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.

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Molecular characterization of signalling complexes involved in G-protein coupled receptor-induced cardiac hypertrophy

In response to pathological stresses, the heart undergoes a remodelling process associated with cardiac hypertrophy. Since sustained hypertrophy can progress to heart failure, there is an intense investigation about the intracellular signalling pathways that control cardiomyocyte growth. Accumulating evidence has demonstrated that most stimuli known to initiate pathological changes associated with the development of cardiac hypertrophy activate G protein-coupled receptors (GPCRs) including the αl-adrenergic- (αl-AR), Angiotensin II- (AT-R) and endothelin-1- (ET-R) receptors. In this context, we have previously identified a cardiac scaffolding protein, called AKAP-Lbc (Α-kinase anchoring protein), with an intrinsic Rho specific guanine nucleotide exchange factor activity, that plays a key role in integrating and transducing hypertrophic signals initiated by these GPCRs (Appert-Collin, Cotecchia et al. 2007). Activated RhoA controls the transcriptional activation of genes involved in cardiomyocyte hypertrophy through signalling pathways that remain to be characterized. Here, we identified the nuclear factor-Kappa Β (NF-κΒ) activating kinase ΙΚΚβ as a novel AKAP-Lbc interacting protein. This raises the hypothesis that AKAP-Lbc might promote cardiomyocyte growth by maintaining a signalling complex that promotes the activation of the pro-hypertrophic transcription factor NF-κΒ. In fact, the activation of NF- κΒ-dependent transcription has been detected in numerous disease contexts, including hypertrophy, ischemia/reperfusion injury, myocardial infarction, allograft rejection, myocarditis, apoptosis, and more (Hall, Hasday et al. 2006). While it is known by more than a decade that NF-κΒ is a critical mediator of cardiac hypertrophy, it is currently poorly understood how pro-hypertrophic signals controlling NF-κΒ transcriptional activity are integrated and coordinated within cardiomyocytes. In this study, we show that AKAP-Lbc and ΙΚΚβ form a transduction complex in cardiomyocytes that couples activation of αl-ARs to NF-κB-mediated transcriptional reprogramming events associated with cardiomyocyte hypertrophy. In particular, we can show that activation of ΙΚΚβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukine-6 (IL-6), which, in turn, enhances foetal gene expression. These findings indicate that the AKAP-Lbc/ΙΚΚβ complex is critical for selectively directing catecholamine signals to the induction of cardiomyocyte hypertrophy.

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.

Photoactive sawhorse-type diruthenium tetracarbonyl complexes

Sawhorse-type diruthenium tetracarbonyl complexes incorporating carboxyphenyl porphyrin bridges and pyridine axial ligands have been prepared, characterized and evaluated as potential photosensitizing and chemotherapeutic agents in several human cancer cells (A2780, A549, Me300, HeLa). The mono carboxyphenyl porphyrin derivatives, 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin (HOOCR1-H2) and 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin-Zn (HOOCR1-Zn), after reaction with Ru-3(CO)(12) and pyridine, give the dinuclear complexes [Ru-2(CO)(4)(OOCR1-H2)(2)(NC5H5)(2)] (1) and [Ru-2(CO)(4)-(OOCR1-Zn)(2)(NC5H5)(2)] (2), respectively. Under the same reaction conditions, the di-carboxyphenyl porphyrin derivatives, 5,10-di(4-carboxyphenyl)-15,20-diphenyl-21,23H-porphyrin (HOOCR2-H2COOH) and 5,10-di(4-carboxyphenyl)-15,20-diphenylporphyrin-Zn (HOOCR2-ZnCOOH), give rise to the tetranuclear complexes, [{Ru-2(CO)(4)(NC5H5)(2)}(2)(OOCR2-H2COO)(2)] (3) and [{Ru-2(CO)(4)(NC5H5)(2! )}(2)(OOCR2-ZnCOO)(2)] (4), in which two sawhorse diruthenium tetracarbonyl units are linked by the di-carboxyphenyl porphyrin ligands. When tested in human cancer cell lines, both Zn(II) metallo-porphyrin derivatives 2 and 4 and the tetranuclear derivative 3 show some degree of cytotoxicity in the dark, but seem to present no phototoxicity upon irradiation at 652 nm. These results demonstrate the effect of the Zn(II) ion insertion into the porphyrin core, resulting in increased cytotoxicity and decreased phototoxicity. On the other hand, complex 1, the less cytotoxic derivative with IC50 > 170 mu M in HeLa cervix and A2780 ovarian cancer cell lines, shows an excellent phototoxicity toward these cancer cell lines with LD50 comprised between 4.5 and 7.5 J/cm(2) (irradiance 30 mW/cm(2)) at 5 mu M concentration (incubation time: 24 h). Overall, an excellent ratio between photo-and cytotoxicity has been found for the metal-free porphyrin derivative [Ru-2(CO)(4)(OOCR1-H2)(2)(! NC5H5)(2)] (1).

Complexité, médecine générale et réformes des systèmes de santé [Complexity, general practice and reforms of health systems].

The family doctor facing complexity must decide in situations of low certainty and low agreement. Complexity is in part subjective but can also be measured. Changes in the health systems aim to reduce health costs. They tend to give priority to simple situations and to neglect complexity. One role of an academic institute of family medicine is to present and promote the results of scientific research supporting the principles of family medicine, taking into account both the local context and health systems reforms. In Switzerland the new challenge is the introduction of managed care.

Antigen-secretory IgA immune complexes are retro-transported into mouse Peyer's patches: immunotargeting to dendritic cells

RÉSUMÉ Les plaques de Peyer (PP) représentent le site d'entrée majeur des pathogènes au niveau des muqueuses intestinales. Après avoir traversé la cellule M, l'antigène est pris en charge par les cellules dendritiques (DC) de la région sub-épithéliale du dôme des PP. Ces dernières activent une réponse immunitaire qui conduit à la production de l'IgA de sécrétion (SIgA), l'anticorps majeur au niveau muqueux. Des études précédentes dans notre laboratoire ont démontré qu'après administration de SIgA dans des anses intestinales de souris, les SIgA se lient spécifiquement aux cellules M, entrent dans les PP, et sont éventuellement internalisées par les DC. Le but de ce travail est de comprendre la relevance biologique de l'entrée des SIgA dans les PP et leur relevance physiologique dans l'homéostasie mucosale. Dans un premier temps, nous avons montré en utilisant une méthode de purification optimisée basée sur une isolation magnétique, que, en plus des DC myéloïdes (CD11c+/CD11b+) et des DC lymphoïdes (CD11c+/CD8+), les PP de souris contiennent un nouveau sous-type de DC exprimant les marqueurs CD11c et CD19. L'utilisation de la microscopie confocale nous a permis de démontrer que les DC myéloïdes internalisent des SIgA, contrairement aux DC lymphoïdes qui n'interagissent pas avec les SIgA, alors que le nouveau sous-type de DC exprimant CD19 lie les SIgA. En plus, nous avons démontré qu'aucune des DC de rate, de ganglion bronchique ou de ganglion inguinal interagit avec les SIgA. Dans le but d'explorer si les SIgA peuvent délivrer des antigènes aux DC des PP in vivo, nous avons administré des complexes immunitaires formés de Shigella flexneri complexées à des SIgA, dans des anses intestinales de souris. Nous avons observé une entrée dans les PP, suivie d'une migration vers les ganglions mésentériques drainants, contrairement aux Shigella flexneri seules, qui n'infectent pas la souris par la voie intestinale. Shigella flexneri délivrée par SIgA n'induit pas de destruction tissulaire au niveau de l'intestin. En plus de l'exclusion immunitaire, ces résultats suggèrent un nouveau rôle des SIgA, qui consiste à transporter des antigènes à l'intérieur des PP dans un contexte non-inflammatoire. RÉSUMÉ DESTINÉ À UN LARGE PUBLIC L'intestin a pour rôle principal d'absorber les nutriments digérés tout au long du tube digestif, et de les faire passer dans le compartiment intérieur sanguin. Du fait de son exposition chronique avec un monde extérieur constitué d'aliments et de bactéries, l'intestin est un endroit susceptible aux infections et a donc besoin d'empêcher l'entrée de microbes. Pour cela, l'intestin est tapissé de "casernes" appelées les plaques de Peyer, qui appartiennent à un système de défense appelé système immunitaire muqueux. Les plaques de Peyer sont composées de différents types de cellules, ayant pour rôle de contrôler l'entrée de microbes et de développer une réaction immunitaire lors d'infection. Cette réaction immunitaire contre les microbes (antigènes) débute par la prise en charge de l'antigène par des sentinelles, les cellules dendritiques. L'antigène est préparé de façon à être reconnu par d'autres cellules appelées lymphocytes T capables d'activer d'autres cellules, les lymphocytes B. La réaction immunitaire résulte dans la production par les lymphocytes B d'un anticorps spécifique appelé IgA de sécrétion (SIgA) au niveau de la lumière intestinale. De manière classique, le rôle de SIgA au niveau de la lumière intestinale consiste à enrober les microbes et donc exclure leur entrée dans le compartiment intérieur. Dans ce travail, nous avons découvert une nouvelle fonction des SIgA qui consiste à introduire des antigènes dans les plaques de Peyer, et de les diriger vers les cellules dendritiques. Sachant que les SIgA sont des anticorps qui ne déclenchent pas de réactions de défense violentes dites inflammatoires, l'entrée des antigènes via SIgA serait en faveur d'une défense intestinale maîtrisée sans qu'il y ait d'inflammation délétère. Ces résultats nous laissent supposer que l'entrée d'antigènes via SIgA pourrait conduire le système immunitaire muqueux à reconnaître ces antigènes de manière appropriée. Ce mécanisme pourrait expliquer les désordres immunitaires de types allergiques et maladies auto-immunitaires que l'on rencontre chez certaines personnes déficientes en IgA, chez qui cette lecture d'antigènes de manière correcte serait inadéquate. ABSTRACT Peyer's patches (PP) represent the primary site for uptake and presentation of ingested antigens in the intestine. Antigens are sampled by M cells, which pass them to underlying antigen-presenting cells including dendritic cells (DC). This leads to the induction of mucosal T cell response that is important for the production of secretory IgA (SIgA), the chief antibody at mucosal surfaces. Previous studies in the laboratory have shown that exogenous SIgA administrated into mouse intestinal loop binds specifically to M cells, enter into PP, and is eventually internalized by DC. The aim of this work is to understand the biological significance of the SIgA uptake by PP DC and its physiological relevance for mucosal homeostasis. As a first step, we have shown by using an optimized MACS method that, in addition to the CD11c+/CD11b+ (myeloid DC) and CD11c+/CD8+ (lymphoid DC) subtypes, mouse PP contain a novel DC subtype exhibiting both CD11c and CD19 markers. By using a combination of MACS isolation and confocal microscopy, we have demonstrated that in contrast to the lymphoid DC which do not interact with SIgA, the myeloid DC internalize SIgA, while the CD19+ subtype binds SIgA on its surface. Neither spleen DC, nor bronchial-lymph node DC, nor inguinal lymph node DC exhibit such a binding specificity. To test whether SIgA could deliver antigens to PP DC in vivo, we administered SIgA-Shigella flexneri immune complexes into mouse intestinal loop containing a PP. We found that (i) SIgA-Shigella flexneri immune complexes enter the PP and are internalized by sub-epithelial dome PP DC, in contrast to Shigella flexneri alone that does not penetrate the intestinal epithelia in mice, (ii) immune complexes migrate to the draining mesenteric lymph node, (iii) Shigella flexneri carried via SIgA do not induce intestinal tissue destruction. Our results suggest that in addition to immune exclusion, SIgA transports antigens back to the PP under non-inflammatory conditions.

Development of a well-defined medium for the in vitro maturation of immature bovine cumulus-oocyte complexes.

PURPOSE: Our purpose was to develop a well-defined medium for the in vitro maturation (IVM) of immature bovine cumulus-oocyte complexes (COC). METHODS: The COC were cultured in the presence of three protein supplementations: fetal bovine serum (FBS), bovine serum albumin, and Synthetic Serum Substitute. The embryos obtained after in vitro fertilization of IVM oocytes were cocultured with Vero cells and their development to the morula and blastocyst stages was studied. RESULTS: When FBS was absent from the IVM medium, a significantly lower fertilization rate was observed, followed by a decrease in the percentage of embryos reaching the blastocyst stage. When FBS was replaced by a defined protein supplementation, the best results were obtained with Synthetic Serum Substitute. CONCLUSIONS: Adequate protein supplementation of the IVM medium optimizes the fertilization rate and the development of bovine IVM oocytes. The implication of these results in the human field is discussed.

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Synthesis and anticancer activity of long-chain isonicotinic ester ligand-containing arene ruthenium complexes and nanoparticles

Arene ruthenium complexes containing long-chain N-ligands L1 = NC5H4-4-COO-C6H4-4-O-(CH2)9-CH3 or L2 = NC5H4-4-COO-(CH2)10-O-C6H4-4-COO-C6H4-4-C6H4-4-CN derived from isonicotinic acid, of the type [(arene)Ru(L)Cl2] (arene = C6H6, L = L1: 1; arene = p-MeC6H4Pr i , L = L1: 2; arene = C6Me6, L = L1: 3; arene = C6H6, L = L2: 4; arene = p-MeC6H4Pr i , L = L2: 5; arene = C6Me6, L = L2: 6) have been synthesized from the corresponding [(arene)RuCl2]2 precursor with the long-chain N-ligand L in dichloromethane. Ruthenium nanoparticles stabilized by L1 have been prepared by the solvent-free reduction of 1 with hydrogen or by reducing [(arene)Ru(H2O)3]SO4 in ethanol in the presence of L1 with hydrogen. These complexes and nanoparticles show a high anticancer activity towards human ovarian cell lines, the highest cytotoxicity being obtained for complex 2 (IC50 = 2 μM for A2780 and 7 μM for A2780cisR)