Periodate treatment reduces the tetrodotoxin-sensitivity of voltage-gated Na+ channels.

(1) Voltage-clamped nerve fibres of the frog Rana esculenta were treated with periodate in the extracellular solution. (2) Periodate treatment irreversibly reduced the effect of tetrodotoxin (TTX) on the Na+ currents. (3) The effect of saxitoxin (STX) was also reduced but less than that of TTX. (4) The presence of STX during the application of periodate to the nerve fibre almost completely prevented the effect of the chemical reagent on the TTX sensitivity of the Na+ channels. (5) The reduction of the TTX effect is not due to the reaction of small amounts of periodate with the diol group of this toxin, because the effect was seen after prolonged washing with reagent-free Ringer solution with or without high amounts of ribose. (6) Carboxyl groups present in the Na+ channel seem to be quite important for the binding of TTX and STX. Periodate modifies several amino acid side chains, however, it does not attack carboxyl groups in a peptide chain. Thus, these results suggest that periodate modifies a further group critically involved in the binding of TTX and STX.

A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application.

Neutralizing effect of hemolymph from the shore crab, Thalamita crenata, on paralytic shellfish toxins.

Several species of crabs are resistant to paralytic shellfish toxins (PSTs) and/or pufferfish toxin, tetrodotoxin, regardless of toxification by the toxins. The shore crab Thalamita crenata, which inhabits Leizhou Peninsula, China, is tolerant to PST toxicity, and the hemolymph has neutralizing effects against the lethal activity of PST. In the present study, we investigated the PST neutralizing factors in the hemolymph from T. crenata and successfully separated PST-binding proteins by PST-ligand affinity chromatography. The neutralization factors, obtained in the fraction with a molecular weight over 10 kDa by ultrafiltration, were susceptible to proteases such as alcalase, animal complex proteases, pancreatin, and papain. The PST-binding protein had high dose-dependent neutralization effects on PST toxicity. The PST-binding activity of the protein was stable at 25 °C and then decreased with an increase in temperature; heating at 65 °C for 60 min eliminated the initial activity by two-thirds. The PST-binding activity was strongly inhibited in the presence of Mg(2+) and Ca(2+), but not Na(+) and K(+). The PST-binding capability of the protein differed among PST components in descending order of neosaxitoxin, gonyautoxins 1 and 4, saxitoxin, and gonyautoxins 2 and 3, suggesting a structure-activity relationship in PST binding.

Recovery from the lethal effects of saxitoxin: a therapeutic window for 4-aminopyridine (4-AP).

We have shown that saxitoxin (STX) induced lethality can be reversed by 4-AP when it is administered at the time of respiratory arrest [Benton, B. J., Spriggs, D. L., Capacio, B. R. and Chang, F.-C. T. (1995) 4-Aminopyridine antagonizes the lethal effects of saxitoxin (STX) and tetrodotoxin (TTX). International Society of Toxicology, 5th Pan American Symposium on Animal, Plant and Microbial Toxins, Frederick, MD. July/August 1995, p. 217]. The purpose of this study was to determine whether 4-AP's efficacy could be enhanced further when administered at different times relative to STX intoxication. The animals used in this study were chronically instrumented for concurrent recordings of diaphragm electromyogram (DEMG), neck skeletal muscle electromyogram, Lead II electrocardiogram, and electrocorticogram (ECoG). There were five groups of unanesthetized guinea pigs. The first group served as 4-AP controls and received a 2 mg/kg i.m. dose of 4-AP. The four remaining groups were given a lethal dose of STX (5 microg/kg i.m.); the second group, STX controls, received no 4-AP; the third group, the 4-AP treatment group, received 4-AP immediately following cardiorespiratory collapse; the fourth group was the 4-AP/STX co-administration group and 4-AP was given concurrently with STX; and the fifth group was the 4-AP pretreatment group in which 4-AP was given 10 min before STX. At the point of STX-induced cardiorespiratory collapse, the guinea pigs were ventilated and given an i.p. injection of sodium bicarbonate. Results showed that 4-AP prevented cardiorespiratory collapse in 3/7 animals in the 4-AP pretreatment group. Also, 4-AP in conjunction with artificial ventilation and sodium bicarbonate accelerated recovery from STX-induced cardiorespiratory collapse in all the treatment groups compared to the STX controls.

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: deconvolution strategy.

Abstract Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.

Mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of the puffer fish, Fugu pardalis.

The mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of Fugu pardalis was investigated by HPLC. The values for the half inhibitory concentration of tetrodotoxin (1.6 microM) binding to this protein (1.2 microM) for saxitoxin, and of saxitoxin (0.47 microM) binding to that (0.30 microM) for tetrodotoxin were 0.35 +/- 0.057 microM and 81 +/- 16 microM (n = 2), respectively.

Effects of 4-aminopyridine on saxitoxin intoxication.

Effects of 4-aminopyridine (4-AP) on neurotoxicity induced by saxitoxin (STX) are investigated in this study. In vitro, twitch tension evoked by nerve stimulation was depressed by STX (1.35 nM) in rat phrenic nerve-diaphragm preparations, and this inhibition was antagonized by 4-AP (0.1 mM). In addition, 4-AP (0.1 mM) restored the firing of membrane action potentials that were suppressed or even abolished by 0.334 nM STX in frog sartorius muscles. In vivo studies showed that 4-AP (0.3 mg/kg, iv) significantly reversed the respiratory rate, tidal volume, and blood pressure to normal values in anesthetized STX-toxicosis rats. Furthermore, 4-AP (0.75-6 mg/kg, ip) not only prolonged the survival time but also decreased the mortality of mice (71-43%) at a normally lethal dose (30 micrograms/kg, ip) of STX. The results suggest that 4-AP may be useful as an antidote for STX intoxication.