Characterization of the role of Rho activating signalling complexes in G protein-coupled receptor induced cardiac fibrosis

Dans certaines conditions pathologiques, telles que l'hypertension artérielle ou l'infarctus du myocarde, le coeur répond à une augmentation de la post-charge par des processus de remodelage aboutissant à une hypertrophie du ventricule gauche. L'hypertrophie cardiaque est caractérisée par une croissance hypertrophique des cardiomyocytes, ainsi que par une différenciation des fibroblastes en un phenotype présentant une capacité accrue de synthèse protéiques, nommés myofibroblastes. Ceci résulte en une accumulation excessive des constituants de la matrice extracellulaire, ou autrement dit fibrose. En raison de son effet délétère sur la contractilité du coeur, menant sur le long terme à une insuffisance cardiaque, de nombreux efforts ont été déployés, afin de définir les mécanismes moléculaires impliqués dans la réponse profibrotique. A ce jour, de nombreuses études indiquent que la petite GTPase RhoA pourrait être un médiateur important de la réponse profibrotique du myocarde. Cependant, les facteurs d'échanges impliqués dans la transduction de signaux profibrotiques, via la régulation de son activité au niveau des fibroblastes cardiaques, n'ont pas encore été identifiés. De précédentes études menées dans le laboratoire, ont identifiées une nouvelle protein d'ancrage de la PKA, exprimée majoritairement dans le coeur, nommée AKAP-Lbc. Il a été montré que cette protéine, en plus de sa fonction de protein d'ancrage, possédait une activité de facteur d'échange de nucléotide guanine (GEF) pour la petite GTPase RhoA. Au niveau des cardiomyocytes, il a été montré que l'AKAP-Lbc participe à une voie de signalisation pro-hypertrophique, incluant la sous-unité alpha de la protéine G hétérotrimerique G12 et RhoA. Chose intéressante, des observations antérieures à cette étude, indiquent que dans le coeur, l'AKAP-Lbc est également exprimée dans les fibroblastes. Cependant aucunes études n'a encore reporté de fonction pour ce facteur d'échange dans les fibroblastes cardiaques. Dans ce travail, les résultats obtenus indiquent que dans les fibroblastes cardiaques, I'activation de RhoA par l'AKAP-Lbc est impliquée dans la transmission de signaux profibrotiques, en aval des récépteurs à l'angiotensine II. En particulier, nous avons observé que la suppression de l'expression de l'AKAP-Lbc dans les fibroblastes ventriculaires de rat adultes, réduisait fortement Γ activation de Rho induite par l'angiotensine II, la déposition de collagène, la capacité migratoire des fibroblastes ainsi que leur différenciation en myofibroblastes. A notre connaissance, l'AKAP-Lbc est le premier RhoGEF identifié comme médiateur de la réponse profibrotique dans les fibroblastes cardiaques. - In pathological conditions such as chronic hypertension or myocardial infarction, the myocardium is subjected to various biomechanical and biochemical stresses, and undergoes an adverse ventricular remodelling process associated with cardiomyocytes hypertrophy and excess deposition of extracellular matrix proteins resulting in fibrosis. During the fibrotic response, cardiac fibroblasts differentiate into a more mobile and contractile phenotype termed myofibroblasts. These cells, possess a greater synthetic ability to produce ECM proteins and have been implicated in diseases with increased ECM deposition including cardiac fibrosis. Because fibrosis impairs myocardial contractility and is associated with the progression to heart failure, a major cause of lethality worldwide, many efforts have been made to define the molecular players involved in this process. During these last years, increasing evidence suggests a role for the small GTPase RhoA in mediating the fibrotic response in CFbs. However the identity of the exchange factors that modulate its activity and transduce fibrotic signals in CFbs is still unknown. Earlier work in our laboratory identified a novel PKA anchoring protein expressed in the heart termed AKAP-Lbc that has been shown to function as anchoring protein as well as a guanine nucleotide exchange factor (GEF) for the small GTPase RhoA. In response to several hypertrophic stimuli we have shown that RhoGEF activity of AKAP-Lbc mediated by Gan promotes the activation of a signaling pathway including RhoA, leading to cardiomyocytes hypertrophy. Within the heart, previous observations made in the laboratory indicated that AKAP-Lbc was also expressed in fibroblasts. However its role in cardiac fibroblasts remained to be determined. In the present study, we show that AKAP-Lbc is critical for activating RhoA and transducing profibrotic signals downstream of angiotensin II receptors in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin RNAs strongly reduces angiotensin II-induced RhoA activation, collagen deposition as well as cell migration and differentiation. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor involved in a profibrotic signalling pathway at the level of cardiac fibroblasts.

Impact of precipitation on the water table level of different ombrotrophic raised bog complexes, central Estonia

Sadannan vaikutus vedenpinnan tasoon kohosuolla

Oeuvres complètes de Alfred de Vigny. [VI]

Collection : Petite bibliothèque littéraire

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Are the light-harvesting I complexes from Rhodospirillum rubrum arranged around the reaction centre in a square geometry?

The basic photosynthetic unit containing the reaction centre and the light-harvesting I complex (RC-LHI) of the purple non-sulphur bacterium Rhodospirillum rubrum was purified and reconstituted into two-dimensional (2D) membrane crystals. Transmission electron microscopy using conventional techniques and cryoelectron microscopy of the purified single particles and of 2D crystals yielded a projection of the RC-LHI complex at a resolution of at least 1.6 nm. In this projection the LHI ring appears to have a square symmetry and packs in a square crystal lattice. The square geometry of the LHI ring was observed also in images of single isolated particles of the RC-LHI complex. However, although the LHI units are packed identically within the crystal lattice, a new rotational analysis developed here showed that the reaction centres take up one of four possible orientations within the ring. This fourfold disorder supports our interpretation of a square ring symmetry and suggests that a hitherto undetected component may be present within the photosynthetic unit.

Reliure de : I Discorsi di M. Pietro Andrea Matthioli,... nelli sei libri di Pedacio Dioscoride,... della materia medicinale, hora di nuovo dal suo istesso autore ricorretti et... aumentati... con due tavole copiosissime... - Del Modo di distillare le acque da tutte le piante, et come vi si possino conservare i loro veri odori et sapori

Numérisation partielle de reliure

Mapeamento pedológico digital da folha Botucatu (SF-22-Z-B-VI-3): treinamento de dados em mapa tradicional e validação de campo

O mapeamento digital de solos permite prever padrões de ocorrência de solos com base em áreas de referência e no uso de técnicas de mineração de dados para modelar associações solo-paisagem. Os objetivos deste trabalho foram produzir um mapa pedológico digital por meio de técnicas de mineração de dados aplicadas a variáveis geomorfométricas e de geologia, com base em áreas de referência; e testar a confiabilidade desse mapa por meio de validação em campo com diferentes sistemas de amostragem. O mapeamento foi realizado na folha Botucatu (SF-22-Z-B-VI-3), utilizando-se as folhas 1:50.000, Dois Córregos e São Pedro, como áreas de referência. Variáveis descritoras do relevo e de geologia associadas às unidades de mapeamento pedológico das áreas de referência compuseram a matriz de dados de treinamento. A matriz foi analisada pelo algoritmo PART de árvore de decisão, do aplicativo Weka (Waikato Environment for Knowledge Analysis), que cria regras de classificação. Essas regras foram aplicadas aos dados geomorfométricos e geológicos da folha Botucatu, para predição de unidades de mapeamento pedológico. A validação de campo dos mapas digitais deu-se por meio de amostragem por transectos em uma unidade de mapeamento da folha São Pedro e de forma aleatório-estratificada na folha Botucatu. A avaliação da unidade de mapeamento na folha São Pedro verificou confiabilidade, respectivamente, de 83 e 66 %, para os mapas pedológicos digital e tradicional com legenda simplificada. Apesar de terem sido geradas regras para todas as unidades de mapeamento pedológico das áreas de treinamento, nem todas as unidades de mapeamento foram preditas na folha Botucatu, o que resultou das diferenças de relevo e geologia entre as áreas de treinamento e de mapeamento. A validação de campo do mapa digital da folha Botucatu verificou exatidão global de 52 %, compatível com levantamentos em nível de reconhecimento de baixa intensidade, e kappa de 0,41, indicando qualidade Boa. Unidades de mapeamento mais extensas geraram mais regras, resultando melhor reprodução dos padrões solo-relevo na área a ser mapeada. A validação por transectos na folha São Pedro indicou compatibilidade do mapa digital com o nível de reconhecimento de alta intensidade e compatibilidade do mapa tradicional, após simplificação de sua legenda, com o nível de reconhecimento de baixa intensidade. O treinamento do algoritmo em mapas e não em observações pontuais reduziu em 14 % a exatidão do mapa pedológico digital da folha Botucatu. A amostragem aleatório-estratificada pelo hipercubo latino é apropriada a mapeamentos com extensa base de dados, o que permite avaliar o mapa como um todo, tornando os trabalhos de campo mais eficientes. A amostragem em transectos é compatível com a avaliação da pureza de unidades de mapeamento individualmente, não necessitando de base de dados detalhada e permitindo estudos de associações solo-paisagem em pedossequências.

Cal.limac h. VI, Euforió fr. 8 Powell i les "tauletes de maledicció" de Cnidos

The scene from Callirnachus' H. VI in which Derneter, under the appearance of her arateira Nicippa, curses Erisichthon rnay be related to the arai of Cnidos. On the other hand, Euphorio fr. 8 Powell can be understood better in the light of the curses of Cnidos, where the culprit's name is not rnentioned ut rhe first stage; a similar interpretation can be applied to Ovid's Ibis 7-10, which was probably inspired on Euphorio

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

The Vi element. A transposon-like repeated DNA sequence interspersed in the vitellogenin locus of Xenopus laevis.

A repeated DNA element in Xenopus laevis is described that is present in about 7500 copies dispersed throughout the genome. It was first identified in the 5' flanking region of one vitellogenin gene and was therefore named the Vi element. Seven copies are present within the vitellogenin gene region, three of them within introns of the genes A1, A2 and B2, and the other four copies in the gene flanking regions. Four of these copies have been sequenced. The Vi element is bounded by a well-conserved 13 base-pair inverted repeat; in addition, it is flanked by a three base-pair direct repeat that appears to be site-specific. The length of these four copies varies from 112 to 469 base-pairs; however, sequence homology between the different copies is very high. Their structural characteristics suggest that length heterogeneity may have arisen by either unequal recombinations, deletions or tandem duplications. Altogether, the characteristics and properties of the Vi element indicate that it might represent a mobile genetic element. One of the four copies sequenced is inserted close (position -535) to the transcription initiation site of the vitellogenin gene B2 in a region otherwise showing considerable homology with the closely related gene B1. Nevertheless, the presence of the Vi element does not seem to influence significantly the estrogen-controlled expression of gene B2. In addition, three alleles of this gene created by length polymorphism in intron 3 and in the Vi element inserted near the transcription initiation site are described.

Visualization of RNA polymerase II ternary transcription complexes formed in vitro on a Xenopus laevis vitellogenin gene.

Stable ternary transcription complexes assembled in vitro, using a HeLa whole-cell extract, have been isolated and visualized by electron microscopy. The formation of these stable complexes on the DNA fragment used as template, the 5' end region of the Xenopus laevis vitellogenin gene B2, depends on factors present in the whole-cell extract, RNA polymerase II and at least two nucleotides. Interestingly, bending in the DNA fragment was frequently observed at the binding site of RNA polymerase II. Dinucleotides that can prime initiation within a short sequence of approximately 10 contiguous nucleotides centered around the initiation site used in vivo, also favour the formation of stable complexes. In addition, pre-initiation complexes were isolated and it was shown that factors in the extract involved in their formation are more abundant than the RNA polymerase II molecules available for binding. The possible implication of this observation relative to the in vivo situation is discussed.

La fortuna di Luciano nel Rinascimento : il volgarizzamento del manoscritto Vaticano Chigiano L.VI.215 : edizione critica dei volgarizzamenti delle "Storie vere"

Résumé II lavoro verte sui volgarizzamenti quattro-cinquecenteschi di Luciano di Samosata, importante capitolo nella fortuna dell'autore greco, che diede l'avvio a quel vasto fenomeno chiamato "lucianesimo", esteso in Europa fino al XIX sec. In particolare fornisco l'edizione critica e commentata delle Storie vere volgarizzate, contenute nella prima, assai ampia (41 opuscoli), e per molto tempo unica, silloge lucianea in volgare, che ho datato a poco prima del 1480. Essa ci è giunta tramite un unico manoscritto, il Vaticano Chigiano L.VI.215, confezionato a Ferrara per Ercole I d'Este, nonché in almeno otto edizioni veneziane apparse fra il 1525 e il 1551. La princeps, da cui dipendono in vario modo tutte le edizioni successive, è pubblicata da Niccolò Zoppino. I1 ms. e le prime due edizioni (1525; 1527 Bindoni e Pasini) tacciono il nome del traduttore, che compare solo nell'edizione del 1529 (Zoppino): Niccolò Leoniceno (1428-1524), medico umanista e valente grecista, attivo a Ferrara dal 1464 al 1524, studioso e traduttore di Ippocrate e Galeno, editore di Aristotele e volgarizzatore di storici per Ercole d'Este. L'edizione ha richiesto uno studio preliminare sulle numerose traduzioni in latino e in volgare di Luciano, per valutare meglio le modalità della sua fortuna umanistica. Confrontando ms. e stampe, per le Storie vere si hanno due volgarizzamenti totalmente diversi, fin dal titolo: La vera historia nel ms., Le vere narrazioni nelle cinquecentine. Ma per l'ultimo quarto di testo, ms. e stampe in sostanza coincidono. La collazione ha coinvolto anche il testo greco (con gli apparati delle edizioni critiche) e la versione latina dell'umanista umbro Lilio Tifernate (1417/18-1486) risalente al 1439-43 ca., intitolata De veris narrationibus, di cui si hanno almeno tre redazioni d'autore; una quarta è invece dovuta probabilmente a Benedetto Bordon, che la inserì nella sua silloge latina di Luciano del 1494. Ho cosa stabilito che il volgarizzamento del ms. Chigiano, La vera historia, è stato eseguito direttamente dal greco, fatto eccezionale nel panorama delle traduzioni umanistiche, mentre quello a stampa, Le vere narrazioni, deriva dalla redazione Bordon del De veris narrationibus. La diversità dei titoli dipende dalle varianti dei codici greci utilizzati dai traduttori: il Vat. gr. 1323, o una sua copia, è utilizzato sia dal volgarizzatore del Chigiano, sia da Bordon, indipendentemente l'uno dall'altro; il Marc. gr. 434, o una sua copia, dal Tifernate. Il titolo latino mantenuto da Bordon risale al Tifernate. Per quanto riguarda l'attribuzione dei due volgarizzamenti, come già per altri due testi della silloge da me studiati (Lucio 01 Asino e Timone), anche per La vera historia del Chigiano è accettabile il nome di Niccolò Leoniceno, poiché: 1) essa è tradotta direttamente dal greco, correttamente e con buona resa in volgare, 2) Paolo Giovio -che conobbe di persona il Leoniceno -, negli Elogia veris clarorum virorum imaginibus apposita ricorda che i volgarizzamenti di Luciano e di Dione eseguiti dal Leoniceno piacquero molto ad Ercole d'Este, 3) nessuno nella prima metà del sec. XVI rivendica, per sé o per un suo maestro, il volgarizzamento di Luciano. Le vere narrazioni a stampa, tradotte dal latino del Bordon, dopo il 1494 e prima del 1525, per la parte che diverge dalla Vera historia rimangono invece anonime. Dato che si tratta di due volgarizzamenti distinti, ho allestito l'edizione a fronte dei due testi fin dove essi divergono, seguendo per l'uno il ms., per l'altro la princeps; per la parte finale, in cui confluiscono, mi baso invece sul manoscritto e relego in apparato le varianti più vistose della princeps (non è emerso un chiaro rapporto di dipendenza fra i due testimoni). Oltre all'apparato critico con le lezioni rifiutate, fornisco un commento con la giustificazione delle scelte e il confronto con i corrispondenti passi greci e latini.