Evaluating resistance to Bt toxin Cry1Ab by F2 screen in European populations of Ostrinia nubilalis (Lepidoptera: Crambidae)

The large-scale cultivation of transgenic crops producing Bacillus thuringiensis (Bt) toxins have already lead to the evolution of Bt resistance in some pest populations targeted by these crops. We used the F2 screening method for further estimating the frequency of resistance alleles of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), to Bt maize, Zea mays L., producing the Cry1Ab toxin. In France, Germany, and Italy, 784, 455, and 80 lines of European corn borer were screened for resistance to Mon810 maize, respectively. In Slovakia, 26 lines were screened for resistance to the Cry1Ab toxin. The cost of F2 screen performed in the four countries varied from U.S. dollars 300 to dollars 1300 per line screened. The major difference in cost was mostly due to a severe loss of univoltine lines during the screen in Germany and Slovakia. In none of the screened lines did we detect alleles conferring resistance to Mon810 maize or to the Cry1Ab toxin. The frequency of resistance alleles were < 1.0 x 10(-3), < 1.6 x 10(-3), < 9.2 x 10(-3), and < 2.6 x 10(-2) in France, Germany, Italy, and Slovakia, with 95% probability, respectively. The average detection probability over all lines was approximately 90%. Making the assumption that European corn borer populations in these countries belong to the same genetic entity, the frequency of alleles conferring resistance to the Cry1Ab produced by the Mon810 maize in western and central Europe was 1.0 x 10(-4), with a 95% confidence interval of 0-3.0 x 10(-4).     

The role of microtubules and microfilaments in the hydrosmotic response to antidiuretic hormone

To test the effects of colchicine and cytochalasin B on the ADH-induced response, unidirectional and net water fluxes were measured at one or two minutes intervals in frog urinary bladder. The action of these agents on the appearance of intramembrane particles aggregates in the luminal membrane of target cells under oxytocin stimulation and the changes in the tissue ultrastructure induced by cytochalasin B were also studied. It was observed that: the time-course of the response to oxytocin was strongly slowed by colchicine while the washout was not affected; the time-course of the 'on and off' of the response to oxytocin was not modified by cytochalasin B; cytochalasin B pretreatment proportionally reduced unidirectional and net water fluxes measured after glutaraldehyde fixation; the combined action of colchicine and cytochalasin B proportionally reduced the net water flux and the number of intramembrane particles aggregates, observed in freeze-fracture studies; after cytochalasin B action the dilation of intercellular spaces classically observed under oxytocin stimulation is strongly reduced. It is concluded that: microtubules probably play an important role in the water channels plug-in, but not in their removal; microfilaments integrity is necessary for the mechanisms inducing intercellular space dilation and the observed results confirm that water permeability is controlled by the number of permeation units present in the luminal border of granular cells and probably represented by the intramembrane particle aggregates.     

N-1H-benzimidazol-5-ylbenzenesulfonamide derivatives as potent hPXR agonists

The Human Pregnane X Receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-metabolizing enzymes, as well as that of drug transporters. Because this receptor plays a critical role in protecting tissues from potentially toxic endo- and xenobiotics, highly active agonists could represent novel therapeutic tools in treating several human diseases. Using an in vitro screening reporter system that allow to characterize hPXR activators and a first step of chemical modifications of an original agonist ligand (C2BA-4, 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide), we identified compounds with a N-1H-benzimidazol-5-ylbenzenesulfonamide scaffold as a potent family of hPXR agonists. Further chemical modifications allowed us to identify enhanced activators, notably N-(1-benzyl-1H-benzimidazol-5-yl)-2,3,4,5,6-pentamethylbenzenesulfonamide (6n) with an EC(50) value in the subnanomolar range. Accordingly to their potent EC(50), these compounds induced an efficient protection of hPXR against proteolytic digestion by trypsin even at very low ligand concentrations and were able to induce the expression of the main target genes of hPXR, CYP3A4 and CYP2B6, in primary cultures of human hepatocytes.     

Crosstalk between H2A variant-specific modifications impacts vital cell functions

Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A.W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.     

Gene flow in the European corn borer Ostrinia nubilalis: implications for the sustainability of transgenic insecticidal maize

Strategies proposed for delaying resistance to Bacillus thuringiensis toxins expressed by transgenic maize require intense gene flow between individuals that grew on transgenic and on normal (referred to as refuges) plants. To investigate gene flow in the European corn borer, Ostrinia nubilalis (Hübner), the genetic variability at 29 sampled sites from France was studied by comparing allozyme frequencies at six polymorphic loci. Almost no deviations from Hardy-Weinberg expectations occurred, and a high stability of allelic distribution was found among samples collected in the same site over two or three different generations, indicating a high stability of the genetic structure over time. The overall genetic differentiation was low at the region and whole country level, suggesting a high and homogeneous gene flow. These results are discussed in relation to the sustainability of transgenic insecticidal maize.     

Glutaraldehyde fixation preserves the permeability properties of the ADH-induced water channels

Summary Unidirectional and net water movements were determined, in frog urinary bladders, before and after glutraldehyde fixation. Experiments were performed in three experimental conditions: 1) in nonstimulated preparations, 2) after the action of antidiuretic hormone (ADH) and 3) in nonstimulated preparations to which amphotericin B was incorporated from the luminal bath. As previously observed for net water fluxes, the increase in the unidirectional water movement induced by ADH was well preserved by glutaraldehyde fixation. After correction for the effects of unstirred layers and nonosmotic pathways, the observed correlation between the ADH-induced increases in the osmotic (Pf) and diffusional (Pd) permeability coefficients was not modified by the fixative action (before glutaraldehyde: slope 11.19,r:0.87±0.07;n=12; after glutaraldehyde: slope 10.67,r:0.86±0.04,n=39). In the case of amphotericin B, Pf/Pd=3.08 (r: 0.83±0.08), a value similar to that observed in lipid bilayers or in nonfixed toad urinary bladders. It is concluded that 1) The experimental approach previously employed to study water channels in artificial lipid membranes and in amphibian urinary bladders, can be applied to the glutaraldehyde-fixed frog urinary bladder. 2) Glutaraldehyde fixation does not modify the permeability properties of the ADH-induced water channels. 3) Any contribution of exo-endocytic processes or cell regulatory mechanisms to the observed permeability parameters can probably be excluded. 4) Glutaraldehyde-fixed preparations are a good model to characterize these water pathways.     

Fusion images and intramembrane particle aggregates during the action of antidiuretic hormone

Summary Antidiuretic hormone (ADH) causes the appearance of water-conducting particle aggregates in the luminal membrane of receptor cells in amphibian bladder and skin, and in the mammalian collecting duct. The aggregates originate from cytoplasmic tubules that fuse with the luminal membrane during ADH stimulation. We have studied the process of fusion and the structure of the particle aggregates by a rapid-freeze technique that renders chemical fixation and glycerol protection unnecessary. Our findings differ in some important respects from previously published work. Aggregate particles, in our study, partition equally between the external (EF) and protoplasmic (PF) membrane leaflets, rather than remaining in the protoplasmic leaflet exlcusively. By including the entire population of fusion images in our survey, we have found that aggregate delivery in ADH-treated cells proceeds preferentially from small fusion images whose diameter is significantly less than the 0.12 m characteristic of the carrier tubules themselves. We have also found that, even in unstimulated preparations, fusion images are numerous, being mostly of small diameter. ADH stimulation produces a moderate increase in the number of fusion images and a significant increase in fusion-image diameter. These findings indicate that the individual particles are mobile within the membrane, lacking interparticle linkage. In addition, contact of cytoplasmic tubules with the luminal membrane may take place even in the absence of ADH, producing small fusion images which are not associated with aggregate delivery to the luminal membrane.Faculty Scholar, Josiah Macey Jr. Foundation     

The molecular basis of dominance relationships: the case of some recent adaptive genes

A controversial debate in evolutionary biology has been to explain why deleterious mutations are usually recessive to their wild-type allele. For Fisher, dominance of the wild-type is the result of selection, whereas for Wright it is a mere consequence of the biochemical properties of physiological pathways. Over time, Wright's theory has appeared as the most appropriate, and Kacser and Burns explained why the widespread occurrence of recessive mutants is the inevitable consequence of the kinetic structure of enzyme networks. Does Wright and Kacser and Burns (W-K-B)'s theory apply for newly arisen adaptive genes? A survey of more than 70 studies shows that pesticide resistance conferred by mutations decreasing the affinity of the pesticide target-sites varies from complete recessivity to complete dominance. This review shows that dominance always has a purely physiological explanation that can be roughly, but not simply, predicted by W-K-B's theory. Thus, although W-K-B's theory remains powerful for predicting the recessivity of deleterious mutations involved in enzymatic pathways, no general theory emerges from the study of other situations, and molecular explanations are to be sought on a case by case basis.