Development of miniaturized immunoassay: Influence of surface chemistry and comparison with enzyme-linked immunosorbent assay and Western blot

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.     

Vibrio cholerae ParE2 Poisons DNA Gyrase via a Mechanism Distinct from Other Gyrase Inhibitors

DNA gyrase is an essential bacterial enzyme required for the maintenance of chromosomal DNA topology. This enzyme is the target of several protein toxins encoded in toxin-antitoxin (TA) loci as well as of man-made antibiotics such as quinolones. The genome of Vibrio cholerae, the cause of cholera, contains three putative TA loci that exhibit modest similarity to the RK2 plasmid-borne parDE TA locus, which is thought to target gyrase although its mechanism of action is uncharacterized. Here we investigated the V. cholerae parDE2 locus. We found that this locus encodes a functional proteic TA pair that is active in Escherichia coli as well as V. cholerae. ParD2 co-purified with ParE2 and interacted with it directly. Unlike many other antitoxins, ParD2 could prevent but not reverse ParE2 toxicity. ParE2, like the unrelated F-encoded toxin CcdB and quinolones, targeted the GyrA subunit and stalled the DNA-gyrase cleavage complex. However, in contrast to other gyrase poisons, ParE2 toxicity required ATP, and it interfered with gyrase-dependent DNA supercoiling but not DNA relaxation. ParE2 did not bind GyrA fragments bound by CcdB and quinolones, and a set of strains resistant to a variety of known gyrase inhibitors all exhibited sensitivity to ParE2. Together, our findings suggest that ParE2 and presumably its many plasmid- and chromosome-encoded homologues inhibit gyrase in a different manner than previously described agents.     

Development of a real-time PCR assay for quantification of Acanthamoeba trophozoites and cysts

Free-living amoebae have been found to be a reservoir for various pathogenic bacteria in aquatic environments. For example, the Acanthamoeba genus renders possible the intracellular multiplication of Legionella pneumophila, which is responsible for legionellosis. It consequently matters to quantify Acanthamoeba cells and thereby enhance our assessment of the risk of contamination. The classical microbiological method of quantification relies on amoebae growth and most probable number calculation. We have developed a real-time PCR assay based on a TaqMan probe that hybridizes onto 18S rDNA. This probe is specific to the Acanthamoeba genus. The assay was successful with both the trophozoite and the cyst forms of Acanthamoeba. Highly sensitive, it proved to permit detection of fewer than 10 cells, even those that are not easily cultivable, such as the cyst forms.     

Using γ-emitting artificial radionuclides,released by nuclear plants,as markers of restricted movements by chub,Leuciscus cephalus,in a large river,the Lower Rhône

Synopsis On the basis that -emitting artificial radionuclides, present in nuclear plant radioactive wastes, can be considered as natural collective markers of fish living downstream from a radioactive discharge, we studied the restricted movements of chub in a reservoir on the Lower Rhône river where the Marcoule nuclear plant is located. A qualitative determination, based on the detection of specific radioelements in our samples originating from radioactive waste, and a quantitative determination of the cesium-137 concentration in the samples, were used to identify fish radioactively marked by the effluent. Individual measurements of -radioactivity in 49 adult chub captured at two stations, each 2 km long and 6 km apart, divided the fish into two distinct sub-units: one living downstream of the discharge pipe, in which 73% were marked, and the other upstream, in which 79% were not marked. Similar results were found in two neighbouring stations when we analysed previous radioecological measurements of -radioactivity in groups of chub. After combining all the data concerning chub, detailed information was obtained on the spatial stability of the chub population in the reservoir. Two spatially different stocks were found and each stock can be divided in two components: a sedentary component that remains in a restricted zone (its home range), and a mobile component that undertakes movements between the two zones.     

Design and biological activity of imidazole-containing peptidomimetics with a broad-spectrum antioxidant activity

Histidine-containing natural dipeptides, such as L-carnosine, were reported to be effective against different oxygen-derived free radicals, and also lipoperoxyl radicals. However, L-carnosine appeared to be uneffective in vivo, due to the presence of ubiquitous specific or semi-specific hydrolytic enzymes. Therefore, a series of peptidomimetics were synthesized in order to confer resistance to enzymatic hydrolysis. Some structural modifications were also done in an attempt to improve the antioxidant power of the molecule, for example, in relation to binding of ferrous ions. The following methods were used for the evaluation of peptidomimetics:– The resistance to enzymatic deactivation was determined using either a purified specific enzyme or a multi-enzymatic system.– The free radical scavenging potential was measured by different in vitro methods involving different free radical species and biological targets.– Deactivation of lipid hydroperoxides was monitored by HPLC and protection of membrane phospholipids and proteins was demonstrated.– The effectiveness of peptidomimetics in vivo was evaluated.It was shown that decarboxylation of L-carnosine results in an important improvement of the resistance towards hydrolytic enzymes. In vitro experiments have demonstrated, for a series of peptidomimetics, free radical scavenging and lipid hydroperoxide deactivating properties similar to or even better than the natural peptide (depending on the experimental design). In addition, the two peptidomimetics -alanylhistamine and L-prolylhistamine proved to be far superior in inhibiting the lipid hydroperoxide-mediated cross-linking of a representative protein. Finally, -alanylhistamine was able to protect skin enzymes from UV-induced degradation in vivo.     

Intraspecific Phylogeography of Lacerta vivipara and the Evolution of Viviparity

The lacertid lizard Lacerta vivipara is one of the few squamate species with two reproductive modes. We present the intraspecific phylogeny obtained from neighbor-joining and maximum-parsimony analyses of the mtDNA cytochrome b sequences for 15 individuals from Slovenian oviparous populations, 34 individuals from western oviparous populations of southern France and northern Spain, 92 specimens from European and Russian viviparous populations, and 3 specimens of the viviparous subspecies L. v. pannonica. The phylogeny indicates that the evolutionary transition from oviparity to viviparity probably occurred once in L. vivipara. The western oviparous group from Spain and southern France is phylogenetically most closely related to the viviparous clade. However, the biarmed W chromosome characterizing the western viviparous populations is an apomorphic character, whereas the uniarmed W chromosome, existing both in the western oviparous populations and in the geographically distant eastern viviparous populations, is a plesiomorphic character. This suggests an eastern origin of viviparity. Various estimates suggest that the oviparous and viviparous clades of L. vivipara split during the Pleistocene. Our results are discussed in the framework of general evolutionary models: the concept of an oviparity–viviparity continuum in squamates, the cold climate model of selection for viviparity in squamates, and the contraction–expansion of ranges in the Pleistocene resulting in allopatric differentiation.     

Two independent estimations of stand-level root respiration on clonal Eucalyptus stands in Congo: up scaling of direct measurements on roots versus the trenched-plot technique

Root respiration at the level of a forest stand, an important component of ecosystem carbon balance, has been estimated in the past using various methods, most of them being indirect and relying on soil respiration measurements. On a 3-yr-old Eucalyptus stand in Congo-Brazzaville, a method involving the upscaling of direct measurements made on roots in situ, was compared with an independent approach using soil respiration measurements conducted on control and trenched plots (i.e. without living roots). The first estimation was based on the knowledge of root-diameter distribution and on a relationship between root diameter and specific respiration rates. The direct technique involving the upscaling of direct measurements on roots resulted in an estimation of 1.53 micromol m(-2) s(-1), c. 50% higher than the mean estimation obtained with the indirect technique (1.05 micromol m(-2) s(-1)). Monte-Carlo simulations showed that the results carried high uncertainty, but this uncertainty was no higher for the direct method than for the trenched-plot method. The reduction of the uncertainties on upscaled results requires more extensive knowledge of temperature sensitivity and more confidence and precision on the respiration rates and biomasses of fine roots.