Molecular interaction of delta-conotoxins with voltage-gated sodium channels

Various neurotoxic peptides modulate voltage-gated sodium (Na(V)) channels and thereby affect cellular excitability. Delta-conotoxins from predatory cone snails slow down inactivation of Na(V) channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that delta-conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain-4 voltage sensor of Na(V) channels. This site overlaps with that of the scorpion alpha-toxin Lqh-2, but not with the alpha-like toxin Lqh-3 site. Delta-SVIE functionally competes with Lqh-2, but exhibits strong cooperativity with Lqh-3, presumably by synergistically trapping the voltage sensor in its "on" position.     

Evaluating the addition of activated carbon to heat-treated mushroom casing for grain-based and compost-based substrates

Two substrates, a non-composted grain spawn substrate and a traditional composted substrate, each covered with peat-based casing that contained varying amounts of activated carbon (AC) and each receiving different heat-treatment durations, were tested for Agaricus bisporus mushroom production. The amounts of AC were 0, 5, 10, 15, and 20% v/v, and the heat treatments were 0, 60, and 180 min at 121 °C and 103.4 kPa. Overall, the addition of AC up to 10–15% of casing for a grain spawn substrate increased mushroom yield. However, the addition of AC to the casing for compost substrates had no significant effect on yield, whereas heat-treating the casing increased yield. The onset of fruiting was retarded in grain spawn treatments not receiving AC with heat-treatment durations of 60 and 180 min, whereas this effect was not as apparent for the compost substrates. On average, mushroom yield was greater for the grain spawn substrate (366 g) than for compost substrate (287 g). For grain spawn substrate, the results show that the addition of AC ranging from 5% to 10% was adequate for maximum mushroom production.     

Determination of DNA-binding parameters for the Bacillus subtilis histone-like HBsu protein through introduction of fluorophores by site-directed mutagenesis of a synthetic gene.

A synthetic gene encoding the histone-like DNA-binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20 mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild-type protein. N-terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli. Site-directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA-binding parameters by fluorescence spectroscopy. According to gel-retardation experiments, the mutant protein [Phe47----Trp]HBsu shows identical DNA binding to wild-type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47----Trp]HBsu binds double-stranded DNA with a dissociation constant in the micromolar range. Computer-assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47----Trp]HBsu appears to contact three or four base pairs of DNA. These results are in partial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.     

A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.     

A modern approach to demineralization of spicules in glass sponges (Porifera: Hexactinellida) for the purpose of extraction and examination of the protein matrix

Glass sponges of the class Hexactinellida are a group of the most ancient multicellular animals, whose fossil remnants from the early Proterozoic have been registered. In order to demineralize the skeletal structures of the glass sponge Hyalonema sieboldi, we have used for the first time a strategy of slow leaching of the silicon-bearing component, based on the usage of alkaline solutions of sodium hydroxide, sodium dodecyl sulfate, and an anionic biosurfactant of a rhamnolipid nature. The obtained data unequivocally corroborate the presence of a fibrillar protein matrix functioning as a basis for silicon biomineralization in the basal spicules of H. sieboldi. Also, it has been found for the first time that the protein matrix is constructed of a collagenous protein. The technical approach proposed here might appear important for the study of the structural organization of skeletons in other silicon-bearing animals and, in an applied aspect, to work out new biomaterials for implantology and biocomposites, in order to use the latter as bioactive additives.