MRX-dependent DNA damage response to short telomeres

Telomere structure allows cells to distinguish the natural chromosome ends from double-strand breaks (DSBs). However, DNA damage response proteins are intimately involved in telomere metabolism, suggesting that functional telomeres may be recognized as DNA damage during a time window. Here we show by two different systems that short telomeres are recognized as DSBs during the time of their replication, because they induce a transient MRX-dependent DNA damage checkpoint response during their prolonged elongation. The MRX complex, which is recruited at telomeres under these conditions, dissociates from telomeres concomitantly with checkpoint switch off when telomeres reach a new equilibrium length. We also show that MRX recruitment to telomeres is sufficient to activate the checkpoint independently of telomere elongation. We propose that MRX can signal checkpoint activation by binding to short telomeres only when they become competent for elongation. Because full-length telomeres are refractory to MRX binding and the shortest telomeres are elongated of only a few base pairs per generation, this limitation may prevent unscheduled checkpoint activation during an unperturbed S phase.     

Novel carbazole derivatives as NPY Y1 antagonists

The synthesis of a series of carbazole derivatives and their SAR at the NPY Y1 receptor is described. Modulation of physicochemical properties by appropriate decoration led to the identification of a high-affinity NPY Y1 antagonist that shows high brain penetration and modest oral bioavailability.     

Intact p53-Dependent Responses in miR-34-Deficient Mice

MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34-deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34-deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc-initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.     

Effect of Treadmill Exercise on Serotonin Immunoreactivity in Medullary Raphe Nuclei and Spinal Cord Following Sciatic Nerve Transection in Rats

The serotoninergic system modulates nociceptive and locomotor spinal cord circuits. Exercise improves motor function and changes dopaminergic, noradrenergic, and serotonergic central systems. However, the direct relationship between serotonin, peripheral nerve lesion and aerobic treadmill exercise has not been studied. Using immunohistochemistry and optic densitometry, this study showed that the sciatic nerve transection increased the serotoninergic immunoreactivity in neuronal cytoplasm of the magnus raphe nuclei of trained and sedentary rats. In the dorsal raphe nucleus the increase only occurred in sedentary-sham-operated rats. In the spinal cord of trained, transected rats, the ventral horn showed significant changes, while the change in dorsal horn was insignificant. Von Frey’s test indicated analgesia in all exercise-trained rats. The sciatic nerve functional index indicated recovery in the trained group. Thus, both the aerobic treadmill exercise training and the nervous lesion appear to contribute to changes in serotonin immunoreactivity.     

beta-lactoglobulin under high pressure studied by small-angle neutron scattering

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of beta-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.     

Rapid and high-throughput genotyping of Staphylococcus epidermidis isolates by automated multilocus variable-number of tandem repeats: a tool for real-time epidemiology

Fast and reliable genotyping methods allowing real-time epidemiology would be instrumental to discriminate Staphylococcus epidermidis isolates, in order to evaluate potential cross-infections or to follow genome content of infecting strains of this important opportunistic pathogen. We describe an automated multilocus variable-number tandem repeat-based assay (MLVA) for the rapid genotyping of S. epidermidis. Multiplex PCR amplifications using 6 primer pairs targeting gene-regions containing variable numbers of tandem repeats and the mecA gene are resolved by micro-capillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for discriminatory power and reproducibility on 2 sequenced strains, on a collection of 21 strains previously characterized using genotyping reference methods and finally on 65 clinical isolates identified in two different institutions. All steps of this new procedure were developed to ensure rapid turn-around time and moderate costs. Our results suggest that this rapid approach is a valuable epidemiological tool to genotype S. epidermidis isolates in real-time. The rapid analysis of a limited number of evolutionary markers showed a power of discrimination similar to that of pulse-field gel electrophoresis (PFGE) or multilocus sequence type (MLST). This type of rapid and high-throughput methodology opens the possibility to rapidly assess long-term nosocomial transmission or to characterize infecting strains in the general procedure of routine laboratories, in real-time.     

Immunolocalization of interleukin-1 receptor antagonist in healthy and infarcted myocardium

Ischemic heart disease is a widespread cause of death. During infarction, myocardial injury is mediated by release of several pro-inflammatory cytokines including multifunctional interleukin-1 (IL-1). In various tissues, IL-1-mediated deleterious effects are known to be attenuated via the over-expression of natural anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra). In the present investigation, IL-1ra distribution in healthy and infarcted myocardium was studied by light and electron microscopy. After immunostaining, weak positivity resulted for cardiomyocytes in normal myocardium and, at higher degrees, in infarction border areas and ischemic ones. In ischemic areas, additional reactivity was displayed by the extracellular matrix and intravascular plasma. Immunogold labelling provided further details on intracytoplasmatic and extracellular distribution; in particular, noticeable gold particle distribution appeared on intercalated discs in normal and hypertrophic cardiomyocytes, as well as on thickened Z-lines for these latter. The present results suggest that cardiomyocytes represent a major source of IL-1ra in vivo, even though additional contribution by blood derived IL-1ra is to be taken in account in ischemic areas. In addition, ischemia-associated intracytoplasmic IL-1ra increase and its additional presence in the extracellular matrix is consistent with the concept that this cytokine plays a cardioprotective role at different levels and by distinct mechanisms.