Inhibition of Notch Pathway Enhances Photoreceptor Commitment From Cultured Retinal Stem Cells

Purpose: Consequently to the principle that photoreceptors have to be at a very precise development stage to be successfully transplanted (MacLaren 2006), we are trying to mimic this development stage in vitro using retinal stem cells. The latter one isolated from the newborn mouse retina, derived from the radial glia population, which were previously isolated and characterized in our laboratory. We developed a protocol to commit these cells to the photoreceptor fate, but even if the percentage of cells expressing photoreceptor markers is high (30%), the differentiation process is incomplete so far (Merhi-Soussi 2006). Methods: In order to ameliorate photoreceptor differentiation, we hypothesized that the Notch pathway may interfere with this process by either promoting glia commitment, or maintaining an undifferentiated state. We are thus using a gamma-secretase inhibitor (DAPT), which inhibits Notch receptor cleavage and thus Notch activation. DAPT was used either during the whole differentiation stimulation, or only during a restricted period in two various retinal stem cell lines (RSC AA and RSC MP1). Results: RT-PCR performed during cell proliferation, showed the same positive expression in both cell lines for the following genes: Math3, Six3, Hes1, NeuroD, Pax6 and Notch1. Additionally, Mash1, Hes5, Prox1, Crx and Otx2 were detected in both cell lines but with a stronger expression in RSC MP1. Opposite results were obtained for Chx10. Nrl, Peripherin/RDS, GFAP and Math5 were detected neither in RSC AA, nor in RSC MP1. The constant presence of DAPT i) leads to a 233% (RSC AA) or 900% (RSC MP1) increase in peripherin/RDS-positive (photoreceptor marker) cells, compared to controls (no DAPT, n=3, P<0.02) along with a 68% (RSC AA) or 80% (RSC MP1) decrease in GFAP- positive cells (n=3, P<0.04), ii) modifies the ratio between uni-/bi- (23%) and multi- (77%) polar peripherin/RDS-positive cells to 45% and 55%, respectively, for both cell lines and iii) reduces by 50% the total cell number during the whole differentiation process for both cell lines. Conclusions: We are now exploring whether this reduction in total cell number is due to inhibition of cell proliferation or to cell death and whether photoreceptor differentiation is promoted instead of glial induction. We also want to confirm the results obtained with DAPT with RSCs isolated from Notch1-loxP mice. Such protocol may help to better mimic photoreceptor development, but this needs to be confirmed by genomic and proteomic profile analyses.

Placental growth factor-1 and epithelial haemato-retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy.

AIMS/HYPOTHESIS: Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. METHODS: ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. RESULTS: In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. CONCLUSIONS/INTERPRETATION: Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

Variabilidade genética de Sugarcane mosaic virus, causando mosaico em milho no Brasil

O objetivo deste trabalho foi caracterizar biológica e molecularmente três isolados de Sugarcane mosaic virus (SCMV) de lavouras de milho, analisá-los filogeneticamente e discriminar polimorfismos do genoma. Plantas com sintomas de mosaico e nanismo foram coletadas em lavouras de milho, no Estado de São Paulo e no Município de Rio Verde, GO, e seus extratos foliares foram inoculados em plantas indicadoras e submetidos à análise sorológica com antissoros contra o SCMV, contra o Maize dwarf mosaic virus (MDMV) e contra o Johnsongrass mosaic virus (JGMV). Mudas de sorgo 'Rio' e 'TX 2786' apresentaram sintomas de mosaico após a inoculação dos três isolados, e o DAS-ELISA confirmou a infecção pelo SCMV. O RNA total foi extraído e usado para amplificação por transcriptase reversa seguida de reação em cadeia de polimerase (RT-PCR). Fragmentos específicos foram amplificados, submetidos à análise por polimorfismo de comprimento de fragmento de restrição (RFLP) e sequenciados. Foi possível discriminar os genótipos de SCMV isolados de milho de outros isolados brasileiros do vírus. Alinhamentos múltiplos e análises dos perfis filogenéticos corroboram esses dados e mostram diversidade nas sequências de nucleotídeos que codificam para a proteína capsidial, o que explica o agrupamento separado desses isolados e sugere sua classificação como estirpes distintas, em lugar de simples isolados geográficos.

Oligonucleotide-polyethylenimine complexes targeting retinal cells: structural analysis and application to anti-TGFbeta-2 therapy.

PURPOSE: The aim of this study was to characterize oligonucleotide-polyethylenimine (ODN/PEI) complex preparation for potential transfection of retinal cells in vitro and in vivo. METHODS: The effect of medium preparation [HEPES-buffered saline (HBS), water] on particle size and morphology was evaluated. Cultured Lewis rat retinal Müller glial (RMG) cells were transfected using fluorescein isothiocyanate (FITC)-ODN/PEI complexes specifically directed at transforming growth factor beta (TGFbeta)-2. Efficacy of transfection was evaluated using confocal microscopy, and regulation of gene expression was assayed using quantitative real-time RT-PCR and ELISA assay. One, 24, and 72 h after injection of FITC-ODN/PEI complexes into the vitreous of rat eyes, their distribution was analyzed on eye sections. RESULTS: Complexes prepared in HBS were smaller than complexes prepared in pure water and presented a core-shell structure. These particles showed a high cellular internalization efficacy, along with a significant and specific down-regulation of TGFbeta-2 expression and production in RMG cells, correlating with specific inhibition of cell growth at 72 h. In vivo, complexes efficiently transfect retinal cells and follow a transretinal migration at 24 h. After 72 h, ODN seems to preferentially target RMG cells without inducing any detectable toxicity. CONCLUSIONS: Specific down-regulation of TGFbeta-2 expression using ODN/PEI complexes may have potential interest for the treatment of retinal diseases associated with glial proliferation.

Proliferative and Osteogenic Differentiation Potentials of Human Fetal Bone Cells

BACKGROUND. Human primary fetal bone cells (hFBC) are being characterized for use in bone tissue regeneration. Unlike human mesenchymal stem cells (hMSC), hFBC are partially differentiated with high expansion and regeneration potential. To date, proliferative and osteoblastic differentiation capacities of fetal bone cells remain poorly examined. The goal of this study was to define an environmental culture conditions for optimal proliferation and production of extracellular bone matrix leading to efficient bone repair. METHODS. Human primary FBC derived from our dedicated, consistent banks of bone cells comprising several fetal donors. For proliferation study, monolayer cultures of both cell types were expanded in DMEM or α- MEM media. Osteoblastic differentiation potentials of both hFBC and hMSC were evaluated through RT-PCR. Regulation of osteogenic differentiation by protein ligands Wnt3a and Wnt5a was studied by ALP enzymatic activity measurement. RESULTS. Evaluation of the proliferation rate demonstrated that hFBC proliferated more rapidly in α-MEM medium. Regarding growth factors that could stimulate cell proliferation rate, we observed that PDGF, FGF2 and Wnt3a had positive effects on proliferation of hFBC. Gene expression analysis demonstrated a higher expression of runx2 in hFBC cultured in basal conditions, which was was similar than that was observed in hMSC in osteoinductive culture conditions. Expression of sox9 was very low in hBFC and hMSC, compared to expression observed in fetal cartilage cells. Looking at osteogenic differentiation capacity, ALP activity was positively regulated byWnt5awhen hFBCwere cultured inα-MEM, but not in DMEM. Conversely, Wnt3a was shown to block the effect of osteogenic inductors on differentiation of both cell types. CONCLUSION. Data presented in this study indicate that the proliferation and differentiation of fetal and mesenchymal stem cells is optimal in α- MEM. Evidence for a pre-differentiated state of hBFC was given by extracellular matrix spontaneous mineralization as well as by higher ALP activity levels observed for these cells in baseline culture conditions, in comparison with hMSC. As we showed that, in vitro, hFBC express a higher capacity to differentiate in osteoblasts, they represent an attractive and promising prospect for fundamental research, and specifically for a new generation of skeletal tissue engineering.

EH-myomesin splice isoform is a novel marker for dilated cardiomyopathy.

The M-band is the prominent cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. To investigate M-band alterations in heart disease, we analyzed the expression of its main components, proteins of the myomesin family, in mouse and human cardiomyopathy. Cardiac function was assessed by echocardiography and compared to the expression pattern of myomesins evaluated with RT-PCR, Western blot, and immunofluorescent analysis. Disease progression in transgenic mouse models for dilated cardiomyopathy (DCM) was accompanied by specific M-band alterations. The dominant splice isoform in the embryonic heart, EH-myomesin, was strongly up-regulated in the failing heart and correlated with a decrease in cardiac function (R = -0.86). In addition, we have analyzed the expressions of myomesins in human myocardial biopsies (N = 40) obtained from DCM patients, DCM patients supported by a left ventricular assist device (LVAD), hypertrophic cardiomyopathy (HCM) patients and controls. Quantitative RT-PCR revealed that the EH-myomesin isoform was up-regulated 41-fold (P < 0.001) in the DCM patients compared to control patients. In DCM hearts supported by a LVAD and HCM hearts, the EH-myomesin expression was comparable to controls. Immunofluorescent analyses indicate that EH-myomesin was enhanced in a cell-specific manner, leading to a higher heterogeneity of the myocytes' cytoskeleton through the myocardial wall. We suggest that the up-regulation of EH-myomesin denotes an adaptive remodeling of the sarcomere cytoskeleton in the dilated heart and might serve as a marker for DCM in mouse and human myocardium.

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.

Les aquaporines 4 et 9 dans le système nerveux central : expression lors de la différenciation cellulaire et implication dans le métabolisme énergétique astrocytaire "in vitro"

RESUME : Les aquaporines (AQPs) sont des protéines membranaires perméables à l'eau (aquaporines strictes) et, pour certaines d'entre elles, également au glycérol (aquaglycéroporines). Ces protéines sont présentes dans les bactéries, les plantes et les différents organes des mammifères. Dans le cerveau, la moindre augmentation de volume hydrique peut avoir de graves conséquences sur son fonctionnement, d'où l'importance de la régulation de l'homéostasie de l'eau grâce aux AQPs. L'AQP4, une aquaporine stricte, est présente dans les astrocytes et est impliquée dans la formation et la résorption des oedèmes cérébraux. En revanche, l'AQP9 est une aquaglycéroporine, qui est localisée non seulement dans les astrocytes mais également dans les neurones catécholaminergiques. Bien que la distribution de l'AQP4 dans le cerveau soit clairement établie, la présence de l'AQP9 est toujours une donnée controversée et son rôle fonctionnel dans le système nerveux central n'est pas connu. Par ailleurs, aucune donnée n'existe sur l'expression des AQP4 et 9 lors de la différenciation de cellules souches neurales foetales (CSNf) en astrocytes ou en neurones catécholaminergiques. Dans la première partie de ce travail, un protocole a été mis au point permettant de différencier des CSNf de souris en astrocytes et neurones, dont des neurones catécholaminergiques. La caractérisation des cultures de CSNf et des cultures mixtes par immunofluorescence a permis de montrer que l'immunomarquage AQP9 est présent dans les CSNf et est conservé lors de leur différenciation en astrocytes ou en neurones catécholaminergiques. Les résultats obtenus ont mis en évidence une très bonne corrélation entre l'expression de la TH (tyrosine hydroxylase: enzyme limitante de la synthèse des catécholamines) et celle de l'AQP9 lors de la différenciation des CSNf en neurones catécholaminergiques. Par contre, l'immunomarquage AQP4 n'est pas présent dans les CSNf alors qu'il est observé dans les astrocytes. De plus, aucun immunomarquage AQP4 ou AQP9 n'a été observé dans les neurones NIAP2-positifs. Dans la deuxième partie de ce travail, l'expression des AQP4 et 9 a été quantifiée dans les CSNf ainsi que dans trois populations d'astrocytes présentant des propriétés métaboliques différentes. Ces trois populations astrocytaires sont issues de la différenciation des CSNf par le CNTF, le LIF ou le sérum de veau foetal. Les analyses par RTPCR quantitative et western blot ont montré une augmentation de l'expression de l'AQP9 et de l'AQP4 corrélée à l'acquisition de propriétés métaboliques spécifiques des astrocytes matures. Dans la dernière partie, la technique d'ARN interférents a permis d'étudier le rôle fonctionnel de l'AQP9 dans le modèle de culture pure d'astrocytes différenciés par le sérum. L'inhibition de l'expression d'AQP9 entraîne une diminution de la perméabilité au glycérol et une augmentation de l'utilisation de glucose, corrélée à une stimulation du métabolisme oxydatif astrocytaire. En revanche, 1a baisse d'expression d'AQP9 n'a aucun effet sur la glycolyse anaérobie ni sur la libération du lactate. En conclusion, dans ce modèle in vitro, seule l'AQP9 est exprimée dans les CSNf et les neurones catécholaminergiques alors que dans Ies astrocytes, à la fois l'AQP9 et l'AQP4 sont exprimées. Cette distribution est identique à celle observée in vivo et confirme la localisation spécifique de l'AQP9 dans les neurones catécholaminergiques. De plus, ces résultats montrent, pour la première fois, l'implication de l'AQP9 dans la perméabilité des astrocytes au glycérol et son implication dans le métabolisme énergétique astrocytaire. ABSTACT : Aquaporins (AQPs) are membrane proteins permeable to water (orthodoxes aquaporins) and some of them are also permeable to glycerol (aquaglyceroporins). These proteins are widely expressed in bacteria, plants and mammals. AQP water homeostasis regulation in brain is of primary importance as the brain volume cannot increase. AQP4, an orthodoxe aquaporin, is present in astrocytes and seems to be involved in edema formation and resorption. On the other hand, AQP9 is an aquaglyceroporin which is localised not only in astrocytes but also in catecholaminergic neurons. Although AQP4 distribution in brain is clearly established, the presence of AQP9 is still a discussed data and its functional role in the central nervous system is unknown. In addition, no data exists on AQP4 or AQP9 expression during fetal neural stem cells (fNSC) differentiation into astrocytes or catecholaminergic neurons. In the first part of this work, a protocol was developed to differentiate mouse fNSC into astrocytes and neurons, with the aim to obtain catecholaminergic neurons. By immunefluorescence, we have shown that AQP9 is expressed in fNSC cultures and also in astrocytes and catecholaminergic neurons in mixt cultures. The results obtained highlighted a very good correlation between TH expression (tyrosin hydroxylase being a limiting enzyme of catecholamines synthesis) and AQP9 in fNSC and all along their differentiation into catecholaminergic neurons. On the other hand, AQP4 immunolabelling is not observed in fNSC whereas it is in astrocytes. Moreover, neitheir AQP4, nor AQP9 immunoreactivity was observed in MAP2-positive neurons. In the second part of this work, AQP4 and AQP9 expression was quantified in fNSC and in three populations of astrocytes presenting different metabolic properties. These three astrocyte populations result from fNSC differentiation by addition of CNTF, LIF or fetal calf serum. Quantitative RT-PCR and western blot analyses have shown an increase in both AQP4 and AQP9 expression, correlated with the acquisition of specific metabolic properties of mature astrocytes. In the last part, siRNA were used to study the functional role of AQP9 in the pure astrocyte culture model differentiated by addition of fetal calf serum. Inhibition of AQP9 expression leads to a decrease of glycerol uptake and to an increase of glucose uptake, correlated with a stimulation of the astrocyte oxydative metabolism. On the other hand, inhibition of AQP9 expression does not have any effect on anaerobic glycolysis nor on lactate release. In conclusion, in this in vitro model, only AQP9 is expressed in fNSC and in catecholaminergic neurons whereas in astrocytes, both AQP9 and AQP4 are expressed. This distribution is identical to that observed in vivo and confirms the specific AQP9 localization in catecholaminergic neurons. IVloreover, these results show, for the first time, that AQP9 is implicated in glycerol uptake and in astrocyte energetic metabolism. Résumé large public : Les aquaporines, des protéines localisées dans les membranes cellulaires sont, comme leur nom l'indique, des canaux à eau. Pendant longtemps, il a été considéré que l'eau diffusait librement dans et à travers les cellules; la caractérisation des AQPs a révolutionné la vision des scientifiques concernant les mouvements d'eau entre les différents compartiments infra et extracellulaires, et a d'ailleurs valu le Prix Nobel à Peter Agre en 1992. Certaines AQPs, dites "strictes", laissent passer uniquement l'eau et participent au contrôle du volume hydrique. Ce contrôle est particulièrement important pour le bon fonctionnement du cerveau en raison de la présence de la boîte crânienne qui limite les variations de volume. D'autres AQPs, les aquaglycéroporines, sont perméables non seulement à l'eau mais également à d'autres molécules comme le glycérol. Elles facilitent, par exemple, la sortie du glycérol des cellules graisseuses et sa capture par les cellules du foie afin de produire du glucose en période de jeûne. Le cerveau est principalement composé de deux types de cellules: les neurones et les cellules gliales, majoritairement des astrocytes. L'AQP4, une AQP stricte, est présente dans les astrocytes et joue un rôle dans la formation et la résorption des oedèmes cérébraux. L'AQP9, une aquaglycéroporine, est également présente dans les astrocytes et dans une population spécifique de neurones, les neurones catécholaminergiques, touchés dans la maladie de Parkinson. A ce jour, la présence de l'AQP9 dans le cerveau est une donnée controversée et son rôle fonctionnel est inconnu. Ce travail de thèse a permis de montrer que l'AQP9 est bien présente d'une part dans les cellules souches neurales foetales et d'autre ,part dans les astrocytes et neurones catécholaminergiques issus de leur différenciation. De plus, ces expériences ont mis en évidence un rôle de l'AQP9 dans l'entrée du glycérol dans les astrocytes, ce qui pourrait être bénéfique dans des conditions d'ischémie. Enfin, les .résultats de cette étude suggèrent également un rôle de l'AQP9 dans le métabolisme énergétique des astrocytes. L'ensemble de ces travaux démontre le rôle important de l'AQP9 dans le cerveau et ouvre de nouvelles perspectives quant aux rôles des AQPs dans des situations pathologiques telles que l'ischémie cérébrale ou encore la maladie de Parkinson.

Identification of cancer/testis-antigen genes by massively parallel signature sequencing.

Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

GENCODE: producing a reference annotation for ENCODE.

BACKGROUND: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results. RESULTS: The GENCODE gene features are divided into eight different categories of which only the first two (known and novel coding sequence) are confidently predicted to be protein-coding genes. 5' rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally verify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been sequenced. They supported 5' extensions of 30 loci and new splice variants in 50 loci. In addition, 46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15 putative transcripts. We assessed the comprehensiveness of the GENCODE annotation by attempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out of 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two of them in intergenic regions. CONCLUSION: In total, 487 loci, of which 434 are coding, have been annotated as part of the GENCODE reference set available from the UCSC browser. Comparison of GENCODE annotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within the two sets, which is a reflection of the high number of alternative splice forms with unique exons annotated. Over 50% of coding loci have been experimentally verified by 5' RACE for EGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human genome with the aid of experimental validation.

The JAK2/STAT3 pathway in the embryonic chick heart submitted to anoxia, reoxygenation and oxidant stress

SUMMARYAim: The embryonic/fetal heart is highly sensitive to oxygenation level and a transient uteroplacental hypoperfusion can lead to oxyradicals overproduction. Information about the molecular mechanisms underlying ischemia-reperfusion (I-R) injury in the developing heart is lacking. The Janus Kinase 2 / Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway, required for cardiogenesis and involved in protection of the adult heart against I-R, could also play a key role in the response of the fetal myocardium to transient oxygen deprivation. The aim of the study was to characterize the involvement of JAK2/STAT3 pathway and its interaction with other signalling pathways in the developing heart transiently submitted to anoxia. Furthermore, the response of the embryonic heart to an exogenous oxidant stress (H2O2) in comparison to reoxygenation-induced endogenous oxyradicals has been investigated.Methods: Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30min) and reoxygenation (80min) with or without the antioxidant MPG, the JAK2/STAT3 inhibitor AG490 or exposed to H202 (50|iM-lmM). The time course of phosphorylation of STAT3atyr0Sine7 and Reperfusion Injury Salvage Kinase (RISK) proteins (PI3K, Akt, GSK3B, Glycogen Synthase and ERK2) was determined in homogenate" and in enriched nuclear and cytoplasmic fractions. The STAT3 DNA-binding was determined by EMSA and the expression of STAT3 specific target genes by RT-PCR. The chrono-, dromo- and inotropic disturbances were also investigated by ECG and mechanical recordings.Results: Phosphorylation of STATSaP (P-Tyr STAT3a) was increased by reoxygenation and reduced by MPG or AG490. STAT3 and GSK36 were detected both in nuclear and cytoplasmic fractions while PI3K, Akt, GS and ERK2 were restricted to cytoplasm. Reoxygenation led to nuclear accumulation of STAT3 but unexpectedly without DNA- binding. AG490 decreased the reoxygenation-induced phosphorylation of STABa^, Akt, GS and ERK2 and phosphorylation/inhibition of GSK3B in the nucleus, exclusively. Inhibition of JAK2/STAT3 delayed recovery of atrial rate, worsened RR. variability and prolonged arrhythmias compared to control hearts. Cardiac activity was altered only at concentrations >500μΜ of H2O2. Moreover, ImM of H2O2 suppressed atrial activity in 45% of the hearts, atrioventricular conduction in 66% and augmented P-Tyr STAT3awhich led to an increase in the DNA-binding but no change in the expression of three STAT3 specific target genes (iNOS, MnSOD, Cox-2).Conclusion: In the developing heart, besides its nuclear translocation without transcriptional activity, ROS-activated STAT3a can rapidly interact with RISK proteins present in nucleus and cytoplasm and reduce the anoxia-reoxygenation-induced arrhythmias. Moreover, the embryonic heart is highly resistant to H2O2 and the atrial region is the less affected. The role of JAK2/STAT3 in the response to reoxygenation-induced oxyradicals is different from the response to strong exogenous oxidant stress where STAT3 DNA-binding activity is increased. Such findings provide a first step in understanding the modulation of signalling cascades in the fetal heart submitted to transient intrauterine oxygen deprivation.RESUMEIntroduction: Le coeur embryonnaire et foetal est très sensible au manque d'oxygène et une hypoperfusion utéroplacentaire transitoire peut conduire à une surproduction d'espèces radicalaires (ROS). Dans le coeur en développement les mécanismes moléculaires impliqués en situation d'ischémie-reperfusion (I-R) ne sont pas connus. La voie de signalisation JAK2/STAT3 (Janus Kinase 2 / Signal Transducer and Activator of Transcription 3), impliquée aussi bien dans la cardiogenèse précoce que dans la protection du coeur adulte contre l'I-R, pourrait jouer un rôle clé dans la réponse du myocarde foetal à un déficit en oxygène. Cette étude a permis d'étudier le rôle de la voie JAK2/STAT3 et son interaction avec d'autres voies de signalisation dans un modèle de coeur embryonnaire soumis à un épisode anoxique. En outre, les effets du stress oxydant endogène provoqué par la réoxygénation ont été comparés à ceux du stress oxydatif exogène induit par du peroxyde d'hydrogène (H2O2).Méthodes: Des coeurs isolés d'embryons de poulet âgés de 4 jours ont été soumis à une anoxie (30min) suivie d'une réoxygénation (80min) en présence ou non de l'antioxydant MPG et de l'inhibiteur de JAK2/STAT3 AG490 ou exposés à de 1Ή202 (50μΜ-1πιΜ). L'évolution temporelle de la phosphorylation de 8ΤΑΤ3α*ΓΟδίη6705 (P-Tyr STAT3a) et celle de la phosphorylation des protéines de la voie RISK (Reperfusion Injury Salvage Kinase: PI3K, Akt, GSK3B, glycogène synthase GS et ERK2) ont été déterminés dans l'homogénat et dans les fractions nucléaire et cytopiasmique du myocarde. La liaison de STAT3 à l'ADN a été déterminée par EMSA et l'expression de gènes cibles de STAT3 (iNOS, MnSOD, Cox2) par RT-PCR. Les effets chrono-, dromo- et inotropes ont été déterminés par les enregistrements de l'ECG et de l'activité contractile ventriculaire.Résultats: STAT3 et GSK3B étaient présents dans les fractions nucléaire et cytopiasmique tandis que PI3K, Akt, GS et ERK2 n'étaient détectées que dans la fraction cytopiasmique. L'augmentation de P-Tyr STAT3a provoquée par la réoxygénation était significativement réduite par le MPG ou PAG490. La réoxygénation entraînait l'accumulation nucléaire de STAT3, mais étonnamment sans liaison avec l'ADN. A la réoxygénation TAG490 diminuait la phosphorylation d'Akt, GS et ERK2 ainsi que celle de GSK36 mais exclusivement dans la fraction nucléaire. L'inhibition de JAK2/STAT3 retardait également la récupération du rythme cardiaque et prolongeait la durée des arythmies. L'activité cardiaque n'était perturbée par de ΓΗ2Ο2 qu'à des concentrations >500μΜ. A ImM, ΓΗ2Ο2 supprimait l'activité auriculaire dans 45% des coeurs et la conduction auriculo-ventriculaire dans 66% et augmentait la formation de P-Tyr STAT3a et sa liaison à l'ADN sans modifier l'expression des gènes cibles.Conclusion: Les ROS produits par l'anoxie-réoxygénation activent STAT3a qui subit une translocation dans le noyau sans se lier à l'ADN et interagit rapidement avec des protéines de la voie RISK dans les compartiments nucléaire et cytopiasmique du coeur embryonnaire. Ce dernier, en particulier au niveau des oreillettes, se révèle très résistant au puissant stress oxydatif de l'H202 qui se différencie du stress lié à la réoxygénation en favorisant la liaison de STAT3 à l'ADN. Ces résultats originaux permettent une meilleure compréhension des mécanismes qui peuvent améliorer la récupération du coeur en développement après un épisode hypoxique intra-utérin.

Expression and secretion of the atrial natriuretic peptide in human adipose tissue and preadipocytes.

OBJECTIVE: Atrial natriuretic peptide (ANP) is a secretory hormone displaying diuretic, natriuretic, and vasorelaxant activities. Recently, its lipolytic activity has been reported. Since the expression of ANP in adipose tissue has not been documented, we used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to investigate the expression of ANP in human adipose tissue and preadipocytes. RESEARCH METHODS AND PROCEDURES: RNA was extracted from the human adipose tissue of severely obese premenopausal women as well as from human preadipocytes. For human preadipocytes, two cell systems were investigated: the human preadipose immortalized (Chub-S7) cells, a well-characterized human preadipose cell line, and primary preadipocytes derived from the stromal vascular fraction of the human adipose tissue. We measured the mRNA of ANP, of corin (a transmembrane serine protease involved in the conversion of pro-ANP to ANP) and of uncoupling protein 2 (UCP2; a control gene known to be ubiquitously expressed). The expression of ANP was also investigated using immunofluorescence and radioimmunoassay in Chub-S7 cells and human primary preadipocytes in culture. RESULTS: Our results indicate that ANP and corin are expressed at the mRNA level in human adipose tissue and preadipocytes. Immunofluorescence experiments demonstrated that pro-ANP was expressed in Chub-S7 cells. In addition, ANP secretion could be measured in Chub-S7 cells and human primary preadipocytes in culture. Rosiglitazone, a selective peroxisome proliferator-activated receptor type gamma (PPAR-gamma) agonist promoting adipocyte differentiation, was found to modulate both ANP expression and secretion in preadipocytes. DISCUSSION: Our findings suggest the existence of an autocrine/paracrine system for ANP in the human adipose tissue whose implications in lipolysis and cardiovascular function need to be further explored.

The role of Receptor Activator of NFKB Ligand (RANKL) in mammary gland development

Abstract : The female reproductive hormones estrogen, progesterone and prolactin control postnatal breast development and are important to breast carcinogenesis. The mechanisms by which they elicit proliferation and morphogenesis remain poorly understood. Using the mouse as a model to study the molecular mechanisms through which hormones elicit morphogenetic changes in the mammary gland in vivo, we found the Receptor Activator of NFκB Ligand, a Tumor Necrosis Factor family member, to be strongly induced by progesterone. Recent publications suggested that hormone dependant RANKURANK signals are involved in the terminal differentiation of mammary gland alveolar buds into lobulo-alveolar structures competent for lactation. I show that in the absence of epithelial RANKL a distinct earlier stage of mammary gland development, side branch formation, is blocked. RANKL acts as a major mediator downstream of progesterone; it is required for progesterone-induced paracrine proliferation and completely rescues the mutant phenotype when ectopically expressed in progesterone receptor (PR) KO mammary epithelia. RANKL is not required for cell autonomous division of estrogen receptor alpha (ERa) /PR positive cells. Cyclin D1, previously implicated as a mediator of RANKL, is not affected by ablation of RANKL and is not required for RANKL-induced paracrine proliferation but for the cell autonomous proliferation. Gene expression arrays to find specific RANKL downstream targets have identified Id4, ElfS and one secreted metalloprotease (Adamtsl8) as potential candidates validated by Q-RT-PCR. Interestingly, Id4 and Adamtsl8 are expressed by the myoepithelial cells. Their expression additionally coincides with RANKL mRNA expression at mid pregnancy, possibly implying a functional contribution of both genes to RANKL mediated sidebranch formation. ElfS in contrast, is found to be strongly expressed by the end of pregnancy supporting recent findings of a prolactin mediated regulation. As for RANKL, this gene was in particular induced in luminal cells. Taken together, I report that progesterone is the major proliferative stimulus in the adult mammary gland eliciting proliferation of ERaJPR positive cells by a cell autonomous, cyclin D1-dependent and a paracrine RANKL-dependent mechanism. My work moreover suggests, that RANKL acts as a major orchestrator affecting different downstream mediators, through which progesterone exerts its effects concomitantly on different cellular compartments. Résumé : Les hormones sexuelles telles que l'oestrogène, la progestérone et la prolactine contrôlent le développement postnatal du sein et sont impliquées dans la cazcinogenèse. Les mécanismes par lesquels elles induisent la prolifération et la morphogénèse demeurent incompris. En utilisant la souris comme modèle, J'ai trouvé que le ligand activateur du récepteur de NFκB, une protéine de la famille du facteur de nécrose des tumeurs, peut être fortement induit par la progestérone. Les publications récentes ont suggéré que cette protéine est nécessaire à la fin de la grossesse, quand les cellules sécrétrices du lait apparaissent. Par des techniques de transplantation d'épithélium, je montre contrairement aux études précédentes, qu'en l'absence de RANKL dans l'épithélium une partie distincte du développement mammaire, la formation de branches latérales, est bloquée. La progestérone agit de manière pazacrine par l'intermédiaire de 12ANKL pour induire la prolifération tandis que la mort cellulaire n'est pas affectée. De plus, l'injection d'une protéine recombinante RANKL dans une souris mutante pour le récepteur à la progestérone induit la prolifération des cellules épithéliales en l'absence de grossesse ; la surexpression de RANKL dans ces mêmes mutants mène à une réversion complète du phénotype. Mes expériences démontrent que la progestérone induit deux types distincts de prolifération. Un premier type direct dans laquelle les cellules positives au récepteur à la progestérone prolifèrent. Cette division cellulaire est alors indépendante de RANKL mais dépendante de la cycline D1. Le second type de prolifération est induit par un mécanisme pazacrine et dépend de RANKL mais pas de la cycline D1. Ici, les cellules négatives au récepteur à la progestérone prolifèrent. Pour détecter des gènes cibles de la voie de signalisation du RANKL, un profil d'expression des gènes a été généré. Les facteurs de transcription Id4, EIf5 et une métalloprotéase sécrétée (Adamtsl8) ont été identifiés en tant que cibles potentielles. D'autres analyses de validation démontrent qu'Id4, Adamtsl8, RANKL mais pas E1f5 sont fortement exprimés au cours de la grossesse, coïncidant avec la formation de branchements latéraux induit par progestérone. EIf5 s'est avéré être exprimé vers la fin de la grossesse appuyant des résultats récents proposant une régulation par la prolactine. Le système canalaire mammaire se compose de couches cellulaires: une couche interne de cellules luminales et une externe de cellules myoépithéliale. Les expériences génétiques d'expression ont révélé que RANKL. et E1f5 sont exprimés dans la partie luminale tandis qu'Id4 et Adamtsl8 sont dans les cellules myoépithéliales. En conclusion, je prouve que la progestérone est le stimulus principal induisant la prolifération dans la glande mammaire d'adulte. Deux mécanismes de prolifération sont impliqués: l'un direct dépendant de la cycline Dl et l'autre paracrine dépendant de RANKI.. Mon travail suggère par ailleurs que RANKL agit en tant que médiateur important, par lequel la progestérone exerce ses effets sur différents compartiments cellulaires tels que la coordination de la prolifération des cellules épithéliales avec la réorganisation de la matrice extracellulaire et de la membrane basale exigées pour la morphogénèse du système canalaire latéral.

Desempenho agronômico de videiras com e sem sintomas de viroses, e comparação molecular de isolados virais

Resumo: O objetivo deste trabalho foi avaliar os efeitos de viroses em videiras sintomáticas e assintomáticas sobre as variáveis agronômicas relacionadas ao vigor das plantas e à qualidade enológica da uva, e comparar os isolados virais obtidos nessas duas condições. Realizaram-se dois experimentos com quatro cultivares. Todas as plantas foram indexadas, por meio da reação em cadeia da polimerase via transcrição reversa (RT-PCR) em tempo real, quanto à provável ocorrência dos seguintes vírus: Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine leafroll-associated virus (GLRaV-1 ao -4, GLRaV-4 estirpe 5), Grapevine rupestris stem pitting-associated virus (GRSPaV) e Grapevine fleck virus (GFkV). As variáveis avaliadas foram: número de gemas brotadas e não brotadas, número de ramos com ou sem cachos, número total de gemas, número de cachos, massa de cachos frescos, massa total de bagas, massa do engaço, número de bagas por cacho, massa média de baga, sólidos solúveis totais, acidez total titulável, pH, massa de ramos podados ou diâmetros do tronco do porta-enxerto e da copa. Os efeitos negativos foram mais pronunciados nas plantas com sintomas de viroses; no entanto, constatou-se frequentemente que plantas sem sintomas também estavam infectadas. A análise molecular de GRSPaV, GVA e GLRaV-2, isolados de plantas sintomáticas e assintomáticas, resultou em alta percentagem de identidade de nucleotídeos entre isolados homólogos.