Inhibition of Sodium Ion Channel Function with Truncated Forms of Batrachotoxin.

A novel family of small molecule inhibitors of voltage-gated sodium channels (NaVs) based on the structure of batrachotoxin (BTX), a well-known channel agonist, is described. Protein mutagenesis and electrophysiology experiments reveal the binding site as the inner pore region of the channel, analogous to BTX, alkaloid toxins, and local anesthetics. Homology modeling of the eukaryotic channel based on recent crystallographic analyses of bacterial NaVs suggests a mechanism of action for ion conduction block.

Bioassay methods for detection of N-palmitoylbrevetoxin-B2 (BTX-B4).

Brevetoxins (BTXs) are a class of cyclic polyether toxins produced by the dinoflagellate Karenia brevis. These substances are subject to extensive conjugative metabolism in shellfish. BTX-B forms a conjugate with cysteine and is oxidized and reduced to yield BTX-B2, which is further modified by fatty acid addition via cysteine amide linkage to give biologically active brevetoxin metabolites. In this study, we evaluated the commonly used in vitro (ELISA, radioimmunoassay, receptor binding assay and N2A cytotoxicity assay) and in vivo mouse brevetoxin bioassays for the detection of the brevetoxin fatty acid conjugate N-palmitoylBTX-B2, and compared the results to those for dihydroBTX-B and BTX-B2. The receptor binding assay for N-palmitoylBTX-B2 showed comparable sensitivity to that for dihydroBTX-B, and an 11-fold higher sensitivity than for BTX-B2. Although the ELISA showed similarly high sensitivity to dihydroBTX-B and BTX-B2, with EC(50) values of ca. 0.26 ng/ml, it was 23 times less sensitive to N-palmitoylBTX-B2. On the other hand, the N2A cytotoxicity assay was highly sensitive to N-palmitoylBTX-B2, with an EC(50) of 0.15 ng/ml, but was 12- and 40-fold less sensitive to dihydroBTX-B and BTX-B2, respectively. The relative sensitivity of the N2A cytotoxicity assay for each of these metabolites paralleled that of the mouse bioassay (relative LD(50) values 1:20:30 for N-palmitoylBTX-B2:dihydroBTX-B:BTX-B2). We conclude that the most sensitive bioassay for dihydroBTX-B and BTX-B2 is the ELISA, whereas the N2A cytotoxicity assay is most sensitive for N-palmitoylBTX-B2.

Accurate quantitative determination of monoaromatic compounds for the monitoring of bioremediation processes.

This work demonstrated that the protocol for sample treatment, necessary to remove the microbial biomass prior to an analysis, is a critical issue for obtaining accurate results when volatile compounds are present. Two phenomena were observed, solute adsorption and stripping in the gas phase in contact with the liquid. It was demonstrated that the best protocol involved centrifugation using poly tetra fluoro ethylene (PTFE) capped tubes completely filled with the liquid suspension, i.e. without any gas phase inside it. This approach allowed a solute loss lower than 1%. The results also indicated that the optimum centrifugation conditions were 10000g at 10 degrees C for 10 min. Alternatively, it was found that the centrifugation technique developed could be used for the experimental determination of the activity coefficient of solubilized volatile compounds. This study additionally highlighted the fact that polyvinylidene fluoride micro filters (PVDF) and propylene GH polypro membranes (GHP) with a pore size of 0.45 microm could be used for biomass separation, although 10-12% monoaromatic adsorption by membrane was still present. In addition, a simple and sensitive method using high performance liquid chromatography (HPLC) with a UV detector set at the optimum point of 208 nm was developed for assessing the concentrations of BTX in samples taken from bioremediation processes. Minimum detection limits of 5, 4 and 10 microg L(-1) were obtained for benzene, toluene and mixed xylenes, respectively.

Homobatrachotoxin in the genus Pitohui: chemical defense in birds?

Three passerine species in the genus Pitohui, endemic to the New Guinea subregion, contain the steroidal alkaloid homobatrachotoxin, apparently as a chemical defense. Toxin concentrations varied among species but were always highest in the skin and feathers. Homobatrachotoxin is a member of a class of compounds collectively called batrachotoxins that were previously considered to be restricted to neotropical poison-dart frogs of the genus Phyllobates. The occurrence of homobatrachotoxin in pitohuis suggests that birds and frogs independently evolved this class of alkaloids.