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2009

A combination of transposable elements and magnetic cell sorting provides a very efficient transgenesis system for chicken primary erythroid progenitors.

Author(s) : Mejia-Pous C, Vinuelas J, Faure C, Koszela J, Kawakami K, Takahashi Y, Gandrillon O,
Journal : BMC Biotechnol
2009
BACKGROUND: Stable transgenesis is an undeniable key to understanding any genetic system. Retrovirus-based insertional strategies, which feature several technicalchallenges when they are used, are often limited to one particular species, and even sometimes to a particular cell type as the infection depends on certain cellular receptors. A universal-like system, which would allow both stable transgene expression independent of the cell type and an efficient sorting of transfected cells, is required when handling cellular models that are incompatible with retroviral strategies. RESULTS: We report here on the combination of a stable insertional transgenesis technique, based on the Tol2 transposon system together with the magnetic cell sorting (MACS) technique, which allows specific selection of cells carrying the transgene in an efficient, reliable and rapid way. CONCLUSION: This new Tol2/MACS system leads to stable expression in a culture of primary chicken erythroid cells highly enriched in cells expressing the transgene of interest. This system could be used in a wide variety of vertebrate species.

Calculation of free-energy differences by confinement simulations. Application to peptide conformers.

Author(s) : Cecchini M, Krivov S, Spichty M, Karplus M,
Journal : J Phys Chem B
2009
Conformational free-energy differences are key quantities for understanding important phenomena in molecular biology that involve large structural changes of macromolecules. In this paper, an improved version of the confinement approach, which is based on earlier developments, is used to determine the free energy of individual molecular states by progressively restraining the corresponding molecular structures to pure harmonic basins, whose absolute free energy can be computed by normal-mode analysis. The method is used to calculate the free-energy difference between two conformational states of the alanine dipeptide in vacuo, and of the beta-hairpin from protein G with an implicit solvation model. In all cases, the confinement results are in excellent agreement with the ones obtainedfrom converged equilibrium molecular dynamics simulations, which have a much larger computational cost. The systematic and statistical errors of the results are evaluated and the origin of the errors is identified. The sensitivity of thecalculated free-energy differences to structure-based definitions of the molecular states is discussed. A variant of the method, which closes the thermodynamic cycle by a quasi-harmonic rather than harmonic analysis, is introduced. The latter is proposed for possible use with explicit solvent simulations.

Cell death: what can we learn from flies? Editorial for the special review issue on Drosophila apoptosis.

Author(s) : Mollereau B,
Journal : Apoptosis
2009

Developmental constraints revealed by co-variation within and among molar rows in two murine rodents.

Author(s) : Renaud S, Pantalacci S, Quere J, Laudet V, Auffray J,
Journal : Evol Dev
2009
Morphological integration corresponds to interdependency between characters thatcan arise from several causes. Proximal causes of integration include that different phenotypic features may share common genetic sets and/or interact during their development. Ultimate causes may be the prolonged effect of selection favoring integration of functionally interacting characters, achieved by the molding of these proximal causes. Strong and direct interactions among successive teeth of a molar row are predicted by genetic and developmental evidences. Functional constraints related to occlusion, however, should have selected more strongly for a morphological integration of occluding teeth and a corresponding evolution of the underlying developmental and genetic pathways. Toinvestigate how these predictions match the patterns of phenotypic integration, we studied the co-variation among the six molars of the murine molar row, focusing on two populations of house mice (Mus musculus domesticus) and wood mice (Apodemus sylvaticus). The size and shape of the three upper and lower molars were quantified and compared. Our results evidenced similar patterns in both species, size being more integrated than shape among all the teeth, and both size and shape co-varying strongly between adjacent teeth, but also between occludingteeth. Strong co-variation within each molar row is in agreement with developmental models showing a cascade influence of the first molar on the subsequent molars. In contrast, the strong co-variation between molars of the occluding tooth rows confirms that functional constraints molded patterns of integration and probably the underlying developmental pathways despite the low level of direct developmental interactions occurring among molar rows. These patterns of co-variation are furthermore conserved between the house mouse and the wood mouse that diverged >10 Ma, suggesting that they may constitute long-running constraints to the diversification of the murine rodent dentition.

Differential regulation of ParaHox genes by retinoic acid in the invertebrate chordate amphioxus (Branchiostoma floridae).

Author(s) : Osborne P, Benoit G, Laudet V, Schubert M, Ferrier D,
Journal : Dev Biol
2009
The ParaHox cluster is the evolutionary sister to the Hox cluster. Like the Hox cluster, the ParaHox cluster displays spatial and temporal regulation of the component genes along the anterior/posterior axis in a manner that correlates with the gene positions within the cluster (a feature called collinearity). The ParaHox cluster is however a simpler system to study because it is composed of only three genes. We provide a detailed analysis of the amphioxus ParaHox cluster and, for the first time in a single species, examine the regulation of the cluster in response to a single developmental signalling molecule, retinoic acid(RA). Embryos treated with either RA or RA antagonist display altered ParaHox gene expression: AmphiGsx expression shifts in the neural tube, and the endodermal boundary between AmphiXlox and AmphiCdx shifts its anterior/posteriorposition. We identified several putative retinoic acid response elements and in vitro assays suggest some may participate in RA regulation of the ParaHox genes.By comparison to vertebrate ParaHox gene regulation we explore the evolutionary implications. This work highlights how insights into the regulation and evolution of more complex vertebrate arrangements can be obtained through studies of a simpler, unduplicated amphioxus gene cluster.

Distinct impacts of Eda and Edar loss of function on the mouse dentition.

Author(s) : Charles C, Pantalacci S, Tafforeau P, Headon D, Laudet V, Viriot L,
Journal : PLoS One
2009
BACKGROUND: The Eda-A1-Edar signaling pathway is involved in the development of organs with an ectodermal origin, including teeth. In mouse, mutants are known for both the ligand, Eda-A1 (Tabby), and the receptor, Edar (Downless). The adult dentitions of these two mutants have classically been considered to be similar. However, previous studies mentioned differences in embryonic dental development between Eda(Ta) and Edar(dl-J) mutants. A detailed study of tooth morphology in mutants bearing losses of functions of these two genes thus appears necessary totest the pattern variability induced by the developmental modifications. METHODOLOGY/PRINCIPAL FINDINGS: 3D-reconstructions of the cheek teeth have been performed at the ESRF (Grenoble, France) by X-ray synchrotron microtomography toassess dental morphology. The morphological variability observed in Eda(Ta) and Edar(dl-J) mutants have then been compared in detail. Despite patchy similarities, our detailed work on cheek teeth in Eda(Ta) and Edar(dl-J) mice show that all dental morphotypes defined in Edar(dl-J) mice resolutely differ from those of Eda(Ta) mice. This study reveals that losses of function of Eda and Edar have distinct impacts on the tooth size and morphology, contrary to what has previously been thought. CONCLUSION/SIGNIFIANCE: The results indicate that unknown mechanisms of the Eda pathway are implicated in tooth morphogenesis. Three hypotheses could explain our results; an unexpected role of the Xedar pathway (which is influenced by the Eda gene product but not that of Edar), a more complex connection than has been appreciated between Edar and another protein, or a ligand-independent activity for Edar. Further work is necessary totest these hypotheses and improve our understanding of the mechanisms of development.

Effect of eda loss of function on upper jugal tooth morphology.

Author(s) : Charles C, Pantalacci S, Peterkova R, Tafforeau P, Laudet V, Viriot L,
Journal : Anat Rec (Hoboken)
2009
The Tabby/eda mice, which bear a loss of function mutation for the eda (ectodysplasinA) gene, are known to display developmental anomalies in organs with an ectodermal origin. Although the lower jugal (cheek) teeth of Tabby/eda mice have been extensively studied, upper teeth have never been investigated in detail. However, this may help us to further understand the function of the eda gene in tooth development. In this work, the shape and size of both the crown and the radicular system were studied in the Tabby/eda mice upper jugal teeth. To deal with the high morphological variability, we defined several morphotypes based on cusp numbers and position. Statistical tests were then performed withinand between the different morphotypes to test the correlation between tooth sizeand morphology. Our analysis reveals that, as in lower teeth, eda is necessary to segment the dental lamina into three teeth with the characteristic size and proportions of the mouse. Nevertheless, since strong effects are observed in heterozygous upper teeth while lower are only mildly affected, it seems that theupper jaw is more sensitive than the lower jaw to the loss of eda function. Modifications in cusp number and the abnormal crown size of the teeth are clearly linked, and our results indicate a role of eda in cusp patterning. Moreover, we found that the Tabby mutation induces variations in the dental root pattern, sometimes associated with hypercementosis, suggesting a newly uncovered role played by eda in root patterning and formation.

ER stress protects from retinal degeneration.

Author(s) : Mendes C, Levet C, Chatelain G, Dourlen P, Fouillet A, Dichtel-Danjoy M, Gambis A, Ryoo H, Steller H, Mollereau B,
Journal : EMBO J
2009
The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivationnor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H(2)O(2), cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissuesfrom harmful exogenous stresses.

Extracting signature motifs from promoter sets of differentially expressed genes.

Author(s) : Mitasiunaite I, Rigotti C, Schicklin S, Meyniel L, Boulicaut J, Gandrillon O,
Journal : In Silico Biol
2009
There is a critical need for new and efficient computational methods aimed at discovering putative transcription factor binding sites (TFBSs) in promoter sequences. Among the existing methods, two families can be distinguished: statistical or stochastic approaches, and combinatorial approaches. Here we focus on a complete approach incorporating a combinatorial exhaustive motif extraction, together with a statistical Twilight Zone Indicator (TZI), in two datasets: a positive set and a negative one, which represents the result of a classical differential expression experiment. Our approach relies on the existence of prior biological information in the form of two sets of promoters of differentially expressed genes. We describe the complete procedure used for extracting either exact or degenerated motifs, ranking these motifs, and finding their known related TFBSs. We exemplify this approach using two different sets of promoters.The first set consists in promoters of genes either repressed or not by the transforming form of the v-erbA oncogene. The second set consists in genes the expression of which varies between self-renewing and differentiating progenitors. The biological meaning of the found TFBSs is discussed and, for one TF, its biological involvement is demonstrated. This study therefore illustrates the power of using relevant biological information, in the form of a set of differentially expressed genes that is a classical outcome in most of transcriptomics studies. This allows to severely reduce the search space and to design an adapted statistical indicator. Taken together, this allows the biologist to concentrate on a small number of putatively interesting TFs.

Heterochronic shifts explain variations in a sequentially developing repeated pattern: palatal ridges of muroid rodents.

Author(s) : Pantalacci S, Semon M, Martin A, Chevret P, Laudet V,
Journal : Evol Dev
2009
Metazoans are largely made of repeated parts, and metazoan evolution is marked by changes in the number of these parts, called meristic evolution. Understanding the mechanisms associated with meristic changes is thus a critical issue to evolutionary developmental biology. Palatal rugae are sensory ridges regularly arranged on the hard palate of mammals. They develop sequentially following mesio-distal growth of the palate, and activation-inhibition mechanisms very likely control spacing and timing of this sequential addition. In this study, wecharacterized trends in rugae number evolution among muroid rodents, showing that most species display 8+/-1 rugae, changes by one being very frequent in the phylogeny. We then compared development of three muroid species: mouse (nine rugae), rat (eight), and golden hamster (seven). We showed that palatal growth rate, spacing, and addition rate in mouse/rat were remarkably similar (with respect to the embryo size difference), and that increase to nine rugae in mouseis achieved by postponing the end of the addition process (hypermorphosis). Sucha heterochronic shift may be typical of +/-1 variations observed among muroid rodents. In contrast, decrease to seven rugae in golden hamster is attributed toearly growth termination (progenesis) of the palate, which correlates with the severe shortening of gestation in this species. Our results provide an experimental support to the intuitive view that heterochronies are especially relevant to meristic evolution of traits that rely on a sequential addition process. We also interpret our results in the light of developmental constraintsspecifically linked to this kind of process.

HPL-2/HP1 prevents inappropriate vulval induction in Caenorhabditis elegans by acting in both HYP7 and vulval precursor cells.

Author(s) : Schott S, Ramos F, Coustham V, Palladino F,
Journal : Genetics
2009
A current model for Caenorhabditis elegans vulval cell fate specification is that SynMuv genes act redundantly in the hyp7 hypodermal syncytium to repress the LIN-3/EGF inducer and prevent ectopic vulval induction of vulva precursor cells (VPCs). Here we show that the SynMuv gene hpl-2/HP1 has an additional function in VPCs, where it may act through target genes including LIN-39/Hox.

Inhibition of the hTERT promoter by the proto-oncogenic protein TAL1.

Author(s) : Terme J, Mocquet V, Kuhlmann A, Zane L, Mortreux F, Wattel E, Duc Dodon M, Jalinot P,
Journal : Leukemia
2009
Telomerase activity, which has fundamental roles in development and carcinogenesis, strongly depends on the expression of human telomerase reverse transcriptase (hTERT), its catalytic subunit. In this report, we show that the basic helix-loop-helix factor, TAL1 (T-cell acute lymphoblastic leukemia 1), is a negative regulator of the hTERT promoter. Indeed, TAL1 overexpression leads to adecrease in hTERT mRNA abundance and hence to reduced telomerase activity. Conversely, suppression of TAL1 by RNA interference in Jurkat cells increases hTERT expression. Analysis by chromatin immunoprecipitation assays showed that TAL1 binds to the hTERT proximal promoter and recruits HDAC1. Considering the relationship recently established between TAL1 and the human T-cell leukemia virus type 1 (HTLV-1) Tax protein, which was confirmed in T lymphocyte clones derived from adult T-cell leukemia patients, we analyzed the effect of TAL1 withrespect to the earlier characterized effects of Tax and HBZ (HTLV-1 basic leucine zipper) on hTERT expression. TAL1 was observed to reinforce the negative effect of Tax, whereas hTERT transactivation by the HBZ-JunD complex was repressed by TAL1 overexpression. Moreover, HBZ was found to induce proteasome-mediated degradation of TAL1. These observations support a model in which Tax and TAL1 byrepressing hTERT would initially favor genomic instability, whereas expression of factors such as HBZ allows at a later stage an increase in hTERT production and consequently in telomerase activity.

MicroRNAs resolve an apparent conflict between annelid systematics and their fossil record.

Author(s) : Sperling E, Vinther J, Moy V, Wheeler B, Semon M, Briggs D, Peterson K,
Journal : Proc Biol Sci
2009
Both the monophyly and inter-relationships of the major annelid groups have remained uncertain, despite intensive research on both morphology and molecular sequences. Morphological cladistic analyses indicate that Annelida is monophyletic and consists of two monophyletic groups, the clitellates and polychaetes, whereas molecular phylogenetic analyses suggest that polychaetes are paraphyletic and that sipunculans are crown-group annelids. Both the monophyly of polychaetes and the placement of sipunculans within annelids are in conflict with the annelid fossil record--the former because Cambrian stem taxa are similar to modern polychaetes in possessing biramous parapodia, suggesting that clitellatesare derived from polychaetes; the latter because although fossil sipunculans areknown from the Early Cambrian, crown-group annelids do not appear until the latest Cambrian. Here we apply a different data source, the presence versus absence of specific microRNAs--genes that encode approximately 22 nucleotide non-coding regulatory RNAs--to the problem of annelid phylogenetics. We show that annelids are monophyletic with respect to sipunculans, and polychaetes are paraphyletic with respect to the clitellate Lumbricus, conclusions that are consistent with the fossil record. Further, sipunculans resolve as the sister group of the annelids, rooting the annelid tree, and revealing the polarity of the morphological change within this diverse lineage of animals.

Molecular analysis of the sex chromosomes of the platyfish Xiphophorus maculatus: Towards the identification of a new type of master sexual regulator in vertebrates.

Author(s) : Bohne A, Schultheis C, Galiana-Arnoux D, Froschauer A, Zhou Q, Schmidt C, Selz Y, Ozouf-Costaz C, Dettai A, Segurens B, Couloux A, Bernard-Samain S, Barbe V, Chilmonczyk S, Brunet F, Darras A, Tomaszkiewicz M, Semon M, Schartl M, Volff J,
Journal : Integr Zool
2009
In contrast to mammals and birds, fish display an amazing diversity of genetic sex determination systems, with frequent changes during evolution possibly associated with the emergence of new sex chromosomes and sex-determining genes. To better understand the molecular and evolutionary mechanisms driving this diversity, several fish models are studied in parallel. Besides the medaka (Oryzias latipes Temminck and Schlegel, 1846) for which the master sex-determination gene has been identified, one of the most advanced models for studying sex determination is the Southern platyfish (Xiphophorus maculatus, Gunther 1966). Xiphophorus maculatus belongs to the Poeciliids, a family of live-bearing freshwater fish, including platyfish, swordtails and guppies that perfectly illustrates the diversity of genetic sex-determination mechanisms observed in teleosts. For X. maculatus, bacterial artificial chromosome contigs covering the sex-determination region of the X and Y sex chromosomes have been constructed. Initial molecular analysis demonstrated that the sex-determination region is very unstable and frequently undergoes duplications, deletions, inversions and other rearrangements. Eleven gene candidates linked to the mastersex-determining gene have been identified, some of them corresponding to pseudogenes. All putative genes are present on both the X and the Y chromosomes,suggesting a poor degree of differentiation and a young evolutionary age for platyfish sex chromosomes. When compared with other fish and tetrapod genomes, syntenies were detected only with autosomes. This observation supports an independent origin of sex chromosomes, not only in different vertebrate lineagesbut also between different fish species.

Monoallelic expression and tissue specificity are associated with high crossover rates.

Author(s) : Necsulea A, Semon M, Duret L, Hurst L,
Journal : Trends Genet
2009
What determines the recombination rate of a gene? Following the observation that, in humans, imprinted genes have unusually high recombination levels, we ask whether increased recombination is seen for other monoallelically expressed genes and, more generally, how transcriptional properties relate to recombination. We find that monoallelically expressed genes do have high crossover rates and discover a striking negative correlation between within-gene crossover rate and expression breadth. We hypothesise that these findings are possibly symptomatic of a more general, adverse relationship between recombination and transcription in the human genome.

Overcoming resistance to conventional drugs in Ewing sarcoma and identification of molecular predictors of outcome.

Author(s) : Scotlandi K, Remondini D, Castellani G, Manara M, Nardi F, Cantiani L, Francesconi M, Mercuri M, Caccuri A, Serra M, Knuutila S, Picci P,
Journal : J Clin Oncol
2009
PURPOSE: The improvement of Ewing sarcoma (EWS) therapy is currently linked to the discovery of strategies to select patients with poor and good prognosis and of modified treatment regimens. In this study, we analyzed the molecular factorsgoverning EWS response to chemotherapy to identify genetic signatures to be usedfor risk-adapted therapy. PATIENTS AND METHODS: Microarray technology was used for profiling 30 primary tumors and seven metastases of patients who were classified according to event-free survival. For selected genes, real-time polymerase chain reaction was applied in 42 EWS primary tumors as validation assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test was used to evaluate in vitro drug sensitivity. RESULTS: We identified molecular signatures that reflect tumor resistance to chemotherapy. Annotation analysis was applied to reveal the biologic functions that critically influenced clinical outcome. The prognostic relevance of glutathione metabolism pathway was validated. The expression of MGST1, the microsomal glutathione S-transferase (GST), was found to clearly predict EWS prognosis. MGST1 expression was associated with doxorubicin chemosensitivity. This prompted us to assess the in vitro effectiveness of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX),a new anticancer agent that efficiently inhibits GST enzymes. Six cell lines were found to be sensitive to this new drug. CONCLUSION: Classification of EWS patients into high- and low-risk groups is feasible with restricted molecular signatures that may have practical value at diagnosis for selecting patients with EWS who are unresponsive to current treatments. Glutathione metabolism pathway emerged as one of the most significantly altered prognosis-associated pathway. NBDHEX is proposed as a new potential therapeutic possibility.

Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 Nuclear Bodies.

Author(s) : Rajan P, Dalgliesh C, Bourgeois C, Heiner M, Emami K, Clark E, Bindereif A, Stevenin J, Robson C, Leung H, Elliott D,
Journal : BMC Cell Biol
2009
BACKGROUND: Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA-binding proteins and their target sequences, sometimes in response to signalling pathways. Sam68 (Src-associated in mitosis 68 kDa) is the prototypic member of the STAR (Signal Transduction and Activation of RNA) family of RNA-binding proteins, which regulate splicing in response to signalling cascades. Nuclear Sam68 protein is concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signalling components and nucleic acids. RESULTS: We used proteomicsto search for the major interacting protein partners of nuclear Sam68. In addition to Sam68 itself and known Sam68-associated proteins (heterogeneous nuclear ribonucleoproteins hnRNP A1, A2/B1 and G), we identified hnRNP L as a novel Sam68-interacting protein partner. hnRNP L protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnRNP L spatially co-localised with Sam68 as a novel component of SNBs and was also observed within the general nucleoplasm. Localisation within SNBs was highly specific to hnRNP L and was not shared by the closely-related hnRNP LL protein, nor any of the other Sam68-interacting proteins we identified by proteomics. Theinteraction between Sam68 and hnRNP L proteins was observed in a cell line whichexhibits low frequency of SNBs suggesting that this association also takes placeoutside SNBs. Although ectopic expression of hnRNP L and Sam68 proteins independently affected splicing of CD44 variable exon v5 and TJP1 exon 20 minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons. CONCLUSION: Here we identify hnRNP L as a novel SNB component. We show that, compared with other identified Sam68-associated hnRNP proteins and hnRNP LL, this co-localisation within SNBs is specific to hnRNP L. Our data suggest that the novel Sam68-hnRNP L protein interaction may have a distinct role within SNBs.

Rapamycin response in tumorigenic and non-tumorigenic hepatic cell lines.

Author(s) : Jimenez R, Boylan J, Lee J, Francesconi M, Castellani G, Sanders J, Gruppuso P,
Journal : PLoS One
2009
BACKGROUND: The mTOR inhibitor rapamycin has anti-tumor activity across a variety of human cancers, including hepatocellular carcinoma. However, resistance to itsgrowth inhibitory effects is common. We hypothesized that hepatic cell lines with varying rapamycin responsiveness would show common characteristics accounting for resistance to the drug. METHODOLOGY/PRINCIPAL FINDINGS: We profiled a total of 13 cell lines for rapamycin-induced growth inhibition. The non-tumorigenic rat liver epithelial cell line WB-F344 was highly sensitive while the tumorigenic WB311 cell line, originally derived from the WB-F344 line, was highly resistant. The other 11 cell lines showed a wide range of sensitivities. Rapamycin induced inhibition of cyclin E-dependent kinase activity in some cell lines, but the ability to do so did not correlate with sensitivity. Inhibition of cyclin E-dependent kinase activity was related to incorporation of p27(Kip1) into cyclin E-containing complexes in some but not all cell lines. Similarly, sensitivity ofglobal protein synthesis to rapamycin did not correlate with its anti-proliferative effect. However, rapamycin potently inhibited phosphorylationof two key substrates, ribosomal protein S6 and 4E-BP1, in all cases, indicatingthat the locus of rapamycin resistance was downstream from inhibition of mTOR Complex 1. Microarray analysis did not disclose a unifying mechanism for rapamycin resistance, although the glycolytic pathway was downregulated in all four cell lines studied. CONCLUSIONS/SIGNIFICANCE: We conclude that the mechanisms of rapamycin resistance in hepatic cells involve alterations of signaling downstream from mTOR and that the mechanisms are highly heterogeneous,thus predicting that maintaining or promoting sensitivity will be highly challenging.

Rev-erbalpha2 mRNA encodes a stable protein with a potential role in circadian clock regulation.

Author(s) : Rambaud J, Triqueneaux G, Masse I, Staels B, Laudet V, Benoit G,
Journal : Mol Endocrinol
2009
Circadian rhythms are observed in nearly all aspects of physiology and behavior.In mammals, such biological rhythms are supported by a complex network of self-sustained transcriptional loops and posttranslational modifications, which regulate timely controlled production and degradation of critical factors on a 24-h basis. Among these factors, the orphan nuclear receptor rev-erbalpha plays an essential role by linking together positive and negative regulatory loops. Asan essential part of the circadian core clock mechanism, REV-ERBalpha expressionshows a precisely scheduled oscillation reflecting the tight control of its production and degradation. In previous studies, we identified two alternative transcripts encoding two protein variants referred to as REV-ERBalpha1 and -alpha2. Interestingly, recent work identified structural elements present only in REV-ERBalpha1 that controls its turnover and thereby influences circadian oscillations. In the present work, we comparatively analyze the two variants andshow that REV-ERBalpha2 exhibits a half-life incompatible with a circadian function, suggesting that this variant exerts different biological functions. However, our comparative study clearly indicates undistinguishable DNA-binding properties and transcriptional repression activity as well as a similar regulation mechanism. The only consistent difference appears to be the relative expression level of the two transcripts, rev-erbalpha1 being one to 100 times more expressed than alpha2 depending on tissue and circadian time. Taking this finding into consideration, we reassessed REV-ERBalpha2 turnover and were able to show that this variant exhibits a reduced half-life when coexpressed with REV-ERBalpha1. We propose that the relative expression levels of the two REV-ERBalpha variants fine-tune the circadian period length by regulating REV-ERBalpha half-life.

Reversible synthesis and characterization of dynamic imino analogues of trimethine and pentamethine cyanine dyes.

Author(s) : Meguellati K, Spichty M, Ladame S,
Journal : Org Lett
2009
A new family of unsymmetrical imine-based trimethine and pentamethine cyanine dye analogues is reported that can form under reversible and thermodynamically controlled conditions from non- or weakly emissive amine and aldehyde building blocks. These dynamic fluorophores show spectroscopic properties comparable to those of their parent cyanine dyes and are responsive to external effectors.

Role of RNA structure and protein factors in the control of HIV-1 splicing.

Author(s) : Saliou J, Bourgeois C, Ayadi-Ben Mena L, Ropers D, Jacquenet S, Marchand V, Stevenin J, Branlant C,
Journal : Front Biosci (Landmark Ed)
2009
Alternative splicing plays a key role in the production of numerous proteins by complex lentiviruses such as HIV-1. The study of HIV-1 RNA splicing has provideduseful information not only about the physiology of the virus, but also about the general mechanisms that regulate mammalian pre-mRNA alternative splicing. Like all retroviruses, a fraction of HIV-1 transcripts remains intact to serve as genomic RNA and to code for Gag and Gag-Pol protein precursors. In addition, splicing is important for controlling the production of some viral proteins, which could otherwise have a negative effect on the infected cell. Here, we summarize how the utilization of HIV-1 splicing sites is limited by the binding of nuclear factors to cis-acting silencer elements, taking into account the roleof RNA secondary structure in these mechanisms. We also describe how the poorly efficient HIV-1 acceptor sites are nevertheless activated by serine/arginine-rich proteins. Finally, we discuss how nuclear factors that interact with both the transcription and splicing machineries also participate in the control of HIV-1 RNA splicing.

Structural and functional diversity of viral IRESes.

Author(s) : Balvay L, Soto Rifo R, Ricci E, Decimo D, Ohlmann T,
Journal : Biochim Biophys Acta
2009
Some 20 years ago, the study of picornaviral RNA translation led to the characterization of an alternative mechanism of initiation by direct ribosome binding to the 5' UTR. By using a bicistronic vector, it was shown that the 5' UTR of the poliovirus (PV) or the Encephalomyelitis virus (EMCV) had the abilityto bind the 43S preinitiation complex in a 5' and cap-independent manner. This is rendered possible by an RNA domain called IRES for Internal Ribosome Entry Site which enables efficient translation of an mRNA lacking a 5' cap structure. IRES elements have now been found in many different viral families where they often confer a selective advantage to allow ribosome recruitment under conditions where cap-dependent protein synthesis is severely repressed. In this review, we compare and contrast the structure and function of IRESes that are found within 4 distinct family of RNA positive stranded viruses which are the (i) Picornaviruses; (ii) Flaviviruses; (iii) Dicistroviruses; and (iv) Lentiviruses.

T-cell tropism of simian T-cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills (Mandrillus sphinx).

Author(s) : Souquiere S, Mouinga-Ondeme A, Makuwa M, Beggio P, Radaelli A, De Giuli Morghen C, Mortreux F, Kazanji M,
Journal : J Med Primatol
2009
BACKGROUND: Although a wide variety of non-human primates are susceptible to simian T-cell leukaemia virus type 1 (STLV-1), little is known about the virological or molecular determinants of natural STLV-1 infection. METHODS: We determined STLV-1 virus tropism in vivo and its relation to the immune response by evaluating cytokine production and T-cell subsets in naturally infected and uninfected mandrills. RESULTS: With real-time PCR methods, we found that STLV-1 in mandrills infects both CD4(+) and CD8(+) T cells; however, proviral loads were significantly higher (P = 0.01) in CD4(+) than in CD8(+) cells (mean STLV-1 copies number per 100 cells (+/- SD) was 7.8 +/- 8 in CD4(+) T cells and 3.9 +/-4.5 in CD8(+) T cells). After culture, STLV-1 provirus was detected in enriched CD4(+) but not in enriched CD8(+) T cells. After 6 months of culture, STLV-1-transformed cell lines expressing CD3(+), CD4(+) and HLADR(+) were established, and STLV-1 proteins and tax/rex mRNA were detected. In STLV-1 infected monkeys, there was a correlation between high proviral load and elevated levels of interleukin (IL)-2, IL-6, IL-10, interferon-gamma and tumour necrosis factor-alpha. The two monkeys with the highest STLV-1 proviral load had activated CD4(+)HLADR(+) and CD8(+)HLADR(+) T-cell subsets and a high percentage of CD25(+) in CD4(+) and CD8(+) T cells. CONCLUSIONS: Our study provides the first cellular, immunological and virological characterization of natural STLV-1 infection in mandrills and shows that they are an appropriate animal model for further physiopathological studies of the natural history of human T-cell leukaemia viruses.

TGF-beta induces degradation of TAL1/SCL by the ubiquitin-proteasome pathway through AKT-mediated phosphorylation.

Author(s) : Terme J, Lhermitte L, Asnafi V, Jalinot P,
Journal : Blood
2009
T-cell acute lymphoblastic leukemia 1 (TAL1), also known as stem cell leukemia (SCL), plays important roles in differentiation of hematopoietic and endothelialcells and is deregulated in a high percentage of T-cell acute lymphoblastic leukemia (T-ALL). In this report we show that the intracellular concentration ofTAL1 is regulated by transforming growth factor beta (TGF-beta), which triggers its polyubiquitylation and degradation by the proteasome. This effect is mediated by AKT1, which phosphorylates TAL1 at threonine 90. Immunoprecipitation experiments showed that this event increases association of TAL1 with the E3 ubiquitin ligase CHIP. The E47 heterodimerization partner of TAL1 hinders this association. Our observations indicate that activation of the TGF-beta and phosphatidylinositol 3-kinase/AKT pathways might reverse overexpression of TAL1 in leukemic cells by inducing proteolysis of this important oncogene.

The evolutionary context of robust and redundant cell biological mechanisms.

Author(s) : Delattre M, Felix M,
Journal : Bioessays
2009
The robustness of biological processes to perturbations has so far been mainly explored in unicellular organisms; multicellular organisms have been studied fordevelopmental processes or in the special case of redundancy between gene duplicates. Here we explore the robustness of cell biological mechanisms of multicellular organisms in an evolutionary context. We propose that the reuse ofsimilar cell biological mechanisms in different cell types of the same organism has evolutionary implications: (1) the maintenance of apparently redundant mechanisms over evolutionary time may in part be explained by their differentialrequirement in various cell types; (2) the relative requirement for two alternative mechanisms may evolve among homologous cells in different organisms.We present examples of cell biological processes, such as centrosome separation in prophase, spindle formation or cleavage furrow positioning, that support the first proposition. We propose experimental tests of these hypotheses.

The germ cell nuclear proteins hnRNP G-T and RBMY activate a testis-specific exon.

Author(s) : Liu Y, Bourgeois C, Pang S, Kudla M, Dreumont N, Kister L, Sun Y, Stevenin J, Elliott D,
Journal : PLoS Genet
2009
The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2beta is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5' splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2beta and hnRNP Gproteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-Tproteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3' splice site normally weakly selected in the testis. Co-expression of Tra2beta with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.

The HSP90 binding mode of a radicicol-like E-oxime determined by docking, binding free energy estimations, and NMR 15N chemical shifts.

Author(s) : Spichty M, Taly A, Hagn F, Kessler H, Barluenga S, Winssinger N, Karplus M,
Journal : Biophys Chem
2009
We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We samplethe macrocyclic scaffold of the unbound ligand by parallel tempering simulationsand dock the most populated conformations to yeast HSP90. Docking poses are thenevaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of backbone (15)N provides an additional evaluation criteria. As a final test we check the binding modes against available structure-activity-relationships. We find that the most likelybinding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol-HSP90 complex.