High-throughput ESI-MS analysis of binding between the Bombyx mori pheromone-binding protein BmorPBP1, its pheromone components and some analogues.

Chip-assisted high-throughput ESI-MS analysis of the pheromone-binding protein of the silkworm moth Bombyx mori, BmorPBP1, incubated with its pheromone components bombykol, bombykal and analogues was developed. The protein bound to bombykol ((10E,12Z)-hexadecadien-1-ol) and all 3 of its geometric isomers to a lesser extent, and showed relaxed specificity toward different chain lengths possessing unsaturation. BmorPBP1 did not bind to bombykal ((10E,12Z)-hexadecadienal), demonstrating molecular recognition of the insect pheromone components.

Characterisation of Bombyx mori Odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components.

In many insect species, odorant-binding proteins (OBPs) are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors (ORs) within the antennal sensilla. In the silkworm Bombyx mori, the OBPs are subdivided into three main subfamilies; pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal-binding proteins (ABPs). We used the MotifSearch algorithm to search for genes encoding putative OBPs in B. mori and found 13, many fewer than are found in the genomes of fruit flies and mosquitoes. The 13 genes include seven new ABP-like OBPs as well as the previously identified PBPs (three), GOBPs (two) and ABPx. Quantitative examination of transcript levels showed that BmorPBP1, BmorGOBP1, BmorGOBP2 and BmorABPx are expressed at very high levels in the antennae and so could be involved in olfaction. A new two-phase binding assay, along with other established assays, showed that BmorPBP1, BmorPBP2, BmorGOBP2 and BmorABPx all bind to the B. mori sex pheromone component (10E,12Z)-hexadecadien-1-ol (bombykol). BmorPBP1, BmorPBP2 and BmorABPx also bind the pheromone component (10E,12Z)-hexadecadienal (bombykal) equally well, whereas BmorGOBP2 can discriminate between bombykol and bombykal. X-ray structures show that when bombykol is bound to BmorGOBP2 it adopts a different conformation from that found when it binds to BmorPBP1. Binding to BmorGOBP2 involves hydrogen bonding to Arg110 rather than to Ser56 as found for BmorPBP1.

Vicia faba-Lygus rugulipennis interactions: induced plant volatiles and sex pheromone enhancement.

The profiles of volatile chemicals emitted by Vicia faba plants damaged by Lygus rugulipennis feeding, and by feeding plus oviposition, were shown to be quantitatively different from those released by undamaged plants. Samples of volatile chemicals collected from healthy plants, plants damaged by males as a consequence of feeding, plants damaged by females as a consequence of feeding and oviposition, plants damaged by feeding with mated males still present, and plants damaged by feeding and oviposition with gravid females still present, showed significant differences in the emission of hexyl acetate, (Z)-beta-ocimene, (E)-beta-ocimene, (E)-beta-caryophyllene, and methyl salicylate. In particular, treatments with mated females present on plants had a significant increase in emission levels of the above compounds, possibly due to eggs laid within plant tissues or active feeding, compared with undamaged plants and plants damaged by males feeding, with or without insects still present. Furthermore, the pheromonal blend released by mated L. rugulipennis females, mainly comprising hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal, was enhanced when females were active on broad bean plants, whereas such an increase was not observed in males. Both sexes gave electroantennogram responses to green leaf volatiles from undamaged plants and to methyl salicylate and (E)-beta-caryophyllene emitted by Lygus-damaged plants, suggesting that these compounds may be involved in colonization of host plants by L. rugulipennis. In addition, mated males and females were responsive to hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal released by mated females on V. faba, indicating that these substances could have a dual function as a possible aggregation pheromone in female-female communication, and as a sex pheromone in female-male communication.

Surface properties of new virginiamycin M(1) derivatives.

Three kinds of derivatives of the M(1) factor of virginiamycin have been synthesised: esters with long chain fatty acids, oximes with modified polar amino acids and bis-derivatives with both the ester and oxime function. The study of the surface tension time dependence of M(1) and its derivatives has shown that it is necessary to enhance simultaneously the hydrophobicity and the hydrophilicity of M(1) to render M(1) surface-active. A structure/function relationship study of the surface-active bis-derivatives has shown that enhancing the hydrophobicity of the molecule led to slower adsorption kinetics, higher stability of the monolayers formed and a better capacity to penetrate a membrane model. The repulsive electrostatic forces due to the presence of charges on the amino acids linked to M(1) lead to higher surface tensions, a greater molecular area at the interface and lower penetration into a membrane model. This study has demonstrated that modifying systematically the hydrophobicity and hydrophilicity of a non surface-active molecule allows the production of surface-active derivatives.

Identification of volatile compounds used in host location by the black bean aphid, Aphis fabae.

Behavioral and electrophysiological responses of winged Aphis fabae to volatiles of faba bean, Vicia faba (var. Sutton dwarf), plants were studied and semiochemicals used in host location were identified. In olfactometer bioassays, aphids spent significantly more time in the region of the olfactometer where V. faba volatiles from an intact plant were present than in control regions with clean air. This response also occurred when an air entrainment sample of a V. faba plant was used as the odor source. Coupled gas chromatography-electroantennography revealed the presence of 16 electrophysiologically active compounds in the air entrainment sample. Fifteen of these were identified as (Z)-3-hexen-1-ol, 1-hexanol, (E)-2-hexenal, benzaldehyde, 6-methyl-5-hepten-2-one, octanal, (Z)-3-hexen-1-yl acetate, (R)-(-)-linalool, methyl salicylate, decanal, undecanal, (E)-caryophyllene, (E)-beta-farnesene, (S)-(-)-germacrene D, and (E,E,)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. An olfactometer response was observed to a 15-component synthetic blend that comprised all identified compounds at the same concentration and ratio as in the natural sample, with the aphids spending significantly more time in the treated regions of the olfactometer where volatiles were present than in the control regions. These data are discussed in the context of insect host location and crop protection.

Nanoscale membrane activity of surfactins: influence of geometry, charge and hydrophobicity.

We used real-time atomic force microscopy (AFM) to visualize the interactions between supported lipid membranes and well-defined surfactin analogs, with the aim to understand the influence of geometry, charge and hydrophobicity. AFM images of mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers recorded after injection of cyclic surfactin at 1 mM, i.e. well-above the critical micelle concentration, revealed a complete solubilization of the bilayers within 30 min. A linear analog having the same charge and acyl chains was able to solubilize DOPC, but not DPPC, and to promote redeposition leading eventually to a new bilayer. Increasing the charge of the polar head or the length of the acyl chains of the analogs lead to the complete solubilization of both DOPC and DPPC, thus to a stronger membrane activity. Lastly, we found that at low surfactin concentrations (40 microM), DPPC domains were always resistant to solubilization. These data demonstrate the crucial role played by geometry, charge and hydrophobicity in modulating the membrane activity (solubilization, redeposition) of surfactin. Also, this study suggests that synthetic analogs are excellent candidates for developing new surfactants with tunable, well-defined properties for medical and biotechnological applications.

The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca.

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.

Dynamic thermodynamic and dynamic kinetic resolution of 2-lithiopyrrolidines.

Dynamic resolution has been studied as a method for the asymmetric synthesis of 2-substituted pyrrolidines. Highly enantioselective electrophilic substitutions of racemic 2-lithiopyrrolidines in the presence of a chiral ligand have been achieved. The organolithium compounds were prepared by tin-lithium exchange from the corresponding tributylstannanes and n-butyllithium or by deprotonation of N-(tert-butyloxycarbonyl)pyrrolidine with sec-butyllithium. A range of N-substituents and chiral ligands were investigated for the dynamic resolution. Electrophilic quench of the resolved diastereomeric 2-lithiopyrrolidine-chiral ligand complexes provided the enantiomerically enriched 2-substituted pyrrolidines. With N-alkyl derivatives, the resolution occurs conveniently at (or just below) room temperature and either enantiomer of the product can be formed by appropriate choice of the chiral ligand. The asymmetric induction occurs as a result of a thermodynamic preference for one of the diastereomeric complexes. The minor complex was found to have a faster rate of reaction with the electrophile. The use of N-allylic derivatives provides a means to prepare the N-unsubstituted pyrrolidine products. Best results were obtained with the N-2,3-dimethylbut-2-enyl derivative, and this N-substituent could be cleaved using 1-chloroethyl chloroformate. With N-Boc-2-lithiopyrrolidine, the enantioselectivity arises by a kinetic resolution and high levels of asymmetric induction in the presence of excess n-butyllithium can be obtained. Dynamic kinetic resolution of the N-Boc derivative is limited in the scope of electrophile that can be used.

Hemolytic activity of new linear surfactin analogs in relation to their physico-chemical properties.

New linear analogs of surfactin have been synthesized. Their physico-chemical parameters were determined. The results indicate that these linear products show surface activities although they are lowered compared to those of cyclic compounds. The hemolytic activities have also been assayed. In contrast with cyclic surfactins, no significant hemolysis occurs for the linear products in the range of concentrations tested. Moreover, a protective effect against Triton X-100 induced hemolysis has been highlighted for linear surfactins. The concentration at which this protective effect happens is correlated directly to the CMC, and inversely to the acyl chain length of the product. In a hypotonic medium, analogs having a long acyl chain tend to increase the hemolysis, meanwhile the product with the shortest chain tends to decrease it.

Barrier to enantiomerization of unstabilized, chelated, and dipole-stabilized 2-lithiopyrrolidines.

Kinetics experiments have been used to establish the free energy, enthalpy, and entropy of activation for the enantiomerization of three structural classes of 2-lithiopyrrolidines. We find that alpha-aminoorganolithiums chelated by a N-methoxyethyl or N-Boc group have a barrier to enantiomerization (DeltaG++) 2-3 kcal/mol lower than that of unstabilized alpha-aminoorganolithiums at 273 K. Density functional calculations were performed to clarify possible ground state and transition structures and to identify possible pathways for inversion of these chiral organolithium species.

Hyperexcitability and epilepsy associated with disruption of the mouse neuronal-specific K-Cl cotransporter gene.

Four genes encode electroneutral, Na+-independent, K-Cl cotransporters. KCC2, is exclusively expressed in neurons where it is thought to drive intracellular Cl- to low concentrations and shift the reversal potential for Cl- conductances such as GABA(A) or glycine receptor channels, thus participating in the postnatal development of inhibitory mechanisms in the brain. Indeed, expression of the cotransporter is low at birth and increases postnatally, at a time when the intracellular Cl- concentration in neurons decreases and gamma-aminobutyric acid switches its effect from excitatory to inhibitory. To assert the significance of KCC2 in neuronal function, we disrupted the mouse gene encoding this neuronal-specific K-Cl cotransporter. We demonstrate that animals deficient in KCC2 exhibit frequent generalized seizures and die shortly after birth. We also show upregulation of Fos, the product of the immediate early gene c-fos, and the significant loss of parvalbumin-positive interneurons, both indicative of brain injury. The regions most affected are the hippocampus and temporal and entorhinal cortices. Extracellular field potential measurements in the CA1 hippocampus exhibited hyperexcitability. Application of picrotoxin, a blocker of the GABA(A) receptor, further increased hyperexcitability in homozygous hippocampal sections. Pharmacological treatment of pups showed that diazepam relieved the seizures while phenytoin prevented them between postnatal ages P4-P12. Finally, we demonstrate that adult heterozygote animals show increased susceptibility for epileptic seizure and increased resistance to the anticonvulsant effect of propofol. Taken together, these results indicate that KCC2 plays an important role in controlling CNS excitability during both postnatal development and adult life.