Yves Boulard

Cell uptake of a biosensor detected by hyperpolarized 129Xe NMR: The transferrin case

For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructs based on the non-selective grafting of cryptophane precursors on holo-transferrin. This biological system was chosen because there are many receptors on the cell surface, and endocytosis further increases this density. The study of these biosensors with K562 cell suspensions via fluorescence microscopy and (129)Xe NMR indicates a strong interaction, as well as interesting features such as the capacity of xenon to enter the cryptophane even when the biosensor is endocytosed, while keeping a high level of polarization. Despite a lack of specificity for transferrin receptors, undoubtedly due to the hydrophobic character of the cryptophane moiety that attracts the biosensor into the cell membrane, these biosensors allow the first in-cell probing of biological events using hyperpolarized xenon.

Cadmium – glutathione solution structures provide new insights into heavy metal detoxification

Cadmium is a heavy metal and a pollutant that can be found in large quantities in the environment from industrial waste. Its toxicity for living organisms could arise from its ability to alter thiol‐containing cellular components. Glutathione is an abundant tripeptide (γ‐Glu‐Cys‐Gly) that is described as the first line of defence against cadmium in many cell types. NMR experiments for structure and dynamics determination, molecular simulations, competition reactions for metal chelation by different metabolites (γ‐Glu‐Cys‐Gly, α‐Glu‐Cys‐Gly and γ‐Glu‐Cys) combined with biochemical and genetics experiments have been performed to propose a full description of bio‐inorganic reactions occurring in the early steps of cadmium detoxification processes. Our results give unambiguous information about the spontaneous formation, under physiological conditions, of the Cd(GS)2 complex, about the nature of ligands involved in cadmium chelation by glutathione, and provide insights on the structures of Cd(GS)2 complexes in solution at different pH. We also show that γ‐Glu‐Cys, the precursor of glutathione, forms a stable complex with cadmium, but biological studies of the first steps of cadmium detoxification reveal that this complex does not seem to be relevant for this purpose.

Structural Determinants for Protein adsorption/non-adsorption to Silica Surface

The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nanotechnology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine) in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many π-π interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption.

Smart Detection of Toxic Metal Ions, Pb2+ and Cd2+, Using a 129Xe NMR-Based Sensor

An approach for sensitive magnetic resonance detection of metal cations is proposed. Combining the use of hyperpolarized (129)Xe NMR and of a cage-molecule functionalized by a ligand able to chelate different cations, we show that simultaneous detection of lead, zinc, and cadmium ions at nanomolar concentration is possible in short time, thanks to fast MRI sequences based on the HyperCEST scheme.

Effect of pH and Counterions on the Encapsulation Properties of Xenon in Water-Soluble Cryptophanes

In the (129)Xe NMR-based biosensing approach in which the hyperpolarized noble gas is transported to biological receptors for a sensitive molecular imaging, cryptophanes are excellent xenon host systems. However to avoid formation of self-organized systems, these hydrophobic cage molecules can be rendered water soluble by introduction of ionic groups. We show that the sensitivity of xenon to its local environment and the presence of these ionic functions can lead to interesting properties. For a first water-soluble cryptophane derivative, we show that a precise monitoring of the local pH can be performed. For a second cryptophane, the presence of ionic groups close to the cryptophane cavity modifies the xenon binding constant and in-out exchange rate. The latter allows the tuning of physical properties of xenon-cryptophane interactions without resorting to a change of the cavity size. These results open new perspectives on the influence of chemical modifications of cryptophanes for optimizing the biosensor properties.

Selective and reversible effects of vinca alkaloids on Trypanosoma cruzi epimastigote forms: Blockage of cytokinesis without inhibition of the organelle duplication

Vinca alkaloids, vincristine and vinblastine, produce differential effects on the cell division of Trypanosoma cruzi epimastigote forms depending on drug concentrations. These effects are related to different microtubule-based mechanisms. For 15 microM vinblastine and 50 microM vincristine, the drugs inhibit both nuclear division and cytokinesis, and affect cell shape. At 3 microM vinblastine and 10 microM vincristine, however, cytokinesis is inhibited without major effect on the progression of the cell cycle; this yields giant cells having multiple nuclei, kinetoplasts and flagella. Cultures maintained over 1 week with daily drug replacement produced cells with more than 16 nuclei and 24 kinetoplasts, indicating that an equivalent of a fifth cell cycle was initiated. The ultrastructure of the multinucleate cells showed a basic organization closely similar to that of trypanosomes. Cytokinesis inhibition by vinca alkaloids seems to result from modulations of interactions between microtubules and associated proteins, rather than from an inhibition of microtubule dynamics as is usually proposed for vinca alkaloids. Cytokinesis inhibition is reversible: after removing the drug, epimastigotes emerge from the multinucleate cells. The emerging process follows a precise axis and polarity which are determined by the position of the flagellum/kinetoplast complex. This region could play an essential role in cell morphogenesis since zoids (cells without a nucleus) are frequently observed.

Hyperpolarized 129Xe NMR signature of living biological cells

We show that the differentiation between internal and external compartments of various biological cells in suspension can be made via simple NMR spectra of hyperpolarized 129Xe. The spectral separation between the signals of 129Xe in these two compartments is already known for red blood cells, because of the strong interaction of the noble gas with hemoglobin. The observation of two separate peaks in the 200‐ppm region can be seen with both eukaryotic and prokaryotic cells, some of which are not known to contain paramagnetic proteins in large quantities. Using different experiments in which the cells are lysed, swell or are blocked in G2 phase, we demonstrate that the low‐field‐shifted peak observed corresponds to xenon in the aqueous pool inside the cells and not in the membranes. The presence of this additional peak is a clear indication of cell integrity, and its integration allows the quantification of the total cell volume. The relaxation time of intracellular xenon is sufficiently long to open up promising perspectives for cell characterization. The exchange time between the inner and outer cell compartments (on the order of 30 ms) renders possible the targeting of intracellular receptors, whereas the observation of chemical shift variations represents a method of revealing the presence of toxic species in the cells. Copyright

A transcriptional-switch model for Slr1738-controlled gene expression in the cyanobacterium Synechocystis

BackgroundProtein-DNA interactions play a crucial role in the life of biological organisms in controlling transcription, regulation, as well as DNA recombination and repair. The deep understanding of these processes, which requires the atomic description of the interactions occurring between the proteins and their DNA partners is often limited by the absence of a 3D structure of such complexes.ResultsIn this study, using a method combining sequence homology, structural analogy modeling and biochemical data, we first build the 3D structure of the complex between the poorly-characterized PerR-like regulator Slr1738 and its target DNA, which controls the defences against metal and oxidative stresses in Synechocystis. In a second step, we propose an expanded version of the Slr1738-DNA structure, which accommodates the DNA binding of Slr1738 multimers, a feature likely operating in the complex Slr1738-mediated regulation of stress responses. Finally, in agreement with experimental data we present a 3D-structure of the Slr1738-DNA complex resulting from the binding of multimers of the FUR-like regulator onto its target DNA that possesses internal repeats.ConclusionUsing a combination of different types of data, we build and validate a relevant model of the tridimensional structure of a biologically important protein-DNA complex. Then, based on published observations, we propose more elaborated multimeric models that may be biologically important to understand molecular mechanisms.

The chemotherapy of rodent malaria, XXXIV.: Causal prophylaxis Part III: Ultrastructural changes induced in exo-erythrocytic schizonts of Plasmodium yoelii yoelii by primaquine

Wistar rats were injected intravenously with sporozoites from Anopheles stephensi infected with Plasmodium yoelii yoelii. The animals were subsequently treated with primaquine at doses of 30, 50 or 100 mg kg-1 at various times. Liver biopsies were made and the exoerythrocytic schizonts examined by light and electron microscopy between 45 and 50 hours. The action of the drug appeared to be principally on the parasite mitochondria, the membranes of which became thickened, then proliferated into multiple layers. Finally the whole organelles swelled up losing all structural organisation. Other parasite membranes were affected similarly and the peripheral enzyme granules disappeared. Following treatment the small, disrupted schizonts were apparently absorbed by their host cells which appeared unaffected by primaquine.

Host Cell and Malarial Targets for Docetaxel (Taxotere™) during the Erythrocytic Development of Plasmodium falciparum

The microtubular stabilizing agent docetaxel (Taxotere™) is known to inhibit the intraerythrocytic development of Plasmodium falciparum. To investigate the mechanism(s) of inhibition, we analyzed the structural organization of the mitotic spindle by immunofluorescence and electron microscopy. When 30 μM docetaxel was applied for five hours on ring forms, alterations in the mitotic spindles leading to abnormal nuclear divisions were observed. At the trophozoite‐ and schizont‐stage, docetaxel pulses prevent mitosis by stabilizing microtubular structures associated with the mitotic apparatus, giving abnormal spindles. However, this inhibition did not interfere with parasite DNA synthesis indicating the absence of a checkpoint that couples exit from mitosis with proper spindle assembly as observed in higher eukaryotic cells. In parallel, intraerythrocytic concentration of docetaxel was measured in parasitized erythrocytes, after incubation of cells with 3H‐docetaxel for five hours. It was found to be 14‐fold increased at the ring‐stage of infected erythrocytes compared to normal ones, 170‐fold increased at the trophozoite‐stage and 1,500‐fold increased at the schizont‐stage. Our data show that, even though the overall intracellular concentration of docetaxel is low in docetaxel‐pulsed rings, the agent might be sufficient to disturb the spindle organization. However, the existence of targets for docetaxel other than mitotic spindle microtubules. i.e. erythrocyte membrane components, could interfere with mitotic spindle formation