A new method for labelling saxitoxin and its binding to non-myelinated fibres of the rabbit vagus, lobster walking leg, and garfish olfactory nerves (original) (raw)
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Determination of Saxitoxin by Receptor Binding Assay: A New Radiolabeled Kit in Thailand
Objective: A receptor binding assay for detection of saxitoxin, a paralytic shellfish poisoning toxin, was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the NOAA National Ocean Service, SC USA, to the Thailand Institute of Nuclear Technology, which uses a Perkin Elmer Microbeta TriLux 1450 microplate scintillation counter. Methods: Validation of method was performed by participating in the regional inter-laboratory program. The result yielded all parameters within the critical control point (i.e. RSD less than 30%). Results: The slope of the calibration graph = 1.2 + 0.26 (RSD = 21.7%) (criteria = 0.8-1.2), half maximal inhibition (IC50) = 3.1 + 0.47 nM (RSD = 15.2%) (criteria = 3.0 nM), dynamic range = 1.2-10 nM and the limit of detection (IC80) from graph = 1.2 nM, equivalent to the limit of quantification of 2.57 mg/100 g shellfish. The result of analysis of unknown samples yielded the RSD between assays ranging from 6-23% and the recovery compared to the expected value ranging from 79-133%. The application of RBA for determination of PSP in shellfish samples yielded RSD in assays less than 30% and between assays ranging from 1.4-15.2%. Conclusion: RBA for STX has showed a valuable for rapid, reliable, cost-effective alternative to live animal testing and high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.
Toxicon, 2007
Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na + channel and for this reason, it has been widely used in the study of Na + channel. The aim of this study was to analyze STX distribution in different rat brain regions after its acute intraperitoneal (i.p.) administration. Male rats (150-200 g) were injected i.p. with STX (5 and 10 mg/kg of body weight). After three time intervals of 30, 60, and 120 min (for 5 mg/kg STX dose) and 30 min (for 10 mg/kg STX dose) animals were sacrificed by cervical dislocation. Brains were removed and dissected in seven regions. STX concentration was measured using a precolumn oxidation high-performance liquid chromatographic method with fluorescence detection (HPLC/FLD). STX was found in all the regions evaluated at ppm levels meaning that STX peripherical administered across the blood-brain barrier and is distributed along the whole brain. r
The Journal of Physiology, 1977
1. The binding of [3H]saxitoxin to innervated and denervated rat diaphragm muscle, and to normal frog muscle, has been measured. 2. A saturable component of saxitoxin binding, which was inhibited by tetrodotoxin, was detected in all preparations, as well as a component of non-saturable binding. The values for the maximum saturable capacity, M, and the equilibrium binding constant, K, for normal rat diaphragm muscle were: M = 24-4 f-mole. mg wet-L, and K = 3-8 nM. 3. Denervation of rat diaphragm muscle reduced the maximum binding capacity per unit weight .to 16-5 f-mole. mg-'.tThe value of K remained virtually unchanged at 4-2 nM. 4. It is suggested that the decrease in density per unit weight does not reflect any change in the density of sodium channels per unit area of membrane. 5. Two varieties of the same species of frog, Rana pipiens, were examined. In one variety (Southern) the value of M was 25-6 f-mole. mg-' and the value of K was 4-3 nM. In the Northern variety the maximum binding capacity was less, M being 14-6 f-mole. mg-'; the value of K was 3-8 nM.
The Journal of general physiology, 1981
The actions of tetrodotoxin (TTX) and saxitoxin (STX) in normal water and in deuterium oxide (D2O) have been studied in frog myelinated nerve. Substitution of D2O for H2O in normal Ringer's solution has no effect on the potency of TTX in blocking action potentials but increases the potency of STX by approximately 50%. Under voltage clamp, the steady-state inhibition of sodium currents by 1 nM STX is doubled in D2O as a result of a halving of the rate of dissociation of STX from the sodium channel; the rate of block by STX is not measurably changed by D2O. Neither steady-state inhibition nor the on- or off-rate constants of TTX are changed by D2O substitution. The isotopic effects on STX binding are observed less than 10 min after the toxin has been added to D2O, thus eliminating the possibility that slow-exchange (t 1/2 greater than 10 h) hydrogen-binding sites on STX are involved. The results are consistent with a hypothesis that attributes receptor-toxin stabilization to isoto...
Saxitoxin binding proteins: biological perspectives
2005
Saxitoxin binding protein (STXBP) is a functional classification which describes all proteins capable of binding to the paralytic shellfish toxin (PST), saxitoxin (STX). Based on this functionality, this group includes the voltage gated sodium channels (VGSCs), pufferfish STX and tetrodotoxin (TTX) binding proteins (PSTBPs) and saxiphilin (SXPN) which was been isolated from the amphibian Rana catesbeiana. Various activities and relationships of bullfrog SXPN have been elucidated including the ability to inhibit papain, human cathepsin Band L and the substantial homology of the amino acid sequence to transferrins (TFs). However, the biological role of SXPN has not been thoroughly examined and remains a mystery. It is likely that a detoxification mechanism exists in animals exposed to PSTs, and may explain the defined STX binding activity of soluble STXBPs. Therefore, the main objective of this thesis was to examine various aspects of the biological relationship between STX and solubl...
Energetic Localization of Saxitoxin in Its Channel Binding Site
Biophysical Journal, 2002
Saxitoxin (STX) selectively blocks the voltage-gated sodium channel at the outer vestibule lined by P-loops of the four domains. Neosaxitoxin has an additional-OH group at the N1 position of the 1,2,3 guanidinium (N1-OH) that interacts with domains I and IV of the Na ϩ channel. Determination of a second toxin interaction with the channel would fix the location of STX. Gonyautoxin 2,3 and Gonyautoxin 1,4 are C-11 sulfated derivatives of saxitoxin and neosaxitoxin, respectively. We used these variants to constrain the STX docking orientation by energetically localizing the C-11 sulfate in the outer vestibule. Interactions between the C-11 sulfate and each of the four domains of the channel were determined by a systematic approach to mutant cycle analysis in which all known carboxyl groups important for site 1 toxin binding were neutralized, allowing energetic triangulation of the toxin sulfate and overcoming some limitations of mutant cycles. Toxin IC 50 s were measured by two-electrode voltage clamp from Xenopus oocytes injected with the channel mRNA. Three unique types of analysis based on the coupling results localized the C-11 sulfate between domains III and IV. Combined with our previous report, the data establish the orientation of STX in the outer vestibule and confirm the clockwise arrangement of the channel domains.
Extended Low-Dose Exposure to Saxitoxin Inhibits Neurite Outgrowth in Model Neuronal Cells
Basic & Clinical Pharmacology & Toxicology, 2016
The potent neurotoxin saxitoxin (STX) belongs to a group of structurally related analogues produced by both marine and freshwater phytoplankton. The toxins act by blocking voltage-gated sodium channels stopping the inflow of sodium ions and the generation of action potentials. Exposure from marine sources occurs as a result of consuming shellfish which have concentrated the toxins, and freshwater exposure can occur from drinking water although there have been no acute poisonings from the latter source to date. Previously, the majority of research into this group of toxins, collectively known as the paralytic shellfish toxins, has focused on acute exposure resulting in paralytic shellfish poisoning. While acute exposure guidelines exist for both sources, there are no chronic exposure guidelines and there has been minimal research into this pattern of exposure despite the known role of electrical activity in neurogenesis. We aimed to investigate this pattern of exposure and its potential effects on neurodevelopment using model neuronal cells. PC12 and SH-SY5Y cells were exposed to STX (0.25-3 lg/l) for 7 days, after which time they were stained with TRITC-Phalloidin, to observe adverse morphological effects. Cells exposed to STX had a significant decrease (18-85%) in long axonlike projections, instead exhibiting a significant increase in shorter projections classified as filopodia (p < 0.05). The results suggest that extended low-dose exposure to STX can inhibit proper neurite outgrowth at concentrations well below guideline levels for both sources of exposure making it a potential public health concern.
Proceedings of The National Academy of Sciences, 1986
The effect of two pL-conotoxin peptides on the specific binding of [3H]saxitoxin was examined in isolated plasma membranes of various excitable tissues. pt-Conotoxins GITIA and GIHIB inhibit [3H]saxitoxin binding inlEkctrophorus electric organ membranes with similar Kds of %50 x 10-9 M in a manner consistent with direct competition for a common binding site. GITIA and GIIIB similarly compete with the majority (80-95%) of [3Hlsaxitoxin binding sites in rat skeletal muscle with Kds of -25 and "140 x 10-9 M, respectively. However, the high-affinity saxitoxin sites in lobster axons, rat