Chips from chips: application to the study of antibody responses to methylated proteins

Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.     

Variability in the bioluminescence response of the dinoflagellate Pyrocystis lunula

Light emission in dinoflagellates is induced by water motions. But although it is known that mechanical stimulations of these organisms trigger the bioluminescent response, the exact mechanism that involves some cell membrane excitations by fluid motions is not yet fully understood and is still controversial. We show in this experimental study that the accelerated shear flow, created by abrupt rotations of one or both co-axial cylinders of a Couette shearing chamber excites the light emission from cultured dinoflagellates Pyrocystis lunula. Following our first results published earlier that state that pure laminar shear does not excite the main bioluminescent response in dinoflagellates, our present experiments show that both shear and acceleration in the flow are needed to trigger the bioluminescent response. Besides, the probability to stimulate this bioluminescent response under acceleration and shear is deduced from the response curves. This response follows a Gaussian distribution that traduces a heterogeneity in individual cell thresholds for the stimulation of bioluminescence in a dinoflagellate population. All these results will have a repercussion in the possible applications of dinoflagellate bioluminescence in flow visualizations and measurements. Moreover, this study opens a new way in studying mechanically-induced stimulus thresholds at the cell level.     

Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia

Globozoospermia is a rare (incidence <0.1% in male infertile patients) form of teratozoospermia, mainly characterized by round-headed spermatozoa that lack an acrosome. It originates from a disturbed spermiogenesis, which is expected to be induced by a genetic factor. Several family cases and recessive mouse models with the same phenotype support this expectation. In this study, we present a consanguineous family with three affected brothers, in whom we have identified a homozygous mutation in the spermatogenesis-specific gene SPATA16. This is the first example of a nonsyndromic male infertility condition in humans caused by an autosomal gene defect, and it could also mean that the identification of other partners like SPATA16 could elucidate acrosome formation.     

Genetic variability in nodulation and root growth affects nitrogen fixation and accumulation in pea

BACKGROUND AND AIMS: Legume nitrogen is derived from two different sources, symbiotically fixed atmospheric N(2) and soil N. The effect of genetic variability of root and nodule establishment on N acquisition and seed protein yield was investigated under field conditions in pea (Pisum sativum). In addition, these parameters were related to the variability in preference for rhizobial genotypes. METHODS: Five different spring pea lines (two hypernodulating mutants and three cultivars), previously identified in artificial conditions as contrasted for both root and nodule development, were characterized under field conditions. Root and nodule establishment was examined from the four-leaf stage up to the beginning of seed filling and was related to the patterns of shoot dry matter and nitrogen accumulation. The genetic structure of rhizobial populations associated with the pea lines was obtained by analysis of nodule samples. The fraction of nitrogen derived from symbiotic fixation was estimated at the beginning of seed filling and at physiological maturity, when seed protein content and yield were determined. KEY RESULTS: The hypernodulating mutants established nodules earlier and maintained them longer than was the case for the three cultivars, whereas their root development and nitrogen accumulation were lower. The seed protein yield was higher in 'Athos' and 'Austin', the two cultivars with increased root development, consistent with their higher N absorption during seed filling. CONCLUSION: The hypernodulating mutants did not accumulate more nitrogen, probably due to the C cost for nodulation being higher than for root development. Enhancing exogenous nitrogen supply at the end of the growth cycle, by increasing the potential for root N uptake from soil, seems a good option for improving pea seed filling.     

Molecularly imprinted polymer films for reflectometric interference spectroscopic sensors

Reflectometric interference spectroscopic measurements were performed on molecularly imprinted polymer (MIP) films with the herbicide atrazine as the template molecule. A conventional imprinting protocol was used relying on non-covalent interactions between the functional monomers and the template. The MIPs were deposited on glass transducers by two different methods: spin-coating followed by in situ polymerization of thin films of monomers containing a sacrificial polymeric porogen, and autoassembly of MIP nanoparticles with the aid of an associative linear polymer. Reproducible results were obtained upon measurements of atrazine solutions in toluene with both films. Atrazine concentrations as low as 1.7 ppm could be detected with the autoassembled particle film. No or very little analyte adsorption was observed onto non-imprinted control films made by spin-coating and by particle assembly, respectively. We believe that these MIP layers in combination with the general reflectrometric transduction scheme could be a versatile sensing tool for the detection of environmentally important and other analytes.     

Improved agarose gel assay for quantification of growth factor-induced cell motility

Cell chemotaxis is frequently required in normal or pathological situations such as invasion, metastasis, and tumor angiogenesis and may involve many different cell types. At present, no device can simultaneously (i) make morphological observations, (ii) quantify cell migration, (iii) test multiple chemoattracting gradients, and (iv) analyze cell-cell interactions. We developed an agarose-based assay to address these questions. Two glass molds were designed, around which agarose gel could be poured to form specific well shapes. Using a vital nuclear stain (Hoechst 33258), we characterized the migration profile of adherent or suspension cells. Cells could be observed during the entire migration process. We were able to follow cells moving toward chemoattractants or being repulsed by other molecules, and we could estimate average migration speed. Using this inexpensive assay, we were able to obtain precise, reproducible results concerning the chemotactic behavior of different cell types. The resulting data differentiated between chemokinetic and chemotactic movement. Chemotactic potencies could be compared using different criteria, such as the number of attracted cells, induced speed, and morphological aspect. This improved agarose assay appears to be a reliable and inexpensive alternative to other available chemotaxis study tools.