Identification of CARDIAK, a RIP-like kinase that associates with caspase-1.

Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.

Molecular basis of interaction between Na,K-ATPase and FXYD proteins.

Résumé au large public Notre corps est constitué de différents types de cellules. La condition minimale ou primordiale pour la survie des cellules est d'avoir de l'énergie. Cette tâche est assumée en partie par une protéine qui se situe dans la membrane de chaque cellule. Nommé Na, K¬ATPase ou pompe à sodium, c'est une protéine pressente dans toutes les cellules chez les mammifères est composée de deux sous-unités, α et β. En transportant 3 ions de sodium hors de la cellule et 2 ions de potassium à l'intérieur de la cellule, elle transforme l'énergie chimique sous forme de l'ATP en énergie motrice, qui permet aux cellules par la suite d'échanger des matériaux entre l'espace intracellulaire et extracellulaire ainsi que d'ingérer des nutriments provenant de son environnement. Le manque de cette protéine chez la souris entraîne la mort de l'embryon. Des défauts fonctionnels de cette protéine sont responsables de plusieurs maladies humaines comme par exemple, un type de migraine. En dehors de sa fonction vitale, cette protéine est également engagée dans diverses activités physiologiques comme la contractilité musculaire, l'activité nerveuse et la régulation du volume sanguin. Vue l'importance de cette protéine, sa découverte par Jens C. Skou en 1957 a été honorée d'un Prix Noble de chimie quarante ans plus tard. Depuis lors, nous connaissons de mieux en mieux les mécanismes de fonctionnement de la Na, K-ATPase. Entre autre, sa régulation par une famille de protéines appelées protéines FXYD. Cette famille contient 7 membres (FXYD 1-7). L'un d'entre eux nommé FXYD 2 est lié à une maladie héréditaire connue sous le nom de hypomagnesemia. Nous disposons actuellement d'informations concernant les conséquences de la régulation par les protéines FXYD sur activité de la Na, K-ATPase, mais nous savons très peu sur le mode d'interaction entre les protéines FXYD et la Na, K-ATPase. Dans ce travail de thèse, nous avons réussi à localiser des zones d'interaction dans la sous- unité a de la Na, K-ATPase et dans FXYD 7. En même temps, nous avons déterminé un 3ème site de liaison spécifique au sodium de la Na, K-ATPase. Une partie de ce site se situe à l'intérieur d'un domaine protéique qui interagit avec les protéines FXYD. De plus, ce site a été démontré comme responsable d'un mécanisme de transport de la Na, K-ATPase caractérisé par un influx ionique. En conclusion, les résultats de ce travail de thèse fournissent de nouvelles preuves sur les régions d'interaction entre la Na, K-ATPase et les protéines FXYD. La détermination d'un 3ème site spécifique au sodium et sa relation avec un influx ionique offrent la possibilité 1) d'explorer les mécanismes avec lesquels les protéines FXYD régulent l'activité de la Na, ATPase et 2) de localiser un site à sodium qui est essentielle pour mieux comprendre l'organisation et le fonctionnement de la Na, K-ATPase. Résumé Les gradients de concentration de Na+ et de K+ à travers la membrane plasmatique des cellules animales sont cruciaux pour la survie et l'homéostasie de cellules. De plus, des fonctions cellulaires spécifiques telles que la reabsorption de Na dans le rein et le côlon, la contraction musculaire et l'excitabilité nerveuse dépendent de ces gradients. La Na, K¬ATPase ou pompe à sodium est une protéine membranaire ubiquitaire. Elle crée et maintient ces gradients en utilisant l'énergie obtenu par l'hydrolyse de l'adénosine triphosphate. L'unité fonctionnelle minimale de cette protéine se compose d'une sous-unité catalytique α et d'une sous-unité régulatrice β. Récemment, il a été montré que des membres de la famille FXYD, sont des régulateurs tissu-spécifiques de la Na, K-ATPase qui influencent ses propriétés de transport. Cependant, on connaît peu de chose au sujet de la nature moléculaire de l'interaction entre les protéines FXYD et la Na, K-ATPase. Dans cette étude, nous fournissons, pour la première fois, l'évidence directe que des résidus du domaine transmembranaire (TM) 9 de la sous-unité α de la Na, K-ATPase sont impliqués dans l'interaction fonctionnelle et structurale avec les protéines FXYD. De plus nous avons identifié des régions dans le domaine transmembranaire de FXYD 7 qui sont importantes pour l'association stable avec la Na, K-ATPase et une série de résidus responsables des régulations fonctionnelles. Nous avons aussi montré les contributions fonctionnelles du TM 9 de la Na, K-ATPase à la translocation de Na + en déterminant un 3ème site spécifique au Na+. Ce site se situe probablement dans un espace entre TM 9, TM 6 et TM 5 de la sous-unité α de la pompe à sodium. De plus, nous avons constaté que le 3ème site de Na + est fonctionnellement lié à un courant entrant de la pompe sensible à l'ouabaïne et activé par le pH acide. En conclusion, ce travail donne de nouvelles perspectives de l'interaction structurale et fonctionnelle entre les protéines FXYD et la Na, K-ATPase. En outre, les contributions fonctionnelles de TM 9 offrent de nouvelles possibilités pour explorer le mécanisme par lequel les protéines FXYD régulent les propriétés fonctionnelles de la Na, K-ATPase. La détermination du 3ème site au Na + fournit une compréhension avancée du site spécifique au Na + de la Na, K-ATPase et du mécanisme de transport de la Na, K-ATPase. Summary The Na+ and K+ gradients across the plasma membrane of animal cells are crucial for cell survival and homeostasis. Moreover, specific tissue functions such as Na+ reabsorption in kidney and colon, muscle contraction and nerve excitability depend on the maintenance of these gradients. Na, K-ATPase or sodium pump, an ubiquitous membrane protein, creates and maintains these gradients by using the energy from the hydrolysis of ATP. The minimal functional unit of this protein is composed of a catalytic α subunit and a regulatory β subunit. Recently, members of the FXYD family, have been reported to be tissue-specific regulators of Na, K-ATPase by influencing its transport properties. However, little is known about the molecular nature of the interaction between FXYD proteins and Na, K-ATPase. In this study, we provide, for the first time, direct evidence that residues from the transmembrane (TM) domain 9 of the α subunit of Na, K-ATPase are implicated in the functional and structural interaction with FXYD proteins. Moreover, we have identified regions in the TM domain of FXYD 7 important for the stable association with Na, K-ATPase and a stretch of residues responsible for the functional regulations. We have further revealed the functional contributions of TM 9 of the Na, K-ATPase α subunit to the Na+ translocation by determining a 3rd Na+-specific cation binding site. This site is likely in a space between TM 9, TM 6 and TM 5 of the a subunit of the sodium pump. Moreover, we have found that the 3rd Na+ binding site is functionally linked to an acidic pH- activated ouabain-sensitive inward pump current. In conclusion, this work gives new insights into the structural and functional interaction between FXYD proteins and Na, K-ATPase. Functional contributions of TM 9 offer new possibilities to explore the mechanism by which FXYD proteins regulate functional properties of Na, K-ATPase. The determination of the 3rd Na+ binding site provides an advanced understanding concerning the Na+ -specific binding site of Na, K-ATPase and the 3rd Na+ site related transport mechanism.

Antigenic proteins involved in occupational rhinitis and asthma caused by obeche wood (Triplochiton scleroxylon).

BACKGROUND Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. OBJECTIVE To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma MATERIALS AND METHODS An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. RESULTS Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA-), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases. CONCLUSIONS Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens.

Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Analysis of c-Myc function in mouse epidermis

Abstract The c-myc gene is one of the most frequently mutated oncogenes found in human tumors. c-Myc has been implicated in the regulation of various biological processes including cell cycle progression, cellular growth, differentiation, angiogenesis, immortalization and apoptosis. To assess the normal role of c-Myc in epithelial cell types in vitro and in vivo we have deleted the c-myc gene in keratinocytes and in the adult skin epidermis by conditional Cre/loxP mediated recombination. Similar to what we have previously shown in mouse embryonic fibroblasts acute elimination of c-Myc activity in cultured keratinocytes causes cells to cease proliferation and adapt a flat cell morphology. Mutant cells accumulate in a diploid Ki67neg stage, indicative of a quiescent Go stage. This demonstrates that c-Myc activity is essential to maintain keratinocytes in a productive cell cycle. In addition, mutant keratinocytes showed a defect in Ca2+ induced induction of the differentiation marker Keratin 1 suggesting a role for c-Myc during differentiation. To assess the in vivo role of c-Myc we used a tamoxifen inducible K5::CreERT transgene to delete the c-myc gene in the adult skin epidermis. Unexpectedly, despite strong c-Myc expression in the basal compartment it is not required for maintenance of the skin epidermis in the adult mouse. The epidermis appeared normal with respect to both proliferation and differentiation. In addition, no selection against c-Myc deficient epidermal cells occurred over many months, further confirming that c-Myc is dispensable for normal skin homeostasis. Even more surprising, TPA induced hyperproliferation also occurred in a c-Myc independent manner. Treatment of the skin with the mutagen DMBA prior to TPA is a classical way to induce papillomas by selecting for mutations that lead to dominant activation of the oncogene Ha-Ras. Most interestingly tumor formation was severely inhibited suggesting that tumor progression requires endogenous c-Myc. Further studies are required to address whether the role of c-Myc in the activation of telomerase or the Werner protein, or its role to induce angiogenesis is required for skin tumor progression, In conclusion, this work shows that while c-Myc is not required for maintenance or hyperplasia of mouse epidermis, it is essential for skin tumor progression in collaboration with Ras. Résumé Le gène c-myc est un des oncogènes les plus fréquemment mutés dans les tumeurs humaines. c-Myc est impliqué dans la régulation de processus biologiques variés, comme la progression du cycle cellulaire, la croissance cellulaire, la différenciation, l'angiogenèse, l'immortalisation et l'apoptose. Pour caractériser le rôle physiologique de c-Myc dans les cellules de type épithélial in vitro et in vivo, le gène c-myc a été délété dans des kératinocytes primaires et dans l'épiderme de peau de souris adultes par des recombinaisons conditionnelles (système Cre/loxP). De la même façon que dans les fibroblastes d'embryon de souris, l'élimination aiguë de l'activité de c-Myc dans les kératinocytes en culture primaire provoque l'arrêt de la prolifération des cellules et leur applatissement morphologique. Les cellules mutantes restent dans un stade diploïde Ki67neg, indiquant un stade quiescent Go. Cela démontre que l'activité de c-Myc est essentielle pour maintenir les kératinocytes dans le cycle cellulaire. De plus, les kératinocytes mutants montrent une déficience pour le marqueur de différenciation Kératine 1 au cours de la différenciation induite par le calcium, suggérant un rôle de c-Myc dans la différenciation cellulaire. Pour comprendre le rôle de c-Myc in vivo, le transgène K5::CreERT inductible par le tamoxifen a été utilisé pour déléter le gène c-inyc dans l'épiderme de souris adultes. Etonnemment, malgré une forte expression de c-Myc dans le compartiment basal de l'épiderme, ce gène n'est pas nécessaire pour la maintenance de l'épiderme de la peau chez la souris adulte. L'épiderme apparait normal avec une prolifération et une différenciation physiologique des cellules. De plus, il n'y a pas de sélection contre les cellules épidennales c-Myc déficientes après plusieurs mois, ce qui confirme que c-Myc n'est pas nécessaire pour l'homéostasie normale de la peau. Encore plus surprenant, une hyperprolifération est également induite par du TPA chez les souris mutantes, impliquant une voie de prolifération indépendante de c-Myc. Le traitement de la peau par le mutagène DMBA avant le traitement au TPA est une voie classique d'induction de papillomes, par sélection de mutations conduisant à l'activation de l'oncogène Ha-Ras. La formation des tumeurs est fortement inhibée chez les souris mutantes, suggérant que la progression des tumeurs nécessite la présence endogène de c-Myc. De nouvelles études sont nécessaires pour savoir si c-Myc a un rôle dans l'activation de la télomérase ou de la protéine de Werner, ou encore dans l'angiogénèse, qui sont nécessaires pour la progression tumorale. En conclusion, ce travail montre que même si c-Myc n'est pas nécessaire pour la maintenance ou l'hyperplasie de la peau de souris, il est essentiel pour la progression des tumeurs de la peau en collaboration avec Ras.

zyg-11and cul-2 promote the metaphase to anaphase transition and M phase exit of meiosis II and prevent polarity establishment in C.elegans

Résumé Les mécanismes qui coordonnent la progression du cycle cellulaire lors de la méiose avec les événements du développement embryonnaire précoce, y compris la formation des axes de polarité embryonnaire, sont peu compris. Dans le zygote du vers Caenorhabditis elegans, les premiers signes de polarité Antéro-Postérieur (A-P) embryonnaire apparaissent après que la méiose soit terminée. La nature des protéines et des mécanismes moléculaires qui cassent la symétrie du zygote n'est pas connue. Nous démontrons que zyg-11 et cul-2 promeuvent la transition métaphase - anaphase et la sortie de la phase M lors de la seconde division méiotique. Nos résultats indiquent que ZYG-11 agit comme unité recrutant le substrat d'une ligase E3 comprennant CUL-2. Nos résultats montrent aussi que le délai de sortie de la phase M dépend de l'accumulation de la Cyclin B, CYB-3. Nous démontrons que dans des embryons zyg-11(RNAi) ou cul-2(RNAi), une polarité inversée est établie lors du délai de méiosis II. Enfin nous montrons que les défauts de cycle cellulaire et ceux de polarité peuvent être séparés. De plus, nous faisons apparaitre que l'établissement d'une polarité inversée pendant le délai de méiose II des embryons zyg-11(RNAi), comme l'établissement de la A-P polarité des embryons sauvage ne semblent pas requérir les microtubules. Nous montrons également les premiers résultats d'un crible deux hybrides ainsi qu'un crible génomique qui vise à identifier des gènes dont l'inactivation augmente ou supprime les défauts de mutants pour le gène zyg-11, afin d'identifier les gènes qui intéragissent avec ZYG-11 pour assumer ses deux fonctions séparables. Par conséquent, nos trouvailles suggèrent un modèle selon lequel ZYG-11 est une sous-unité qui recrute les substrats d'une ligase E3 basée sur CUL-2 qui promeut la progression du cycle cellulaire et empêche l'établissement de la polarité pendant la méiose II, et où le centrosome agit comme la clé qui polarise l'embryon à la fin de la méiose. Summary The mechanisms that couple meiotic cell cycle progression to subsequent developmental events, including specification of embryonic axes, are poorly understood. In the one cell stage embryos of Caenorhabditis elegans, the first signs of Antero-Posterior (A-P) polarity appear after meiosis completion. A centrosome ¬derived component breaks symmetry of the embryo, but the molecular nature of this polarity signal is not known. We established that zyg-11 and cul-2 promote the metaphase to anaphase transition and M phase exit at meiosis II. Our results indicate that ZYG-11 acts as a substrate recruitment subunit of a CUL-2-based E3 ligase. Moreover, we find that the delayed meiosis II exit of embryos lacking zyg-11 is caused by accumulation of the B-type cyclin, CYB-3. We demonstrate that inverted A-P polarity is established during the meiosis II delay in zyg-11(RNAi) and cul¬2(RNAi) embryos. Importantly, we demonstrate that the polarity defects following zyg-11 or cul-2 inactivation can be uncoupled from the cell cycle defects. Furthermore, we found that microtubules appear dispensable for inverted polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for A-P polarity during the first mitotic cell cycle in wild-type embryos. We also show the initial results from a comprehensive yeast two hybrid, as well as an RNAi-based functional genomic enhancer and suppressor screen, that may lead to identification of proteins that interact with zyg-11 to ensure the two functions. Our findings suggest a model in which ZYG-11 is a substrate recruitment subunit of an CUL-2-based E3 ligase that promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.

Use of fluorescent proteins to monitor the regulation of antifungal activity in root-associated Pseudomonas fluorescens CHA0

SUMMARY Roots of crop plants are the target of soil-borne pathogens, mainly fungi that cause considerable damage to plant health. By antagonizing these pathogens, some root-colonizing pseudomonads provide plants with efficient biological protection from disease. Pseudomonas fluorescens CHAO is a soil bacterium with the ability to suppress a considerable range of root diseases. A major characteristic conferring biocontrol capacity to this strain is the production of antifungal compounds, in particular 2,4-diacetyphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the biosyntheses of these metabolites is complex and involves several regulatory systems responding to multiple environmental signals. In the present work, we have developed reporter systems based on green (GFP) and red fluorescent (DsRed) proteins to monitor regulation of antifungal gene expression in vitro and on plant roots. Stable and unstable GFP-based reporter fusions to the DAPG and PLT biosynthetic genes allowed us to demonstrate that P. fluorescens CHAO keeps the two antifungal compounds at a fine-tuned balance that can be affected by environmental signals. A GFP-based screening technique helped us to identify two novel regulators of balanced antibiotic production, i.e. MvaT and MvaV that are functionally and structurally related to the nucleoid-binding protein H-NS. They act in concert as global regulators of DAPG and PLT production and other biocontrol-related traits in P. fluorescens CHAO, and are essential for the bacterium's capacity to control a root disease caused by Pythium. The combined use of autofluorescent reporters, flow cytometry, and epifluorescence microscopy permitted us to visualize and quantify the expression of DAPG and PLT biosynthetic genes on roots. A GFP- and DsRed-based two-color approach was then developed to further improve the sensitivity of the flow cytometric quantitation method. The findings of this study shed more light on the complex regulatory mechanisms controlling antifungal activity of P. filuorescens in the rhizosphere. RESUME 4 e Les racines de plantes de culture sont la cible de divers pathogènes, principalement des champignons, qui nuisent gravement à la santé des plantes. Certains pseudomonades colonisant les racines peuvent avoir un effet antagoniste sur les pathogènes et protéger ainsi les plantes de manière efficace. Pseudomonas fluorescens CHAO est une bactérie du sol ayant la capacité de supprimer une gamme considérable de maladies racinaires. Une des caractéristiques principales conférant la capacité de biocontrôle à cette souche, est la production de composés antifongiques, en particulier le 2,4-diacétyphloroglucinol (DAPG) et la pyolutéorine (PLT). La régulation de la biosynthèse de ces métabolites est complexe et implique plusieurs systèmes régulateurs répondant à de multiples signaux environnementaux. Dans ce travail, nous avons développé des systèmes rapporteurs basés sur des protéines fluorescentes verte (GFP) et rouge (DsRed), afin d'étudier la régulation de l'expression des gènes d'antifongiques in vitro et sur les racines des plantes. Des fusions GFP stables et instables rapportrices de l'expression des gènes de biosynthèse du DAPG et de la PLT nous ont permis de démontrer que P. fluorescens CHAO gère les deux antifongiques dans une balance finement régulée pouvant être affectée par des signaux environnementaux. Une technique de criblage basée sur la GFP nous a permis d'identifier deux nouveaux régulateurs de la production d'antibiotiques, MvaT et MvaV, apparentés à la protéine H-NS liant l'ADN, Elles agissent de concert en tant que régulateurs globaux sur la production de DAPG et de PLT, ainsi que sur d'autres éléments relatifs au biocontrôle chez P. fluorescens CHAO. De plus, elles sont essentielles à la bactérie pour contrôler une maladie racinaire causée par Pythium. L'utilisation combinée de rapporteurs autofluorescents, de cytométrie de flux et de microscopie à épifluorescence nous a permis de visualiser et de quantifier l'expression des gènes de biosynthèse du DAPG et de la PLT sur les racines. Une approche utilisant simultanément la GFP et la DsRed a ensuite été développée afin d'améliorer la sensibilité de la méthode de quantification par cytométrie de flux. Les résultats de cette étude ont apporté plus de lumière sur les mécanismes régulateurs complexes contrôlant l'activité antifongique de P. fluorescens dans la rizosphère.

Functional Characterization of a Novel Frameshift Mutation in the C-terminus of the Nav1.5 Channel Underlying a Brugada Syndrome with Variable Expression in a Spanish Family.

INTRODUCTION We functionally analyzed a frameshift mutation in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) found in a proband with repeated episodes of ventricular fibrillation who presented bradycardia and paroxysmal atrial fibrillation. Seven relatives also carry the mutation and showed a Brugada syndrome with an incomplete and variable expression. The mutation (p.D1816VfsX7) resulted in a severe truncation (201 residues) of the Nav1.5 C-terminus. METHODS AND RESULTS Wild-type (WT) and mutated Nav1.5 channels together with hNavβ1 were expressed in CHO cells and currents were recorded at room temperature using the whole-cell patch-clamp. Expression of p.D1816VfsX7 alone resulted in a marked reduction (≈90%) in peak Na(+) current density compared with WT channels. Peak current density generated by p.D1816VfsX7+WT was ≈50% of that generated by WT channels. p.D1816VfsX7 positively shifted activation and inactivation curves, leading to a significant reduction of the window current. The mutation accelerated current activation and reactivation kinetics and increased the fraction of channels developing slow inactivation with prolonged depolarizations. However, late INa was not modified by the mutation. p.D1816VfsX7 produced a marked reduction of channel trafficking toward the membrane that was not restored by decreasing incubation temperature during cell culture or by incubation with 300 μM mexiletine and 5 mM 4-phenylbutirate. CONCLUSION Despite a severe truncation of the C-terminus, the resulting mutated channels generate currents, albeit with reduced amplitude and altered biophysical properties, confirming the key role of the C-terminal domain in the expression and function of the cardiac Na(+) channel.

Neurofilament triplet proteins are restricted to a subset of neurons in the rat neocortex.

The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.

Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.

Parenteral is more efficient than mucosal immunization to induce regression of human papillomavirus-associated genital tumors.

Cervical cancer is a public health concern as it represents the second cause of cancer death in women worldwide. High-risk human papillomaviruses (HPV) are the etiologic agents, and HPV E6 and/or E7 oncogene-specific therapeutic vaccines are under development to treat HPV-related lesions in women. Whether the use of mucosal routes of immunization may be preferable for inducing cell-mediated immune responses able to eradicate genital tumors is still debated because of the uniqueness of the female genital mucosa (GM) and the limited experimentation. Here, we compared the protective activity resulting from immunization of mice via intranasal (i.n.), intravaginal (IVAG) or subcutaneous (s.c.) routes with an adjuvanted HPV type 16 E7 polypeptide vaccine. Our data show that s.c. and i.n. immunizations elicited similar frequencies and avidity of TetE71CD81 and E7-specific Interferon-gamma-secreting cells in the GM, whereas slightly lower immune responses were induced by IVAG immunization. In a novel orthotopic murine model, both s.c. and i.n. immunizations allowed for complete long-term protection against genital E7-expressing tumor challenge. However, only s.c. immunization induced complete regression of already established genital tumors. This suggests that the higher E7-specific systemic response observed after s.c. immunization may contribute to the regression of growing genital tumors, whereas local immune responses may be sufficient to impede genital challenges. Thus, our data show that for an efficiently adjuvanted protein-based vaccine, parenteral vaccination route is superior to mucosal vaccination route for inducing regression of established genital tumors in a murine model of HPV-associated genital cancer.

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

A dynamic view of hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large structures, representing membranous webs, showed restricted motility, were stable over many hours, were partitioned among daughter cells during cell division, and displayed a static internal architecture without detectable exchange of NS5A-GFP. (ii) In contrast, small structures, presumably representing small RCs, showed fast, saltatory movements over long distances. Both populations were associated with endoplasmic reticulum (ER) tubules, but only small RCs showed ER-independent, microtubule (MT)-dependent transport. We suggest that this MT-dependent transport sustains two distinct RC populations, which are both required during the HCV life cycle.