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Accueil > Publications > Recherche par années > Années 2010 > 2014

2014

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A Bis(pyridine N-oxide) Analogue of DOTA : Relaxometric Properties of the Gd-III Complex and Efficient Sensitization of Visible and NIR-Emitting Lanthanide(III) Cations Including Pr-III and Ho-III

We report the synthesis of a cyclen-based ligand (4,10-bis[(1-oxidopyridin-2-yl)methyl]-1,4,7,10-tetraazacyclododecane-1,7-diacetic acid = L1) containing two acetate and two 2-methylpyridine N-oxide arms anchored on the nitrogen atoms of the cyclen platform, which has been designed for stable complexation of lanthanide(III) ions in aqueous solution. Relaxometric studies suggest that the thermodynamic stability and kinetic inertness of the Gd-III complex may be sufficient for biological applications. A detailed structural study of the complexes by H-1 NMR spectroscopy and DFT calculations indicates that they adopt an anti-Delta(lambda lambda lambda lambda) conformation in aqueous solution, that is, an anti-square antiprismatic (anti-SAP) isomeric form, as demonstrated by analysis of the H-1 NMR paramagnetic shifts induced by Yb-III. The water-exchange rate of the Gd-III complex is k(ex)(298) = 6.7 x 10(6) s(-1), about a quarter of that for the mono-oxidopyridine analogue, but still about 50% higher than the k(ex)(298) of GdDOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The 2-methylpyridine N-oxide chromophores can be used to sensitize a wide range of Ln(III) ions emitting in both the visible (Eu-III and Tb-III) and NIR (Pr-III, Nd-III, Ho-III, Yb-III) spectral regions. The emission quantum yield determined for the Yb-III complex (Q(Yb)(L) = 7.3(1) x 10(-3)) is among the highest ever reported for complexes of this metal ion in aqueous solution. The sensitization ability of the ligand, together with the spectroscopic and relaxometric properties of its complexes, constitute a useful step forward on the way to efficient dual probes for optical imaging (OI) and MRI.

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A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1

Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 x 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 mug/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000, and gene expression is higher than observed with FreeStyle and JetPEI(R). Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.

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Agents de contraste pour l’IRM

L’imagerie par résonance magnétique (IRM) est maintenant entrée dans la vie courante, la moindre blessure aux sports d’hiver conduit à passer une IRM. Son caractère atraumatique et non invasif constitue un avantage décisif. La majorité des hôpitaux en sont équipés. L’IRM ne nécessite pas obligatoirement l’utilisation d’agents d’imagerie pour fournir une image structurelle de l’intérieur de l’organisme. Les réglages physiques de l’expérimentation permettent même d’accéder à différents paramètres des tissus (détection de tumeurs, oedèmes...). Ce n’est que si l’on veut aller plus loin dans la spécificité de la détection, qu’il est intéressant et même indispensable de faire appel à des agents de contraste injectables, même si l’on perd alors partiellement le caractère non invasif de l’IRM. Comme nous allons le montrer, la complexité des molécules va de l’agent chimiquement non spécifique jusqu’à l’agent de contraste dit « intelligent » (smart agent).

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Anomalous lateral diffusion in lipid bilayers observed by molecular dynamics simulations with atomistic and coarse-grained force fields

In the present article we study the lateral molecular di-usion in homogenous lipid (POPC) bilayers by molecular dynamics simulations with the all-atom OPLS and the coarse-grained MARTINI force fields. On the statistically relevant time scales the center-of-mass mean-square displacement exhibits in both cases the subdiffusive asymptotic form W (t) 2Dαtα , with α ≈ 0.67 and α ≈ 0.57 , respectively. The diffusive dynamics obtained by the MARTINI force field is, however, faster by a factor of about 3. The subdiffusive characteristics of the di-usion process is confirmed by comparing the integral of the center-of-mass velocity autocorrelation function with its analytical long-time tail. The agreement is particularly good for the MARTINI force field, which permits to extend the simulation length and the system size considerably. Our results are in agreement with experimental observations of subdi-usion in lipid bilayers on longer time scales and do not support the finding of some authors that the latter should be considered as a transient phenomenon.

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Bacterial Rho helicase : a new tool to dissect mRNP biogenesis and quality control in yeast

In eukaryotic cells, the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export competent messenger ribonucleoprotein particles (mRNPs) are under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from faulty events are detected by the QC apparatus and retained in the nucleus with ensuing elimination of their mRNA component by the RNA degradation machinery. A decade of biochemical and genetic experiments in yeast allowed the identification of the nuclear degradation machinery including the core exosome and its two associated catalytic subunits Rrp6p and Rrp44p, its cofactors Rrp47p and Mpp6p as well as the activator complex TRAMP. Similarly, studies of the THO-Sub2 complex of the mRNP assembly and export apparatus have provided valuable information on the nuclear retention and degradation of a particular class of aberrant mRNPs. However, a unifying mechanism of action underlying the QC process remains elusive. Here, we review the implementation of a new experimental approach whereby the production of aberrant mRNPs is massively increased upon heterologous expression of the bacterial Rho helicase in yeast. Using this methodology, we have shown that the QC process is coordinated by Nrd1p (a component of the early termination complex) whose increased co-transcriptional recruitment promotes the attachment of the 3’-5’ exonuclease Rrp6p along with the co-factors Rrp47p and Mpp6p. Interestingly, we established that Rrp6p functions independently from the core exosome, yet is stimulated by two forms of the TRAMP complex that include Trf4p or Trf5p and Air2p but not Air1p. The results suggest that specific substrates could be primed for decay via various QC pathways owing to the versatility of the mRNA degradation apparatus. In this context, the bacterial Rho helicase provides a valuable tool to decipher the QC molecular process in yeast and possibly the homologous process in mammalian cells.

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Bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through transcriptional derepression of ramA in Salmonella enterica serovar Typhimurium

OBJECTIVES : In Salmonella Typhimurium, the genes encoding the AcrAB-TolC multidrug efflux system are mainly regulated by the ramRA locus, composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, the gene for which is itself transcriptionally repressed by RamR. Previous studies have reported that bile induces acrAB in a ramA-dependent manner, but none provided evidence for an induction of ramA expression by bile. Therefore, the objective of this study was to clarify the regulatory mechanism by which bile activates acrAB and tolC. METHODS : qRT-PCR was used to address the effects of bile (using choleate, an ox-bile extract) on the expression of ramA, ramR, acrB and tolC. Electrophoretic mobility shift assays and surface plasmon resonance experiments were used to measure the effect of bile on RamR binding to the ramA promoter (PramA) region. RESULTS : We show that ramA is transcriptionally activated by bile and is strictly required for the bile-mediated activation of acrB and tolC. Additionally, bile is shown to specifically inhibit the binding of RamR to the PramA region, which overlaps the putative divergent ramR promoter, thereby explaining our observation that bile also activates ramR transcription. *

CONCLUSIONS : We propose a regulation model whereby the bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through the transcriptional derepression of the ramA activator gene.

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cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity : evidences from mass spectrometry and biochemical analyses

Genomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. In order to minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is down regulated by PKA phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that PKA phosphorylation may play a double role in the early stages of genome invasion by mariner elements.

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Cdk5 induces constitutive activation of 5-HT6 receptors to promote neurite growth

The serotonin6 receptor (5-HT6R) is a promising target for treating cognitive deficits of schizophrenia often linked to alterations of neuronal development. This receptor controls neurodevelopmental processes, but the signaling mechanisms involved remain poorly understood. Using a proteomic strategy, we show that 5-HT6Rs constitutively interact with cyclin-dependent kinase 5 (Cdk5). Expression of 5-HT6Rs in NG108-15 cells induced neurite growth and expression of voltage-gated Ca2+ channels, two hallmarks of neuronal differentiation. 5-HT6R–elicited neurite growth was agonist independent and prevented by the 5-HT6R antagonist SB258585, which behaved as an inverse agonist. Moreover, it required receptor phosphorylation at Ser350 by Cdk5 and Cdc42 activity. Supporting a role of native 5-HT6Rs in neuronal differentiation, neurite growth of primary neurons was reduced by SB258585, by silencing 5-HT6R expression or by mutating Ser350 into alanine. These results reveal a functional interplay between Cdk5 and a G protein–coupled receptor to control neuronal differentiation.

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Changes in Metabolic Chemical Reporter Structure Yield a Selective Probe of O-GlcNAc Modification

Metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain bioorthogonal functionalities and enable the direct visualization and identification of glycoproteins from living cells. Each MCR was initially thought to report on specific types of glycosylation. We and others have demonstrated that several MCRs are metabolically transformed and enter multiple glycosylation pathways. Therefore, the development of selective MCRs remains a key unmet goal. We demonstrate here that 6-azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and comparative proteomics with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz), and N-azidoacetyl-galactosamine (GalNAz) revealed that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz and GalNAz are incorporated into a combination of intracellular and extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be biosynthetically transformed into the corresponding UDP sugar-donor by the canonical salvage-pathway that requires phosphorylation at the 6-hydroxyl. In vitro experiments showed that 6AzGlcNAc can bypass this roadblock through direct phosphorylation of its 1-hydroxyl by the enzyme phosphoacetylglucosamine mutase (AGM1). Taken together, 6AzGlcNAc enables the specific analysis of O-GlcNAcylated proteins, and these results suggest that specific MCRs for other types of glycosylation can be developed. Additionally, our data demonstrate that cells are equipped with a somewhat unappreciated metabolic flexibility with important implications for the biosynthesis of natural and unnatural carbohydrates.

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Cherts of the Barberton Greenstone Belt, South Africa : Petrology and Trace-element Geochemistry of 3.5 to 3.3 Ga Old Silicified Volcaniclastic Sediments

A suite of green and carbonaceous black chert interbedded with submarine volcanic rocks from several stratigraphic levels of the Onverwacht Group of the Barberton greenstone belt, from the top of the 3.48 Ga Komati Formation to the base of the 3.26 Ga Fig Tree Group have been investigated petrologically and geochemically. Provenance analysis was undertaken using immobile trace element ratios in comparison with potential source rocks from the Barberton granitoid-greenstone terrain. Raman spectroscopy was used to better characterise carbonaceous matter in different chert varieties. Green cherts consist predominantly of silicified mafic to ultramafic volcaniclastic material. Black cherts contain volcaniclastic and/or epiclastic material of ultramafic to felsic composition, admixed with carbonaceous matter. In several cases, the clastic sediment is compositionally distinct to the volcanic substrate, suggesting that it represents volcanic material of relatively distal sources. Soon after settling out of the water column, lithification due to silicification took place. It gave rise to excellent textural preservation, but strongly modified the mineralogical and chemical composition of the seafloor sediments. Zircon, Cr-spinel and rare phosphate minerals are the only primary minerals remaining, while clastic grains have been largely replaced by a fine intergrowth of microquartz, Ti-oxide and K-bearing mica. Chemical changes are similar to those reported previously for silicification of volcanic rocks from the barberton belt and are consistent with low-temperature hydrothermal processes common to the Palaeoarchaean seafloor environment.

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