Investigation of the Protonation Site in the Dialanine Peptide by Infrared Multiphoton Dissociation Spectroscopy

Protonated dialanine cations have been isolated in a Fourier transform ion cyclotron resonance mass-spectrometer (FT-ICR-MS) and subjected to infrared multiphoton dissociation (IRMPD) at the free electron laser facility CLIO in Orsay (France). The spectral dependence of the IR induced fragmentation pattern in the mid-infrared region (800-2000 cm-1) is interpreted with the help of structure and vibrational spectrum calculations of the different protonated conformers. This comparison allows for the assignment of the proton on the terminal amino group, as the most favourable proton site, the neighbouring amide bond being in the trans conformation

The domestic basis of the scientific career: gender inequalities in ecology in France and Norway

International audienceGender-related inequalities in scientific careers are widespread, evidenced by the attrition of women along the different stages of the promotion ladder. We studied the interwoven personal and professional trajectories of researchers in ecology and compared these trajectories between France and Norway. Given their differing welfare state policies and work/family regimes, we expected contrasts in the depth and modalities of the gender gap. We focused on the career consequences of time-use inequalities in the workplace and in the private sphere (domestic tasks and parental care). We find a more frequent assignment of women to less-valued tasks at work (e.g. teaching) and pronounced gender differences in the involvement in domestic and parental tasks, especially in France. Age at promotion and probability to be promoted differed between gender in both countries and more so in France, women being less promoted and promoted later than men. This gender gap was particularly discriminating women with children, when they were either single or with a partner who also was a researcher. These differences are mainly due to a lower scientific productivity of women when they get children. These analyses raise a number of questions on welfare policies and on the definition of academic standards of peer judgment within local employment policies in universities

Functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood-brain barrier model.

International audienceUNLABELLED: ABSTRACT: BACKGROUND: Knowledge of the molecular basis and transport function of the human blood-brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model. METHODS: Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions. RESULTS: Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibited by metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptake was increased by extracellular alkalization and intracellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB. CONCLUSIONS: The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells

Functional expression of a proton-coupled organic cation (H⁺/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood–brain barrier model

Abstract Background Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model. Methods Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions. Results Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibited by metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptake was increased by extracellular alkalization and intracellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB. Conclusions The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.</p

IR and UV spectroscopic signatures of DNA higher-order structures in the gas phase

Introduction Electrospray mass spectrometry (ESI-MS) can be used to transfer large biomolecular complexes from the solution to the gas phase. However, a longstanding question is whether the gas-phase multiply-charged ions produced by ESI-MS keep a folded conformation in the absence of solvent. Nucleic acid secondary structures are determined by hydrogen bonding interactions between nucleic bases and by stacking interactions between neighboring base pairs. Here we will show that infrared (IR) and ultraviolet (UV) action spectroscopies provide useful and complementary information on the structure of nucleic acid ions in the gas phase. Methods IR spectroscopy experiments on DNA negative ions were carried out at the CLIO free electron laser (FEL) center (Orsay, France) using an Esquire 3000 (Bruker) mass spectrometer modified to inject the IR beam through the ring electrode. IRMPD spectra are recorded by monitoring the fragmentation of mass-selected parent ions as a function of the excitation wavenumber, in the range 1000-2000 cm-1. UV spectroscopy experiments were carried out using a tunable OPO laser (Continuum Lasers) with frequency doubling. The laser is interfaced with either a Finnigan LCQ ESI-QIT mass spectrometer or a Bruker Apex-Qe 9.4 T ESI-FTICR mass spectrometer. The UV action spectra were recorded by monitoring electron detachment as a function of the wavelength between 220 and 300 nm. Preliminary results First, DNA oligonucleotide ions forming G-quadruplex structures were studied in the gas phase using IR multiple-photon dissociation spectroscopy. Data interpretation on these large biomolecule ions is made using carefully chosen control experiments. The IR spectrum of the (dTG4T)4 quadruplex has been recorded, and compared to that of the single strand. Given the strand stoichiometry and the selective incorporation of three ammonium cations, there is little doubt about the quadruplex structure of [(dTG4T)4•(NH4+)3]5-. The major finding is a fingerprint of hydrogen bonding in the gas phase in the guanine C6=O6 stretching mode, that allows probing the conservation of G-quartets in the gas phase. Further experiments also demonstrate the conservation of G-quadruplex hydrogen bonds in the human telomeric sequence d(TTAGGG)4 [Gabelica et al., JACS, accepted]. Second, we also studied DNA duplexes and G-quadruplex ions in the gas phase by UV spectroscopy. We recorded the UV spectra of the (dTG4T)4 quadruplex, with and without ammonium ions. Molecular modeling [Rueda et al., JACS, 2006, p3608] and ion mobility spectrometry data [Gabelica et al., JACS, 2007, 895] showed that G-quadruplexes keep their hydrogen-bonded structure but become more floppy if inner cations are removed. We found that the UV spectra differ dramatically with and without inner cations, suggesting that UV spectroscopy is very sensitive to stacking interactions between neighboring G-quartets. We also used UV spectroscopy to probe the structure of 12-mer DNA duplexes, by comparing the duplex spectra to those obtained on single strands. Preliminary results show that stacking interactions may be preserved in duplexes containing GC base pairs, but not in duplexes containing AT base pairs. Altogether, these results show the complementarities between IR and UV spectroscopy to characterize DNA structures in the gas phase: IR data mainly give access to information on hydrogen bonding of bases, and UV spectroscopy provides information on stacking interactions

IR and UV spectroscopy of DNA ions stored in a quadrupole ion trap mass spectrometer

Electrospray mass spectrometry (ESI-MS) can be used to transfer large biomolecular complexes from the solution to the gas phase. However, a longstanding question is whether the gas-phase multiply-charged ions produced by ESI-MS keep a folded conformation in the absence of solvent. Nucleic acid secondary structures are determined by hydrogen bonding interactions between nucleic bases and by stacking interactions between neighboring base pairs. In solution, infrared (IR) and ultraviolet (UV) spectroscopies provide information on hydrogen bonding and stacking interactions in nucleic acids, respectively. Here we will show how IR and UV spectra of gas-phase ions can be recorded, and what can be learned on the structure of nucleic acids (double helices and quadruple helices) in the gas phase. The IR spectroscopy experiments on DNA negative ions were carried out at the CLIO free electron laser (FEL) center using an electrospray quadrupole ion trap mass spectrometer (Esquire 3000, Bruker Daltonics, Germany) modified to inject the IR beam in the trap through the ring electrode. IRMPD spectra are recorded by monitoring the relative fragmentation efficiency of mass-selected parent ions as a function of the excitation wavenumber, in the range 1000-2000 cm-1. Data interpretation on these large biomolecule ions is made using carefully chosen control experiments. The major finding is a fingerprint of hydrogen bonding in the gas phase in the guanine C6=O6 stretching mode, that allows probing the conservation of G-quartets in the gas phase. The experiments demonstrate the conservation of G-quadruplex hydrogen bonds in the human telomeric sequence d(TTAGGG)4. The UV spectroscopy experiments were carried out using a tunable OPO laser (Continuum Lasers, Santa Clara, CA, USA) with frequency doubling. The laser is interfaced with a Finnigan LCQ ESI-QIT mass spectrometer. The UV action spectra were recorded by monitoring electron detachment from DNA multiply charged anions as a function of the wavelength between 220 and 300 nm. Preliminary results suggest that stacking interactions are preserved in duplexes containing GC base pairs, and in G-quadruplexes containing inner cations