Quantitation of polar analytes using column-switching: Application to oxycodone and three metabolites in human plasma

We present herein a sensitive and selective assay for the determination of oxycodone and its main metabolites, oxymorphone, noroxycodone and noroxymorphone in human plasma, using column-switching and liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Sample preparation comprised protein precipitation with perchloric acid. After neutralization, the supernatant was injected without any evaporation step onto a polymeric, pH-resistant cartridge (HySphere Resin GP 10–12 μm) for sample clean-up (Prospekt II). The latter operation was achieved by using alkaline conditions to ensure retention of analytes and methanol for matrix interference removal. More than two hundred plasma samples could be analyzed with a single cartridge. Analytes were desorbed in the backflush mode and were separated on a conventional reversed phase column (XTerra MS 4.6 × 50 mm, 3.5 μm), using an acidic mobile phase (i.e. containing 0.1% of formic acid). Mass spectrometric detection was achieved with a 4000 Q TRAP equipped with an atmospheric pressure chemical ionization (APCI) source, in positive ionization mode, operated in the selected reaction monitoring mode (SRM). Starting from a plasma volume of 250 μl, quantification ranges were 25–10,000 pg/ml for OXM and NOXM and 50–10,000 pg/ml for OXC and NOXC. Accuracy was found to be within 98% and 108% and precision better than 7%. Replicate determination of incurred or study samples ensured the method to be reproducible and usable for clinical studies.     

Differential regulation of B-raf isoforms by phosphorylation and autoinhibitory mechanisms

The B-Raf proto-oncogene encodes several isoforms resulting from alternative splicing in the hinge region upstream of the kinase domain. The presence of exon 8b in the B2-Raf(8b) isoform and exon 9b in the B3-Raf(9b) isoform differentially regulates B-Raf by decreasing and increasing MEK activating and oncogenic activities, respectively. Using different cell systems, we investigated here the molecular basis of this regulation. We show that exons 8b and 9b interfere with the ability of the B-Raf N-terminal region to interact with and inhibit the C-terminal kinase domain, thus modulating the autoinhibition mechanism in an opposite manner. Exons 8b and 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. The S365A mutation increased the activity of all B-Raf isoforms, but the effect on B2-Raf(8b) was more pronounced. This was correlated to the high level of S365 phosphorylation in this isoform, whereas the B3-Raf(9b) isoform was poorly phosphorylated on this residue. In contrast, S429 was equally phosphorylated in all B-Raf isoforms, but the S429A mutation activated B2-Raf(8b), whereas it inhibited B3-Raf(9b). These results indicate that phosphorylation on both S365 and S429 participate in the differential regulation of B-Raf isoforms through distinct mechanisms. Finally, we show that autoinhibition and phosphorylation represent independent but convergent mechanisms accounting for B-Raf regulation by alternative splicing.     

Specific GFP-binding artificial proteins (αRep): a new tool for in?vitro to live cell applications

A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes.     

Use of the NEO strategy (Nucleophilic addition/Epoxide Opening) for the synthesis of a new C-galactoside ester analogue of KRN 7000

Our goal in the search for potentially bioactive analogues of KRN 7000 was to design an easy synthetic approach to a library of analogues using a strategy recently developed in our laboratory based on a Nucleophilic addition followed by an Epoxide Opening (the NEO strategy). Through the use of a common pivotal structure, a new C-galactoside ester analogue (23) was synthesized which showed an encouraging T_H2 biased response during preliminary biological tests.     

Variation in cooperative behaviour within a single city

Human cooperative behaviour, as assayed by decisions in experimental economic dilemmas such as the Dictator Game, is variable across human populations. Within-population variation has been less well studied, especially within industrial societies. Moreover, little is known about the extent to which community-level variation in Dictator Game behaviour relates to community-level variation in real-world social behaviour. We chose two neighbourhoods of the city of Newcastle upon Tyne that were similar in most regards, but at opposite ends of the spectrum in terms of level of socioeconomic deprivation. We administered Dictator Games to randomly-selected residents, and also gathered a large number of more naturalistic measures of cooperativeness. There were dramatic differences in Dictator Game behaviour between the two neighbourhoods, with the mean allocation to the other player close to half the stake in the affluent neighbourhood, and close to one tenth of the stake in the deprived neighbourhood. Moreover, the deprived neighbourhood was also characterised by lower self-reported social capital, higher frequencies of crime and antisocial behaviour, a higher frequency of littering, and less willingness to take part in a survey or return a lost letter. On the other hand, there were no differences between the neighbourhoods in terms of the probability of helping a person who dropped an object, needed directions to a hospital, or needed to make change for a coin, and people on the streets were less likely to be alone in the deprived neighbourhood than the affluent one. We conclude that there can be dramatic local differences in cooperative behaviour within the same city, and that these need further theoretical explanation.     

COMMD1-mediated ubiquitination regulates CFTR trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.     

The Xenopus ELAV protein ElrB represses Vg1 mRNA translation during oogenesis

Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis.     

A directed screen for chlamydial proteins secreted by a type III mechanism identifies a translocated protein and numerous other new candidates

Chlamydiae are strict intracellular parasites that induce their internalization upon contact with the host cell and grow inside an intracellular compartment called an inclusion. They possess a type III secretion (TTS) apparatus, which allows for the translocation of specific proteins in the host cell cytosol. In particular, chlamydial proteins of the Inc family are secreted to the inclusion membrane by a TTS mechanism; other TTS substrates are mostly unknown. Using a secretion assay based on the recognition of TTS signals in Shigella flexneri, we searched for TTS signals in the proteins of unknown function, conserved between three different chlamydial species, Chlamydia pneumoniae, C. trachomatis and C. caviae. We identified 24 new candidate proteins which did not belong to the Inc family. Four of these proteins were also secreted as full-length proteins by a TTS mechanism in S. flexneri, indicating that their translocation does not require other chlamydial proteins. One of these proteins was detected in the cytosol of infected cells using specific antibodies, directly demonstrating that it is translocated in the host cell during bacterial proliferation. More generally, this work represents the first directed search for TTS effectors not based on genetic information or sequence similarity. It reveals the abundance of proteins secreted in the host cell by chlamydiae.