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Accueil > Publications > Recherche par années > Années 2010 > 2012

2012

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A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line

Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line : CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using ‘‘in situ’’ proximity ligation assay (PLA).

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A new metallostar complex based on an aluminum(iii) 8-hydroxyquinoline core as a potential bimodal contrast agent

A ditopic DTPA monoamide derivative containing an 8-hydroxyquinoline moiety was synthesized and the corresponding gadolinium(iii) complex ([Gd(H5)(H2O)]-) was prepared. After adding aluminum(iii), the 8-hydroxyquinoline part self-assembled into a heteropolymetallic triscomplex [(Gd5)3Al(H2O)3]3-. The magnetic and optical properties of this metallostar compound were investigated in order to classify it as a potential in vitro bimodal contrast agent. The proton nuclear magnetic relaxation dispersion measurements indicated that the relaxivity r1 of [Gd(H5)(H2O)]- and [(Gd5)3Al(H2O)3]3- at 20 MHz and 310 K equaled 6.17 s-1 mM-1 and 10.9 s-1 mM-1 per Gd(iii) ion respectively. This corresponds to a relaxivity value of 32.7 s-1 mM-1 for the supramolecular complex containing three Gd(iii) ions. The high relaxivity value is prominently caused by an increase of the rotational tumbling time [small tau]R by a factor of 2.7 and 5.5 respectively, in comparison with the commercially used MRI contrast agent Gd(iii)-DTPA (Magnevist[registered sign]). Furthermore, upon UV irradiation, [(Gd5)3Al(H2O)3]3- exposes green broad-band emission with a maximum at 543 nm. Regarding the high relaxivity and the photophysical properties of the [(Gd5)3Al(H2O)3]3- metallostar compound, it can be considered as a lead compound for in vitro bimodal applications.

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A path-integral Langevin equation treatment of low-temperature doped helium clusters

We present an implementation of path integral molecular dynamics for sampling low temperature properties of doped helium clusters using Langevin dynamics. The robustness of the path integral Langevin equation and white-noise Langevin equation [M. Ceriotti, M. Parrinello, T. E. Markland, and D. E. Manolopoulos, J. Chem. Phys. 133, 124104 (2010)] sampling methods are considered for those weakly bound systems with comparison to path integral Monte Carlo (PIMC) in terms of efficiency and accuracy. Using these techniques, convergence studies are performed to confirm the systematic error reduction introduced by increasing the number of discretization steps of the path integral. We comment on the structural and energetic evolution of HeN−CO2 clusters from N = 1 to 20. To quantify the importance of both rotations and exchange in our simulations, we present a chemical potential and calculated band origin shifts as a function of cluster size utilizing PIMC sampling that includes these effects. This work also serves to showcase the implementation of path integral simulation techniques within the molecular modelling toolkit [K. Hinsen, J. Comp. Chem. 21, 79 (2000)], an open-sourcemolecular simulation package.

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A role for Rho-dependent polarity in gene regulation by a noncoding small RNA

Gene regulation by bacterial trans-encoded small RNAs (sRNAs) is generally regarded as a post-transcriptional process bearing exclusively on the translation and/or the stability of target messenger RNA (mRNA). The work presented here revealed the existence of a transcriptional component in the regulation of a bicistronic operon-the chiPQ locus-by the ChiX sRNA in Salmonella. By studying the mechanism by which ChiX, upon pairing near the 5’ end of the transcript, represses the distal gene in the operon, we discovered that the action of the sRNA induces Rho-dependent transcription termination within the chiP cistron. Apparently, by inhibiting chiP mRNA translation cotranscriptionally, ChiX uncouples translation from transcription, causing the nascent mRNA to become susceptible to Rho action. A Rho utilization (rut) site was identified in vivo through mutational analysis, and the termination pattern was characterized in vitro with a purified system. Remarkably, Rho activity at this site was found to be completely dependent on the function of the NusG protein both in vivo and in vitro. The recognition that trans-encoded sRNA act cotranscriptionally unveils a hitherto neglected aspect of sRNA function in bacteria.

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A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate

The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex ; it is a reversible competitive inhibitor with a K-i comparable to the K-m of the parent substrate. Low concentrations (60 mu M) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 mu L) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2 h.

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A tripodal ruthenium-gadolinium metallostar as a potential α(v)β(3) integrin specific bimodal imaging contrast agent

Gd-III-containing metallostar contrast agents are gaining increased attention, because their architecture allows for a slower tumbling rate, which, in turn, results in larger relaxivities. So far, these metallostars find possible applications as blood pool contrast agents. In this work, the first example of a tissue-selective metallostar contrast agent is described. This RGD-peptide decorated Ru-II(Gd-III)(3) metallostar is synthesized as an alpha(v)beta(3)-integrin specific contrast agent, with possible applications in the detection of atherosclerotic plaques and tumor angiogenesis. The contrast agent showed a relaxivity of 9.65 s(-1) mM(-1), which represents an increase of 170%, compared to a low-molecular-weight analogue, because of a decreased tumbling rate (tau(R) = 470 ps). The presence of the MLCT band (absorption 375-500 nm, emission 525-850 nm) of the central Ru-II(Ph-Phen)(3)-based complex grants the metallostar attractive luminescent properties. The (MLCT)-M-3 emission is characterized by a quantum yield of 4.69% and a lifetime of 804 ns, which makes it an interesting candidate for time-gated luminescence imaging. The potential application as a selective MRI contrast agent for alpha(v)beta(3)-integrin expressing tissues is shown by an in vitro relaxometric analysis, as well as an in vitro T-1-weighted MR image.

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A Water Soluble CuI–NHC for CuAAC ligation of unprotected peptides under open air conditions

A reducing agent–free version of CuAAC able to operate
under open air conditions is reported. A readily–synthesizable, hydrophilic and highly stable CuI–NHC allows the clean ligations of unprotected peptides comprising sensitive side chains, at millimolar concentrations.

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Alanine Screening Mutagenesis Establishes the Critical Inactivating Damage of Irradiated E. coli Lactose Repressor.

The function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor.

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Analysis of Helicase–RNA Interactions Using Nucleotide Analog Interference Mapping

Nucleotide analog interference mapping (NAIM) is a combinatorial approach that probes individual atoms and functional groups in an RNA molecule and identifies those that are important for a specific biochemical function. Here, we show how NAIM can be adapted to reveal functionally important atoms and groups on RNA substrates of helicases. We explain how NAIM can be used to investigate translocation and unwinding mechanisms of helicases and discuss the advantages and limitations of this powerful chemogenetic approach.

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Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium

The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (P(ramA)). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of P(ramA), including the -10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P(ramA) as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (K(D) [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted P(ramA) of an MDR S. Typhimurium clinical isolate than for the wild-type P(ramA) (K(D) = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.

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