Papers by Béatrice Schaack
This paper highlights the way in which eukaryotic cell and bacteria based biochips are relevant f... more This paper highlights the way in which eukaryotic cell and bacteria based biochips are relevant for nanotoxicological risk evaluation. Here we define NP-biochips as formatted surfaces containing nanoparticles (NPs). They are simple devices which can easily be used to generate quantitative data expressing the effects of NPs on biological material in parallelized medium throughput assays. Firstly we dropped NPs and bacteria onto an agarose layer, using fluorescent bacteria in order to follow by imaging the effects of these NPs on bacterial growth. Secondly we embedded the targeted NPs at precise spot locations in a matrix on which eukaryotic cells can adhere, and followed cell growth. We used titanium dioxide as model NPs for the concept validation. Both types of NP-biochip are realized in order to pattern NPs in 50 or 100 dried 400 mm diameter spots on a glass plate, with less than 0.3% variation in concentration between spots. For anatase TiO 2 NPs, we were able to record a non-toxic effect by measuring bacteria or eukaryotic cell survival. NPs are not internalized in bacteria; we thus used hyperspectral imaging to observe NPs on their surfaces. In contrast, NPs translocate in eukaryotic cells so we used fluorescent TiO 2 and quantum dots to verify that NPs migrate from the NP-biochip matrix into bronchial cells. In order to illustrate the release of NP from the chip into the cell, we present the dose–response curve in the case of a toxic rutile TiO 2 NP. These devices prevent cell and bacteria suffocation that is often observed in standard assays in wells due to NP precipitation. We believe that these tests realized on gel coated biochips are a rather realistic model for NP exposure in situ, imitating bacterial growth in biofilms and eukaryotic cells in tissues.
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Methods in Enzymology, 2015
Analytical ultracentrifugation is a key tool to assess homogeneity of membrane protein samples, t... more Analytical ultracentrifugation is a key tool to assess homogeneity of membrane protein samples, to determine protein association state and detergent concentration, and to characterize protein-protein equilibrium. Combining absorbance and interference detections gives information on the amount of the detergent and lipid bound to proteins. Changing the solvent density affects specifically the buoyancy of each of the different components, and can also be used to gain information on particle composition and interaction. We will present the related tools, recently implemented in the softwares Sedphat (sedfitsedphat.nibib.nih.gov/software) and Gussi (http://biophysics.swmed.edu/MBR/software.html), which help to measure the amount of detergent bound to the protein, and ascertain the protein association state within the protein-detergent complex. In addition, fluorescence detection allows focusing specifically on a labeled component within a complex mixture. We present two examples of sedimentation velocity experiments, allowing on one hand to evidence complex formation between an unpurified GFP-labeled protein and a membrane protein, and on the other hand to characterize fluorescent lipid vesicles. Small-angle X-ray and neutron scattering are techniques that give insights into the structure and conformation of macromolecules in solution. However, the detergents used to purify membrane protein are often imperfectly masked due to their amphipathic character. Particular strategies addressing membrane proteins were recently proposed, which are shortly presented.
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Current Opinion in Pharmacology, 2009
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Journal de physiologie
The relative importance of synaptic versus paracrine dopamine transmission for the occurrence of ... more The relative importance of synaptic versus paracrine dopamine transmission for the occurrence of functional effects following intrastriatal grafting is not fully established. In the present study we grafted cell lines, expressing the form I of human tyrosine hydroxylase after infection with a recombinant retrovirus and selection in tyrosine-free-medium, to the denervated striatum in order to analyse the extent to which extracellular dopamine levels can be restored and the effect of a diffuse release of dopamine on motor impairement in a rat model of Parkinson's disease. In petri dish, the modified fibroblast cells (NIH.3T3) release DOPA constitutively whereas the modified endocrine cells (RIN) store and release dopamine in a regulated way. Interestingly, in denervated striatum, grafts of modified fibroblast cells produce DOPA which was efficiently converted into dopamine by the host striatal tissue. In the grafted striatum, both fibroblast and endocrine cells restore subnormal levels of diffuse release of dopamine which is notably unaffected and stimulated, respectively, by high concentration of potassium, in connection with the in vitro properties of the grafted cells. The intrastriatal grafts of modified cells partially reversed the apomorphine-induced but not the amphetamine-induced motor asymmetry. We discuss the implications of these results in the context of Parkinson disease.
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Molecular Pharmacology
The mammalian N-methyl-D-aspartate (NMDA) receptor complex is though to consist of an NR1 subunit... more The mammalian N-methyl-D-aspartate (NMDA) receptor complex is though to consist of an NR1 subunit in combination with one or more of the four NR2 subunits (A, B, C, and D). When corresponding cDNAs are expressed in Xenopus oocytes, ion channels with the characteristic profile of NMDA receptors are formed. The receptor is unique in requiring two coagonists, glutamate and glycine, for activation of the channel. We have used site-directed mutagenesis to study amino acids in the human NR1 subunit that contribute to the glycine binding site of the NMDA receptor without affecting the agonist site for glutamate. Mutations to D481 and K483 produced receptors with up to 160-fold lower affinities for glycine, as well as other agonists and partial agonists, without affecting maximum current size or the degree of agonist efficacy. The D481A mutation also led to 40-50-fold lower affinities for two structurally diverse glycine site antagonists. From these data we propose that the carboxyl group of this aspartate interacts with the amino moiety of glycine and the equivalent group contained in other agonists and antagonists.
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Journal of Biological Chemistry
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Neuroscience
The expression of the messenger RNAs encoding N-methyl-D-aspartate receptor subunits in neurologi... more The expression of the messenger RNAs encoding N-methyl-D-aspartate receptor subunits in neurologically normal post-mortem human brain was studied by in situ hybridization. In the caudate, putamen and nucleus accumbens strong hybridization signals were observed for N-methyl-D-aspartate R1-1 messenger RNA but much weaker signals for N-methyl-D-aspartate R1-3 and N-methyl-D-aspartate R1-4, N-Methyl-D-aspartate R1-2 was not detectable. N-methyl-D-aspartate R2B was the only N-methyl-D-aspartate R2 subunit detected in these nuclei. In the hippocampus the messenger RNAs for both N-methyl-D-aspartate R1-1 and N-methyl-D-aspartate R1-4 were strongly expressed in the dentate gyrus, CA3-CA1 pyramidal cells, subiculum, entorhinal cortex and perirhinal cortex. Much lower expression was seen for N-methyl-D-aspartate R1-2 and N-methyl-D-aspartate R1-3. The messenger RNAs for both N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2B, but not N-methyl-D-aspartate R2C, subunits were expressed in the...
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Miniaturization of biology-dedicated microsystems, called labs-on-a-chip, provides a better sensi... more Miniaturization of biology-dedicated microsystems, called labs-on-a-chip, provides a better sensitivity for molecular or cellular detection and reduces the volume of solvent and the quantity of handled active principles. However, the resulting small number of molecules or cells and the high surface-to-volume ratio can lead to significant contamination and stiction problems, respectively. Such phenomena raise major issues for samples in the micro- to picolitre (μl-pl) range. For these reasons, contactless handling methods are widely investigated these days. Diamagnetic levitation is one of the rare natural repulsive forces capable of compensating gravity. Although this kind of repulsion is negligible at our scale, at the microscale (< ~1 mm) it becomes significant and diamagnetic levitation of micro-objects above micro-magnets can be achieved. Widely investigated at the macroscale with superconducting coils [1] or bulk magnets [2], diamagnetic levitation has been little explored a...
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Biosensors & Bioelectronics, 2005
In microarrays experiments, a serious limitation is the unreliability of low signal intensities d... more In microarrays experiments, a serious limitation is the unreliability of low signal intensities data and the lack of reproducibility for the resulting ratios between samples and controls. Most of the light emitted by a fluorophore at the air/glass interface of a glass slide is absorbed by the glass so just a part of the emitted fluorescence is detected. To improve
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European journal of biochemistry / FEBS, 1992
Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia ... more Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data...
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Alternatives to laboratory animals : ATLA, 2006
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European Journal of Biochemistry, 1993
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European Journal of Biochemistry, 1991
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Proceedings of the National Academy of Sciences, 1999
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Proceedings of the National Academy of Sciences, 1992
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Papers by Béatrice Schaack