Design of an artificial phage-display library based on a new scaffold improved for average stability of the randomized proteins.

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the µM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site.

Involvement of cytochrome P450 monooxygenases in the response of mosquito larvae to dietary plant xenobiotics.

The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed.

Molecular approach to aquatic environmental bioreporting: differential response to environmental inducers of cytochrome P450 monooxygenase genes in the detritivorous subalpine planktonic Crustacea, Daphnia pulex.

In order to examine the usefulness of detoxifying genes as molecular markers in different chemical environments, isolation of cytochrome P450 genes (CYPs) belonging to the CYP4 family was performed in different samples from two subalpine populations of Daphnia pulex. The use of degenerate primers allowed us to isolate seven cDNAs. Four of them were assigned to the CYP4C subfamily, and were closely related to previously isolated crustacean CYP4s while the others were assigned to new CYP4AN and CYP4AP subfamilies. Expression studies, using semiquantitative polymerase chain reaction (PCR) followed by Southern blot hybridization with specific probes revealed differences in CYP4C32 and CYP4AP1 expressions between the two populations, which differ in the polyphenol richness of the vegetation surrounding their aquatic habitat. Further exposure to toxic dietary polyphenols showed different CYP induction patterns. Taken together, these preliminary results suggest a possible involvement of CYP4s in the ecological differentiation of subalpine D. pulex populations related to the polyphenol richness of the environmental vegetation. CYP4s may thus be considered as possible molecular markers in aquatic environmental bioreporting.

Effects of tamoxifen on the electron transport chain of isolated rat liver mitochondria.

Tamoxifen (and 4-hydroxytamoxifen), a nonsteroidal triphenylethylene antiestrogenic drug widely used in the treatment of breast cancer, interacts strongly with the respiratory chain of isolated rat liver mitochondria. The drug acts as both an uncoupling agent and a powerful inhibitor of electron transport. Tamoxifen brings about a collapse of the membrane potential. Enzymatic assays and spectroscopic studies indicate that tamoxifen inhibits electron transfer in the respiratory chain at the levels of complex III (ubiquinol-cytochrome-c reductase) and, to a lesser extent, of complex IV (cytochrome-c oxidase). The activities can be restored by the addition of diphosphatidylglycerol, a phospholipid implicated in the functioning of the respiratory chain complexes.