α-Syntrophin Modulates Myogenin Expression in Differentiating Myoblasts

α-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, α-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of α-syntrophin and myogenin, a key muscle regulatory factor.The absence of α-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from α-syntrophin null mice, muscle regeneration studies in α-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of α-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from α-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the α-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing α-syntrophin and in Sol8 cells transfected with a vector to over-express α-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of α-syntrophin and increased by α-syntrophin over-expression. Immunofluorescence microscopy shows that α-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that α-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression.We conclude that α-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression

Integration of Expressed Sequence Tag Data Flanking Predicted RNA Secondary Structures Facilitates Novel Non-Coding RNA Discovery

Many computational methods have been used to predict novel non-coding RNAs (ncRNAs), but none, to our knowledge, have explicitly investigated the impact of integrating existing cDNA-based Expressed Sequence Tag (EST) data that flank structural RNA predictions. To determine whether flanking EST data can assist in microRNA (miRNA) prediction, we identified genomic sites encoding putative miRNAs by combining functional RNA predictions with flanking ESTs data in a model consistent with miRNAs undergoing cleavage during maturation. In both human and mouse genomes, we observed that the inclusion of flanking ESTs adjacent to and not overlapping predicted miRNAs significantly improved the performance of various methods of miRNA prediction, including direct high-throughput sequencing of small RNA libraries. We analyzed the expression of hundreds of miRNAs predicted to be expressed during myogenic differentiation using a customized microarray and identified several known and predicted myogenic miRNA hairpins. Our results indicate that integrating ESTs flanking structural RNA predictions improves the quality of cleaved miRNA predictions and suggest that this strategy can be used to predict other non-coding RNAs undergoing cleavage during maturation

ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

Le droit international priv\ue9 communautaire, un nouveau syst\ue8me - Table Ronde, Universit\ue0 Cattolica del Sacro Cuore, Milan (Italie), 7 mai 2004 (compte rendu)

L'autore, con la collaborazione della collega francese P. Daubas, presenta una sintesi della tavola rotonda svoltasi presso l'Universit\ue0 Cattolica di Milano il 7 maggio 2004 allo scopo di riflettere se la comunitarizzazione del diritto internazionale privato avviata dal Trattato di Amsterdam mediante l'introduzione nel Trattato CE della disciplina in materia di cooperazione giudiziaria civile si sia tradotta nell'elaborazione di un sistema coerente di disciplina della materia. Dalle relazioni e dal dibattito avutosi in seno alla tavola rotonda, si \ue8 potuta trarre la conclusione che se si pu\uf2 dire che un primo corpo di principi suscettibile di costituire la base di un sistema comunitario di diritto internazionale privato sia in via di formazione, nondimeno i metodi seguiti dalle istituzioni comunitarie nei diversi strumenti sinora adottati in materia appaiono ispirati a metodi differenti di coordinamento tra ordinamenti

Le droit international privé communautaire, un nouveau système - Table Ronde, Università Cattolica del Sacro Cuore, Milan (Italie), 7 mai 2004 (compte rendu)

L'autore, con la collaborazione della collega francese P. Daubas, presenta una sintesi della tavola rotonda svoltasi presso l'Università Cattolica di Milano il 7 maggio 2004 allo scopo di riflettere se la comunitarizzazione del diritto internazionale privato avviata dal Trattato di Amsterdam mediante l'introduzione nel Trattato CE della disciplina in materia di cooperazione giudiziaria civile si sia tradotta nell'elaborazione di un sistema coerente di disciplina della materia. Dalle relazioni e dal dibattito avutosi in seno alla tavola rotonda, si è potuta trarre la conclusione che se si può dire che un primo corpo di principi suscettibile di costituire la base di un sistema comunitario di diritto internazionale privato sia in via di formazione, nondimeno i metodi seguiti dalle istituzioni comunitarie nei diversi strumenti sinora adottati in materia appaiono ispirati a metodi differenti di coordinamento tra ordinamenti