Analysis of an intact G-protein coupled receptor by MALDI-TOF mass spectrometry: molecular heterogeneity of the tachykinin NK-1 receptor

Integral membrane proteins are among the most challenging targets for biomedical research as most important cellular functions are tied to these proteins. To analyze intrinsically their structure/function, their transduction mechanism, or both, these proteins are commonly expressed in cultured cells as recombinant proteins. However, it is not possible to check whether these recombinant proteins are homogeneously or heterogeneously expressed. Owing to difficulties in their purification, very few mass spectrometry studies have been performed with those proteins and even less with G-protein coupled receptors. Here we have set up a procedure that is highly compatible with MALDI-TOF mass spectrometry to analyze an intact histidine-tagged G-protein coupled, namely, the tachykinin NK-1 receptor expressed in CHO cells, solubilized and purified using cobalt or nickel chelating magnetic beads. The metal-chelating magnetic beads containing the receptor were directly spotted on the MALDI plate for analysis. SDS-PAGE, combined with in-gel digestion analyzed by mass spectrometry, Western blot ((His)6 and FLAG M2 tags), photoaffinity labeling with a radioactive agonist, and Edman sequencing, confirmed the identity of the purified protein as the human tachykinin NK-1 receptor. Mass spectrometry study of both the glycosylated and deglycosylated intact protein forms revealed the existence of several receptor species that is tempting to correlate with the unusual pharmacological behavior of the receptor.     

Solid fraction modelling for CO2 and CO2–THF hydrate slurries used as secondary refrigerants

In the present paper, the suitability of hydrate slurries in secondary refrigeration was investigated by the means of a new hydrate solid-fraction model. Considering the high melting enthalpy of CO₂-containing hydrates, slurries presenting high hydrate solid fractions can carry sufficient latent heat to be useful for a two-phase secondary-refrigerant application. The model presented in this paper allowed to calculate the solid fraction of CO₂ and CO₂–THF hydrate from thermodynamic conditions of pressure and temperature. Contrary to a previous work on single CO₂ hydrates in a closed system, the present model can take into account hydrate mixture and is well adapted to additional CO₂ injections (opened system). By relying on the hydrate-conversion model results, the study of hydrates in suspension in a carrying liquid was also studied in an experimental loop and was based on a formation process by CO₂ injection in a cooled aqueous solution.     

Deciphering Differential Life Stage Radioinduced Reproductive Decline in Caenorhabditis elegans through Lipid Analysis

Wildlife is chronically exposed to various sources of ionizing radiations, both environmental or anthropic, due to nuclear energy use, which can induce several defects in organisms. In invertebrates, reproduction, which directly impacts population dynamics, has been found to be the most radiosensitive endpoint. Understanding the underlying molecular pathways inducing this reproduction decrease can help in predicting the effects at larger scales (i.e., population). In this study, we used a life stage dependent approach in order to better understand the molecular determinants of reproduction decrease in the roundworm C. elegans. Worms were chronically exposed to 50 mGy·h⁻¹ external gamma ionizing radiations throughout different developmental periods (namely embryogenesis, gametogenesis, and full development). Then, in addition to reproduction parameters, we performed a wide analysis of lipids (different class and fatty acid via FAMES), which are both important signaling molecules for reproduction and molecular targets of oxidative stress. Our results showed that reproductive defects are life stage dependent, that lipids are differently misregulated according to the considered exposure (e.g., upon embryogenesis and full development) and do not fully explain radiation induced reproductive defects. Finally, our results enable us to propose a conceptual model of lipid signaling after radiation stress in which both the soma and the germline participate.     

The Lack of Amyloidogenic Activity Is Persistent in Old WT and APPswe/PS1ΔE9 Mouse Retinae

We have previously reported that vision decline was not associated with amyloidogenesis processing in aging C57BL/6J wild-type (WT) mice and in a mouse model of Alzheimer’s disease, the APP_swe/PS1ΔE9 transgenic mouse model (APP/PS1). This conclusion was drawn using middle-aged (10–13 months old) mice. Here, we hypothesized that compared with hippocampal and cortical neurons, the weak amyloidogenic activity of retinal neurons may result in a detectable release of amyloid β (Aβ) only in aged mice, i.e., between 14 and 24 months of age. The aim of the present study was thus to follow potential activity changes in the amyloidogenic and nonamyloidogenic pathways of young (4 months) and old (20–24 months) WT and APP/PS1 mice. Our results showed that in spite of retinal activity loss reported by electroretinogram (ERG) recordings, the level of amyloid beta precursor protein (APP) and its derivatives did not significantly vary in the eyes of old vs. young mice. Strikingly, the ectopic expression of human APP_swe in APP/PS1 mice did not allow us to detect Aβ monomers at 23 months. In contrast, Aβ was observed in hippocampal and cortical tissues at this age but not at 4 months of life. In contrast, optic nerve transection-induced retinal ganglion cell injury significantly affected the level of retinal APP and the secretion of soluble APP alpha in the vitreous. Collectively, these results suggest that the amyloidogenic and nonamyloidogenic pathways are not involved in visual function decline in aging mice. In WT and APP/PS1 mice, it is proposed that retinal neurons do not have the capacity to secrete Aβ in contrast with other cortical and hippocampal neurons.     

Study of the Role of the Tyrosine Kinase Receptor MerTK in the Development of Kidney Ischemia-Reperfusion Injury in RCS Rats

Renal ischaemia reperfusion (I/R) triggers a cascade of events including oxidative stress, apoptotic body and microparticle (MP) formation as well as an acute inflammatory process that may contribute to organ failure. Macrophages are recruited to phagocytose cell debris and MPs. The tyrosine kinase receptor MerTK is a major player in the phagocytosis process. Experimental models of renal I/R events are of major importance for identifying I/R key players and for elaborating novel therapeutical approaches. A major aim of our study was to investigate possible involvement of MerTK in renal I/R. We performed our study on both natural mutant rats for MerTK (referred to as RCS) and on wild type rats referred to as WT. I/R was established by of bilateral clamping of the renal pedicles for 30′ followed by three days of reperfusion. Plasma samples were analysed for creatinine, aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), kidney injury molecule -1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) levels and for MPs. Kidney tissue damage and CD68-positive cell requirement were analysed by histochemistry. monocyte chemoattractant protein-1 (MCP-1), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and histone 3A (H3A) levels in kidney tissue lysates were analysed by western blotting. The phagocytic activity of blood-isolated monocytes collected from RCS or WT towards annexin-V positive bodies derived from cultured renal cell was assessed by fluorescence-activated single cell sorting (FACS) and confocal microscopy analyses. The renal I/R model for RCS rat described for the first time here paves the way for further investigations of MerTK-dependent events in renal tissue injury and repair mechanisms.     

First direct synthesis of highly ordered bifunctionalized mesoporous silica thin films

Optically transparent and highly ordered mesoporous organosilica thin films functionalized with two different organic groups in various proportions were synthesized by templated-directed cocondensation of tetraethylorthosilicate (TEOS) and a mixture of two distinct and functional organotriethoxysilanes [NC(CH2)3Si(OEt)3 and O=P(OEt)2(CH2)3Si(OEt)3]. The mesostructured films obtained by evaporation induced self-assembly (EISA) approach were deposited on glass or silicon substrates by dip-coating. They were characterized by Grazing Incidence Small Angle X-ray Scattering (GISAXS) and X-ray reflectivity. We showed that whatever the proportion in organic groups, only 2D hexagonal phase having p6m symmetry was observed for all the materials indicating a good compatibility between the organic groups. The bi-functionalization of the internal pores surface by the organotriethoxysilanes groups was clearly evidenced by using micro-Raman spectroscopy.     

Thin chromium nitride PVD coatings on stainless steel for conductive component as bipolar plates of PEM fuel cells: Ex-situ and in-situ performances evaluation

In this paper, two types of chromium PVD coatings (100 nm) have been elaborated on 316L stainless steel (SS) by adjusting the nitrogen flow rate. The first coating is a mixture of Cr₂N and Cr, the second one is a single phase CrN. It is shown that the performances of the material are strongly dependant of the nature of the passive film formed on the chromium nitride layers due to the galvanic coupling between the coating and the substrate. The CrN coated SS shows very good corrosion resistance in simulated PEMFC media. The surface conductivity of the SS is also greatly improved and the CrN coated SS shows an interfacial contact resistance of 10 mΩ cm² at 140 N cm⁻². Five single cells of stainless steel bipolar plates coated with the CrN film were assembled for performance test. This 5 cell stack does not show any mean voltage degradation over 200 h dynamic cycling. Moreover, the performances of the CrN coated SS bipolar plates are very close to the Au-coated SS bipolar plates.     

The effect of expectancy of a threatening event on time perception in human adults.

Two experiments were conducted to investigate the effect of a threatening stimulus in human adults in a temporal bisection task. In Experiment 1, for two anchor duration conditions (400/800 vs. 800/1600 ms), the participants completed trials in which the probe duration was followed by an aversive stimulus or a nonaversive stimulus. The results showed that the duration was judged longer when the participants expected an aversive rather than a nonaversive stimulus. In Experiment 2, the effect of the temporal localization of the aversive stimulus was also tested, with the aversive stimulus being presented at the beginning or at the end of the probe duration. The results revealed a temporal overestimation in each condition compared to the trials in which no aversive stimulus was presented. Furthermore, the temporal overestimation was greater when the expectation for the forthcoming threatening stimulus was longer. This temporal overestimation is explained in terms of a speeding-up of the neural timing system in response to the increase in the arousal level produced by the expectation of a threatening stimulus. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

激光诱导击穿光谱法连续测定高炉流道中的组分和温度

介绍了用于炼铁和炼钢领域过程控制的传感器。用激光诱导击穿光谱法（LIBS）连续测定高炉（BF）流道中的组分和温度,此传感器主要用于高炉流道的在线测量,监测了两次浇铸过程中元素硅、锰和碳的浓度,结果满意。