A mathematical model of exposure of non-target Lepidoptera to Bt-maize pollen expressing Cry1Ab within Europe.

Genetically modified (GM) maize MON810 expresses a Cry1Ab insecticidal protein, derived from Bacillus thuringiensis (Bt), toxic to lepidopteran target pests such as Ostrinia nubilalis. An environmental risk to non-target Lepidoptera from this GM crop is exposure to harmful amounts of Bt-containing pollen deposited on host plants in or near MON810 fields. An 11-parameter mathematical model analysed exposure of larvae of three non-target species: the butterflies Inachis io (L.), Vanessa atalanta (L.) and moth Plutella xylostella (L.), in 11 representative maize cultivation regions in four European countries. A mortality-dose relationship was integrated with a dose-distance relationship to estimate mortality both within the maize MON810 crop and within the field margin at varying distances from the crop edge. Mortality estimates were adjusted to allow for physical effects; the lack of temporal coincidence between the susceptible larval stage concerned and the period over which maize MON810 pollen is shed; and seven further parameters concerned with maize agronomy and host-plant ecology. Sublethal effects were estimated and allowance made for aggregated pollen deposition. Estimated environmental impact was low: in all regions, the calculated mortality rate for worst-case scenarios was less than one individual in every 1572 for the butterflies and one in 392 for the moth.

Comparative studies of the monomeric and filamentous actin-myosin head complexes.

The functional and structural properties of the monomeric and filamentous actin-myosin head (S1) complexes were compared under strictly controlled conditions which avoid the S1-induced polymerization of monomeric actin. Under these conditions, monomeric (G) and filamentous (F) actin were found to activate S1 Mg(2+)-ATPase by 3- and 120-fold, respectively, in the presence of a 5-fold excess of actin over S1. Using the change in fluorescence intensity of pyrene-G-actin induced by S1 binding in the presence of various nucleotide analogues, we discovered that the ternary G-actin-S1-AMPPNP complex could not be formed. Moreover, the association constants of G-actin to S1-ADP or of ADP to the G-actin-S1 complex were at least an order of magnitude lower than in the filamentous state. Such a low affinity between G-actin and the S1-nucleotide intermediates can reasonably explain the lack of ATPase activation by the monomeric complex. Analysis of the G-actin-S1 interface by chemical cross-linking and limited proteolytic experiments showed that, in the monomeric complex, S1 interacted almost exclusively by its positively charged segment 636-642 with the patch of negative residues located on the actin flexible loops 1-7, 20-28, and 90-100. Moreover, the variation in the cross-linking pattern and in the proteolytic susceptibility of S1 segment 636-642 demonstrated that this electrostatic interface was different in the monomeric and the filamentous complexes. Taken together, the results suggested that the G-actin-S1 interaction encompasses only a small fraction of the strong as well as of the weak F-actin-S1 interface. The monomeric complex would in fact resemble more the collision complex which takes place early in the F-actin-S1 interaction.

Functional significance of the binding of one myosin head to two actin monomers.

The functional significance of the interaction of one myosin head (S1) with two actin monomers was investigated by comparing the properties of the cross-linked monomeric and filamentous actin-S1 complexes. S1 was cross-linked to monomeric actin (G-actin) either in the absence or in the presence of DNase I by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The binary G-actin-S1 and ternary DNase I-G-actin-S1 complexes were then purified by a combination of ion exchange and gel filtration columns. Both the binary and the ternary complexes were characterized by negligible, though different, Mg(2+)-ATPase activities of 0.018 and 0.006 s-1s(-1), respectively. Using fluorescence, light-scattering, and ultracentrifugation experiments, we found that only the binary G-actin-S1 complex could be polymerized in the presence of 2 mM MgCl2. Electron microscopic analysis of the cross-linked filamentous complex showed fully decorated filaments with the arrowhead pattern characteristic of the non-covalent complex in the rigor state. Such a 100% cross-linked F-actin-S1 complex exhibited a Mg(2+)-ATPase activity of 6.2 +/- 0.8 s-1, slightly lower than the maximum velocity of the non-cross-linked complex of 8.6 +/- 0.8 s-1, but comparable to the 6.9 +/- 0.6 s-1 obtained for a partially (35%) cross-linked complex. These results implied that the activation of S1 ATPase by actin requires the interaction of S1 with a second actin monomer within the thin filament. They also suggested that the full activation of the filamentous complex is not dependent on the degree of saturation of the thin filament by myosin.

Interaction and polymerization of the G-actin-myosin head complex: effect of DNase I.

The properties of polymerization and interaction of the G-actin-myosin S1 complexes (formed with either the S1(A1) or the S1(A2) isoform) have been studied by light-scattering and fluorescence measurements in the absence and in the presence of DNase I. In the absence of DNase I, the G-actin-S1(A1) and G-actin-S1(A2) complexes were found to be characterized by different limiting concentrations (l.c.), defined as the complex concentrations above which the polymerization occurs spontaneously within 20 h at 20 degrees C in a "no salt" buffer (l.c. = 0.42 and 8.8 microM for G-actin-S1(A1) and G-actin-S1(A2), respectively). The occurrence of a limiting concentration for either complex together with the kinetic properties of the polymerization led us to conclude that the G-actin-S1 polymerization occurs via a nucleation-elongation process. Fluorescence titrations and proteolysis experiments revealed that G-actin interacts with S1 with a 1:1 stoichiometry (independently of the presence of ATP) with dissociation constants, in the absence of nucleotide, of 20 and 50 nM for the G-actin-S1(A1) and G-actin-S1(A2) complexes, respectively. In the presence of at least a 1.5-fold excess of DNase I, the polymerization of the G-actin-S1 complexes was blocked even at high protein concentration or in the presence of salts. In addition, the affinity of either S1 isoform to actin was reduced 4-5-fold by DNase I, while the stoichiometry of the G-actin-S1 complexes was not changed. However, since the dissociation constants remain in the submicromolar range, we could demonstrate the existence of ternary DNase I-G-actin-S1 complexes stable under polymerizing conditions. Finally, the study of the effect of nucleotides and of various salts on the G-actin-S1 interaction further showed significant differences between the G-actin-S1 and F-actin-S1 interactions.