively) implied in the genesis of the Brugada syndrome by fever. Our results established two mechanisms implied in Nav1.5 regulation. The first one implies that following the interaction between the PY-motif of Nav1.5 and Nedd4- 2 Nav1.5 is ubiquitinated by Nedd4-2. This ubiquitination leads to the internalization of Nav1 .5. The second mechanism is a phenomenon called the "dominant negative" effect of Nav1.5 L325R on Nay1.5 where the decrease of 'Na is potentially due to the retention of Nav1.5 by Nav1.5 L325R in an undefined intracellular compartment. These studies defined two mechanisms of Nav1.5 regulation, which could play an important role for the genesis of cardiac arrhythmias where molecular processes are still poorly understood. Résumé La genèse du potentiel d'action cardiaque, permettant la contraction cardiaque, est due au courant sodique INa issu des canaux sodiques cardiaques dépendants du voltage Nav1.5. Nombreuses arythmies cardiaques telles que le syndrome de Brugada sont connues pour être liées à des mutations du gène SCN5A, codant pour Nav1.5. L'étude de ces mutations a permis une meilleure compréhension des propriétés structurelles et fonctionnelles de Nav1.5 et leurs implications dans la genèse de ces pathologies. Néanmoins peu d'études ont été menées afin de comprendre les mécanismes de régulation de Nav1.5. Mon travail de thèse a consisté à étudier des mécanismes de régulation de Nav1.5 en utilisant un système d'expression hétérologue, les cellules HEK293, couplé à une technique d'enregistrement des courants sodiques, le "patch clamp" en configuration cellule entière. La présence sur Nav1.5 d'un motif-PY similaire à ceux nécessaires pour la régulation d'un canal épithélial sodique par une enzyme de la famille de Nedd4, nous a amenée à étudier le rôle de ces ubiquitine-ligases, en particulier Nedd4-2, dans la régulation de Nav1.5. La seconde étude s'est intéressée aux conséquences de deux mutations de SCN5A codant pour deux mutants peu ou pas fonctionnels (Nav1.5 L325R et Nav1.5 R535X respectivement) retrouvées chez des patients présentant un syndrome de Brugada exacerbé par un état fébrile. Nos résultats ont permis d'établir deux mécanismes de régulation de Nav1.5 L'un par Nedd4-2 qui implique rubiquitination de Nav1.5 par cette ligase suite à l'interaction entre le motif-PY de Nav1.5 et Nedd4-2. Cette modification déclenche l'internalisation du canal impliquée dans la diminution d'INa. Le second mécanisme quant à lui est un effet "dominant négatif" de Nav1.5 L325R sur Nav1.5 aboutissant à une diminution d'INa suite à la séquestration intracellulaire potentielle de Nav1.5 par Nav1.5 L325R. Ces études ont mis en évidence deux mécanismes de régulation de Nav1.5 pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des arythmies cardiaques dont les processus moléculaires au sein des cardiomyocytes, impliquant des modifications du courant sodiques, sont encore mal compris. Résumé destiné à un large public La dépolarisation électrique de la membrane des cellules cardiaques permet la contraction du coeur. La génèse de cette activité électrique est due au courant sodique issu d'un type de canal à sodium situé dans la membrane des cellules cardiaques. De nombreuses pathologies provoquant des troubles du rythme cardiaque sont issues de mutations du gène qui code pour ce canal à sodium. Ces canaux mutants, entrainant diverses pathologies cardiaques telles que le syndrome de Brugada, ont été largement étudiées. Néanmoins, peu de travaux ont été réalisés sur les mécanismes de régulation de ce canal à sodium non muté. Mon travail de thèse a consisté à étudier certains des mécanismes de régulation de ce canal à sodium en utilisant une technique permettant l'enregistrement des courants sodiques issus de l'expression de ces canaux à sodium à la membrane de cellules mammifères. La présence sur ce canal à sodium d'une structure spécifique, similaire à celle nécessaire pour la régulation d'un canal épithélial à sodium par une enzyme appelée Nedd4-2, nous a amenée à étudier le rôle de cette enzyme dans la régulation de ce canal à sodium. La seconde étude s'est intéressée aux rôles de deux mutations du gène codant pour ce canal à sodium retrouvées chez des patients présentant un syndrome de Brugada exacerbé par la fièvre. Nos résultats nous ont permis d'établir deux mécanismes de régulation de ce canal à sodium diminuant le courant sodique l'un par l'action de l'enzyme Nedd4-2, suite à son interaction avec ce canal, qui modifie ce canal à sodium (ubiquitination) diminuant de ce fait la densité membranaire du canal. L'autre par un mécanisme suggérant un effet négatif de l'un des canaux mutants sur l'expression à la membrane du canal à sodium non muté. Ces études ont mis en évidence deux mécanismes de régulation de ce canal à sodium pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des troubles du rythme cardiaques dont les mécanismes cellulaires sont encore incompris.

A study of the translational regulation exerted by some neuroactive substances on the expression of the monocarboxylate transporter MCT2 in cultured cortical neurons

Summary of the thesis Glucose has been considered the major, if not the exclusive, energy substrate for the brain. But under certain conditions other substrates, namely monocarboxylates (lactate, pyruvate, and ketone bodies), can contribute significantly to satisfy brain energy demands. These monocarboxylates need to be transported across the blood brain barrier as well as out of astrocytes into the extracellular space and taken up into neurons. It has been shown that monocarboxylates are transported by a family of proton-linked transporters called monocarboxylate transporters (MCTs). In the central nervous system, MCT2 is the predominant neuronal form and little is known about the regulation of its expression. The neurotransmitter noradrenaline (NA) was shown previously to enhance the expression of MCT2 in cultured cortical neurons via a translational mechanism. Here, we demonstrate that two other substances, namely, insulin and IGF-1 enhance MCT2 protein expression in cultured mouse cortical neurons in a time- and concentrationdependent manner without affecting MCT2 mRNA levels. This result confirmed that MCT2 protein expression is translationally regulated and extend the observation to different types of neuroactive substances. Then we sought to determine by which signaling pathway(s) NA, insulin and IGF-1 can induce MCT2 protein expression. First, we observed by Western blot that all three substances cause activation of the MAP kinase ERK as well as the kinase Akt via their phosphorylation. Moreover, the mTOR/S6K pathway which is known to play an important role in translation initiation regulation was also strongly stimulated by all three substances. Second, we sought to determine the implication of these signaling pathways on the NA-, insulin- and IGF-1-induced enhancement of MCT2 protein expression and used specific inhibitors of these signaling pathways. We observed that the Pia kinase and mTOR inhibitors LY294002 and rapamycin respectively, strongly prevent the enhancement. of MCT2 expression caused by either NA, insulin ar IGF-1. In contrast, the MEK inhibitor PD98059 and the p38 MAP kinase inhibitor SB202190 had only a slight effect on the enhancement of MCT2 expression in all three cases. These results suggest that NA, insulin and IGF-1 regulate MCT2 protein expression by a common mechanism most likely involving the Akt/PKB pathway and translational activation via mTOR. In conclusion, considering the roles of NA, insulin and IGF-1 in synaptic plasticity, the tight translational regulation of MCT2 expression by these substances may represent a common mechanism through which supply of potentiated synapses with nonglucose energy substrates can be adapted to the level of activity. Résumé du travail de thèse Le glucose représente le substrat énergétique majeur pour le cerveau. Cependant, dans certaines conditions physiologiques ou pathologiques, le cerveau a la capacité d'utiliser des substrats énergétiques appartenant à la classe des monocarboxylates (lactate, pyruvate et corps cétoniques) afin de satisfaire ses besoins énergétiques. Ces monocarboxylates doivent être transportés à travers la barrière hématoencéphalique mais aussi hors des astrocytes vers l'espace extracellulaire puis re-captés par les neurones. Leur transport est assuré par une famille de transporteurs spécifiques, protons-dépendants, appelés transporteurs aux monocarboxylates (MCTs). Dans le système nerveux central, les neurones expriment principalement l'isoforme MCT2 mais peu d'informations sont disponibles concernant la régulation de son expression. Il a été montré que le neurotransmetteur noradrénaline (NA) augmente l'expression de MCT2 dans les cultures de neurones corticaux de souris par le biais d'un mécanisme de régulation traductionnel. La présente étude nous a permis de démontrer que deux autres substances, l'insuline et 17GF-1, induisent une augmentation de la protéine MCT2 dans ces mêmes cultures selon un décours temporel et une gamme de concentrations particulière. Etonnamment, aucun changement n'a été observé concernant les niveaux d'ARNm de MCT2. Ce résultat .confirme que la protéine MCT2 est régulée de manière traductionnelle et révèle que différentes substances neuro-actives peuvent réguler l'expression de MCT2. Compte tenu de ces observations, nous avons voulu déterminer par quelle(s) voie(s) de signalisation la NA, l'insuline et l'IGF-1 exercent leur effet sur l'expression de MCT2. Dans un premier temps, nous avons pu observer par Western blot que ces trois substances activent la MAP kinase ERK ainsi que la kinase Akt via leur phasphorylation. De plus, la voie mTOR/S6K, connue pour son implication dans la régulation de l'initiation de la traduction est aussi fortement activée par ces trois substances. Dans un second temps, nous avons voulu déterminer I implication de chacune de ces voies de signalisation dans l'augmentation de l'expression de la protéine MCT2 observée après stimulation à la NA, à l'insuline et à l'IGF-1. Pour ce faire, nous avons utilisé des inhibiteurs spécifiques de chacune de ces voies. (Vous avons observé que les inhibiteurs des voies PI3 kinase et mTOR (LY294002 et rapamycin respectivement), prévenaient fortement l'augmentation de l'expression de MCT2 induite par la NA, l'insuline ou (IGF-1. A l'inverse, les inhibitions de la MAP kinase .kinase MEK ainsi que de la MAP kinase p38 (par l'utilisation des inhibiteurs spécifiques PD98059 et SB202190 respectivement) n'ont eu qu'un léger effet dans ces mêmes conditions. Ces résultats suggèrent que la NA, 'l'insuline et I~GF-1 régulent l'expression de la protéine MCT2 par un mécanisme commun impliquant probablement la voie Akt/PKB et l'activation de la traduction via mTOR. En conclusion, considérant l'implication de la NA, de l'insuline et de I`IGF-1 dans la plasticité synaptique, le contrôle traductionnel étroit exercé par ces substances sur l'expression de MCT2 pourrait être un moyen d'alimenter en substrats énergétiques autres que le glucose les synapses activées et également d'adapter l'approvisionnement en substrats énergétiques au niveau d'activité. Résumé « grand public » Le cerveau est un organe qui réalise des tâches complexes nécessitant un apport important en énergie. La principale source d'énergie du cerveau est le glucose. Bien que le cerveau ne représente que 2% de la masse corporelle, il consomme à lui seul plus de 25% du glucose et 20% de l'oxygène provenant de la circulation sanguine. La nécessité d'un tel apport en énergie réside dans la nature -même du fonctionnement des milliards de neurones qui utilisent des signaux électriques et chimiques pour communiquer entre eux. Hormis l'utilisation massive du glucose comme source d'énergie, le cerveau est capable de consommer d'autres substrats énergétiques dans certaines conditions physiologiques ou pathologiques. Les monocarboxylates (lactate, pyruvate et corps cétoniques) font partie de ces autres sources d'énergie. Contrairement au glucose, les monocarboxylates ne diffusent pas facilement de la circulation sanguine vers les neurones. Afin de pouvoir être consommés par les neurones, ils doivent être transportés par un système adapté. Ce sont des transporteurs appelés transporteurs aux monocarboxylates ou MCT qui permettent le passage de ces substrats énergétiques du sang vers les neurones. Le but de ce travail de thèse a été de comprendre comment est régulée l'expression de MCT2, l'un de ces transporteurs exprimé spécifiquement à la surface des neurones. Cette étude nous a permis de mettre en évidence que le neurotransmetteur noradrénaline ainsi que les hormones insuline et IGF-1 (insulinlike growth factor-1) sont capables d'induire une augmentation d'expression de MCT2 à la surface des neurones en culture. Nous avons ensuite voulu déterminer par quels mécanismes de signalisation ces substances agissent sur l'expression de MCT2. Nous avons pu observer que la surexpression de la protéine MCT2 est due à une augmentation d'activité traductionnelle (la traduction étant une des étapes qui permet la synthèse des protéines) induite par le biais d'une voie de signalisation particulière. En conclusion, lorsque la noradrénaline, l'insuline ou 17GF-1 agissent sur les neurones, la traduction de la protéine MCT2 est activée et on observe une augmentation de l'expression de MCT2. Ce mécanisme pourrait permettre d'augmenter l'apport énergétique au niveau des neurones en augmentant le nombre de transporteurs pour les substrats énergétiques que sont les monocarboxylates. D'un point de vue physiologique, cette régulation d'expression pourrait jouer un rôle primordial dans des situations d'apprentissage et de mémorisation. Sur le plan pathologique, cela pourrait permettre de prévenir les dommages causes aux neurones dans certains cas d'atteintes cérébrales.

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silicoana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and SmBAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin.

Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3(PAR2/+)), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2-null background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.

HERV-W polymorphism in chromosome X is associated with multiple sclerosis risk and with differential expression of MSRV.

BACKGROUND Multiple Sclerosis (MS) is an autoimmune demyelinating disease that occurs more frequently in women than in men. Multiple Sclerosis Associated Retrovirus (MSRV) is a member of HERV-W, a multicopy human endogenous retroviral family repeatedly implicated in MS pathogenesis. MSRV envelope protein is elevated in the serum of MS patients and induces inflammation and demyelination but, in spite of this pathogenic potential, its exact genomic origin and mechanism of generation are unknown. A possible link between the HERV-W copy on chromosome Xq22.3, that contains an almost complete open reading frame, and the gender differential prevalence in MS has been suggested. RESULTS MSRV transcription levels were higher in MS patients than in controls (U-Mann-Whitney; p = 0.004). Also, they were associated with the clinical forms (Spearman; p = 0.0003) and with the Multiple Sclerosis Severity Score (MSSS) (Spearman; p = 0.016). By mapping a 3 kb region in Xq22.3, including the HERV-W locus, we identified three polymorphisms: rs6622139 (T/C), rs6622140 (G/A) and rs1290413 (G/A). After genotyping 3127 individuals (1669 patients and 1458 controls) from two different Spanish cohorts, we found that in women rs6622139 T/C was associated with MS susceptibility: [χ2; p = 0.004; OR (95% CI) = 0.50 (0.31-0.81)] and severity, since CC women presented lower MSSS scores than CT (U-Mann-Whitney; p = 0.039) or TT patients (U-Mann-Whitney; p = 0.031). Concordantly with the susceptibility conferred in women, rs6622139*T was associated with higher MSRV expression (U-Mann-Whitney; p = 0.003). CONCLUSIONS Our present work supports the hypothesis of a direct involvement of HERV-W/MSRV in MS pathogenesis, identifying a genetic marker on chromosome X that could be one of the causes underlying the gender differences in MS.

Functional and molecular evolutionary analysis of the "Brevix Radix" gene family in mono-and dicotyledonous plants

ABSTRACTIn contrast to animals, plants cannot move from their place of birth and, therefore, need to adapt to their particular habitat in order to survive. Thus, plant development is remarkably plastic, making plants an ideal system for the isolation of genes that account for intraspecific natural variation and possibly environmental adaptation. However, to date, this approach mostly identified null alleles and missed mutations with subtle effects. For instance, BREVIS RADIX (BRX) has been isolated as a key regulator of root growth through a naturally occurring loss-of-function allele in the Arabidopsis thaliana accession Uk-1 and is the founding member of a highly-conserved plant-specific gene family.In this work, we show that a strong selective pressure is acting on the BRX gene family and dates back before the monocot-dicot divergence. However, functional diversification is observed mainly in dicotyledon BRX family genes and is correlated with acceleration in the evolutionary rates in the N-terminal regions. Population genetic data revealed that BRX is highly conserved across Arabidopsis accessions and presents signatures of adaptation. Interestingly, a seven amino acid deletion polymorphism in BRX sequence was found in a few accessions, which seems to be responsible for their enhanced primary root growth. Nevertheless, BRX might not only be active in the root, as suggested by its expression in the shoot. Indeed, leaves and cotyledons of brx mutants are significantly smaller than wild- type. This phenotype is a direct consequence of the absence of BRX function in the shoot rather than an indirect effect of an altered root system growth. Interestingly, cotyledons of brx plants reflect the same physiological defects as the root. Moreover, phenotypes in BRX gain-of-function plants, such as epinastic leaves and increased epidermal cell size, could be associated with an increase in leaf brassinosteroid content.Collectively, these results indicate that BRX contributes to local adaptation by ubiquitously regulating plant growth, probably through the modulation of brassinosteroid biosynthesis.RÉSUMÉContrairement à la plupart des animaux, les plantes ne peuvent se mouvoir et doivent ainsi s'adapter à leur environnement pour survivre. Pour cette raison, elles représentent un système idéal pour l'identification de gènes contribuant à la variation naturelle intra- spécifique, ainsi qu'à l'adaptation. Cependant, cette approche a, jusqu'à présent, surtout permis d'isoler des allèles nuls et non des mutations conférant des effets plus subtiles. C'est le cas du gène Β REVIS RADIX (BRX), un régulateur clé de la croissance racinaire, qui a été identifié grâce à un allèle non-fonctionnel présent dans l'accession naturelle d'Arabidopsis thaliana Uk-1. BRX et ses homologues des plantes mono- et dicotylédones forment une famille très conservée et spécifique aux plantes.Dans ce travail, nous démontrons que la famille de gènes BRX est soumise à une forte pression de sélection qui remonte avant la divergence entre mono- et dicotylédones. Cependant, une diversification fonctionnelle a été observée chez les gènes des dicotylédones et corrèle avec une accélération de la vitesse d'évolution dans leur région N- terminale. Une analyse génétique de différentes accessions naturelles d'Arabidopsis a révélé que BRX est hautement conservé et présente des signatures d'adaptation. Remarquablement, un polymorphisme de délétion de sept acides aminés a été détecté dans quelques accessions et a pour conséquence une plus forte croissance de la racine primaire. Néanmoins, il semble que le rôle de BRX ne se limite pas qu'à la racine, comme indiqué par son expression dans les parties aériennes de la plante. En effet, les mutants brx présentent des cotylédons et des feuilles significativement plus petits que le type sauvage, une conséquence directe de l'absence d'activité de BRX dans ces organes. Nous avons aussi noté que les cotylédons des mutants brx, à l'instar des racines, ont une perception altérée de l'auxine et peuvent être complémentés par l'application exogène de brassinostéroïdes. De plus, dans des plantes présentant un gain de fonction BRX, les feuilles sont épinastiques et les cellules de leur épiderme plus grandes. Ces phénotypes sont accompagnés d'une augmentation de la concentration de brassinostéroïdes dans les feuilles. Conjointement, ces résultats démontrent que BRX contribue à une adaptation locale de la plante par la régulation générale de sa croissance, probablement en modulant la biosynthèse des brassinostéroïdes.

Immunotherapy reduces allergen-mediated CD66b expression and myeloperoxidase levels on human neutrophils from allergic patients.

CD66b is a member of the carcinoembryonic antigen family, which mediates the adhesion between neutrophils and to endothelial cells. Allergen-specific immunotherapy is widely used to treat allergic diseases, and the molecular mechanisms underlying this therapy are poorly understood. The present work was undertaken to analyze A) the in vitro effect of allergens and immunotherapy on cell-surface CD66b expression of neutrophils from patients with allergic asthma and rhinitis and B) the in vivo effect of immunotherapy on cell-surface CD66b expression of neutrophils from nasal lavage fluid during the spring season. Myeloperoxidase expression and activity was also analyzed in nasal lavage fluid as a general marker of neutrophil activation. RESULTS CD66b cell-surface expression is upregulated in vitro in response to allergens, and significantly reduced by immunotherapy (p<0.001). Myeloperoxidase activity in nasal lavage fluid was also significantly reduced by immunotherapy, as were the neutrophil cell-surface expression of CD66b and myeloperoxidase (p<0.001). Interestingly, CD66b expression was higher in neutrophils from nasal lavage fluid than those from peripheral blood, and immunotherapy reduced the number of CD66+MPO+ cells in nasal lavage fluid. Thus, immunotherapy positive effects might, at least in part, be mediated by the negative regulation of the CD66b and myeloperoxidase activity in human neutrophils.

Results from a multicentre international registry of familial Mediterranean fever: impact of environment on the expression of a monogenic disease in children.

BACKGROUND AND AIM: Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations of the MEFV gene. We analyse the impact of ethnic, environmental and genetic factors on the severity of disease presentation in a large international registry. METHODS: Demographic, genetic and clinical data from validated paediatric FMF patients enrolled in the Eurofever registry were analysed. Three subgroups were considered: (i) patients living in the eastern Mediterranean countries; (ii) patients with an eastern Mediterranean ancestry living in western Europe; (iii) Caucasian patients living in western European countries. A score for disease severity at presentation was elaborated. RESULTS: Since November 2009, 346 FMF paediatric patients were enrolled in the Eurofever registry. The genetic and demographic features (ethnicity, age of onset, age at diagnosis) were similar among eastern Mediterranean patients whether they lived in their countries or western European countries. European patients had a lower frequency of the high penetrance M694V mutation and a significant delay of diagnosis (p<0.002). Patients living in eastern Mediterranean countries had a higher frequency of fever episodes/year and more frequent arthritis, pericarditis, chest pain, abdominal pain and vomiting compared to the other two groups. Multivariate analysis showed that the variables independently associated with severity of disease presentation were country of residence, presence of M694V mutation and positive family history. CONCLUSIONS: Eastern Mediterranean FMF patients have a milder disease phenotype once they migrate to Europe, reflecting the effect of environment on the expression of a monogenic disease.

Evolution and expression of vitellogenin genes.

A major event during the growth period of oocytes in nonmammalian animals is the accumulation of yolk. The genes coding for the yolk protein precursor, known as vitallogenin, are well characterized in a few vertebrate and invertebrate species. Studies on the evolution of these genes and on the regulatory mechanisms involved in their time, tissue- and hormone-specific expression are presented and discussed in this review.