Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering.

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.

Surface modification of biomaterials for conjugation with human fetal osteoblasts.

Surface functionalization of hydroxyapatite (HA) and beta-tricalcium phosphate (TCP) bioceramics with chemical ligands containing a pyrrogallol moiety was developed to improve the adhesion of bone cell precursors to the biomaterials. Fast and biocompatible copper-free click reaction with azido-modified human fetal osteoblasts resulted in improved cell binding to both HA and TCP bioceramics, opening the way for using this methodology in the preparation of cell-engineered bone implants.

T cell receptor-ligand interactions: a conformational preequilibrium or an induced fit.

Kinetic parameters of T cell receptor (TCR) interactions with its ligand have been proposed to control T cell activation. Analysis of kinetic data obtained has so far produced conflicting insights; here, we offer a consideration of this problem. As a model system, association and dissociation of a soluble TCR (sT1) and its specific ligand, an azidobenzoic acid derivative of the peptide SYIPSAEK-(ABA)I (residues 252-260 from Plasmodium berghei circumsporozoite protein), bound to class I MHC H-2K(d)-encoded molecule (MHCp) were studied by surface plasmon resonance. The association time courses exhibited biphasic patterns. The fast and dominant phase was assigned to ligand association with the major fraction of TCR molecules, whereas the slow component was attributed to the presence of traces of TCR dimers. The association rate constant derived for the fast phase, assuming a reversible, single-step reaction mechanism, was relatively slow and markedly temperature-dependent, decreasing from 7.0 x 10(3) at 25 degrees C to 1.8 x 10(2) M(-1).s(-1) at 4 degrees C. Hence, it is suggested that these observed slow rate constants are the result of unresolved elementary steps of the process. Indeed, our analysis of the kinetic data shows that the time courses of TCR-MHCp interaction fit well to two different, yet closely related mechanisms, where an induced fit or a preequilibrium of two unbound TCR conformers are operational. These mechanisms may provide a rationale for the reported conformational flexibility of the TCR and its unusual ligand recognition properties, which combine high specificity with considerable crossreactivity.

Differential stress reaction of human colon cells to oleic-acid-stabilized and unstabilized ultrasmall iron oxide nanoparticles.

Therapeutic engineered nanoparticles (NPs), including ultrasmall superparamagnetic iron oxide (USPIO) NPs, may accumulate in the lower digestive tract following ingestion or injection. In order to evaluate the reaction of human colon cells to USPIO NPs, the effects of non-stabilized USPIO NPs (NS-USPIO NPs), oleic-acid-stabilized USPIO NPs (OA-USPIO NPs), and free oleic acid (OA) were compared in human HT29 and CaCo2 colon epithelial cancer cells. First the biophysical characteristics of NS-USPIO NPs and OA-USPIO NPs in water, in cell culture medium supplemented with fetal calf serum, and in cell culture medium preconditioned by HT29 and CaCo₂ cells were determined. Then, stress responses of the cells were evaluated following exposure to NS-USPIO NPs, OA-USPIO NPs, and free OA. No modification of the cytoskeletal actin network was observed. Cell response to stress, including markers of apoptosis and DNA repair, oxidative stress and degradative/autophagic stress, induction of heat shock protein, or lipid metabolism was determined in cells exposed to the two NPs. Induction of an autophagic response was observed in the two cell lines for both NPs but not free OA, while the other stress responses were cell- and NP-specific. The formation of lipid vacuoles/droplets was demonstrated in HT29 and CaCo₂ cells exposed to OA-USPIO NPs but not to NS-USPIO NPs, and to a much lower level in cells exposed to equimolar concentrations of free OA. Therefore, the induction of lipid vacuoles in colon cells exposed to OA utilized as a stabilizer for USPIO NPs is higly amplified compared to free OA, and is not observed in the absence of this lipid in NS-USPIO NPs.

On the diurnal soil water content dynamics during evaporation using dielectric methods

The water content dynamics in the upper soil surface during evaporation is a key element in land-atmosphere exchanges. Previous experimental studies have suggested that the soil water content increases at the depth of 5 to 15 cm below the soil surface during evapo- ration, while the layer in the immediate vicinity of the soil surface is drying. In this study, the dynamics of water content profiles exposed to solar radiative forcing was monitored at a high temporal resolution using dielectric methods both in the presence and absence of evaporation. A 4-d comparison of reported moisture content in coarse sand in covered and uncovered buckets using a commercial dielectric-based probe (70 MHz ECH2O-5TE, Decagon Devices, Pullman, WA) and the standard 1-GHz time domain reflectometry method. Both sensors reported a positive correlation between temperature and water content in the 5- to 10-cm depth, most pronounced in the morning during heating and in the afternoon during cooling. Such positive correlation might have a physical origin induced by evaporation at the surface and redistribution due to liquid water fluxes resulting from the temperature- gradient dynamics within the sand profile at those depths. Our experimental data suggest that the combined effect of surface evaporation and temperature-gradient dynamics should be considered to analyze experimental soil water profiles. Additional effects related to the frequency of operation and to protocols for temperature compensation of the dielectric sensors may also affect the probes' response during large temperature changes.

The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface.

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.